Heightened awareness of polycyclic aromatic hydrocarbons (PAHs) has become prevalent due to urban background levels found in surface water, soil, air, cosmetics and food. They are generated by the combustion of fossil fuels and are always found as a mixture of individual compounds that differ in behavior, environmental distribution, and their effect on biological systems. PAHs encompass a wide molecular weight range, differing based on their physical, chemical, and biological characteristics. PAHs in surface water result from a variety of sources including residential, industrial and commercial outlets, streets and parking lots, and atmospheric fallout. In this application, via a spiking experiment, we explore the levels at which PAHs in surface water can be monitored by UHPLC with a sub-2 μm particle sized column combined with photo diode array (PDA) and fluorescence (FL) detection.

1. Introduction
Heightened awareness of polycyclic aromatic
hydrocarbons (PAHs) has become prevalent due
to urban background levels found in surface
water, soil, air, cosmetics and food. They are
generated by the combustion of fossil fuels and are always found as a mixture of individual
compounds that differ in behavior, environmental distribution, and their effect on biological
systems. PAHs encompass a wide molecular weight range, differing based on their physical,
chemical, and biological characteristics.
Lower molecular weight PAHs, in general, are quite water soluble and have significant acute
toxicity to invertebrate aquatic organisms. Naphthalene is structurally the simplest PAH and is
especially water soluble. It is the most abundant component of road, driveway and parking
lot sealcoating. The U.S. Environmental Protection Agency (EPA) has classified naphthalene as
a Group C possible human carcinogen.1
PAHs in Surface
Water by PDA and
Fluorescence Detection
A P P L I C A T I O N N O T E
Authors:
Catharine Layton
Wilhad M. Reuter
PerkinElmer, Inc.
Shelton, CT
Liquid Chromatography

2. 2
High molecular weight PAHs are expected to bio-accumulate in
aquatic organisms such as oysters, rainbow trout, bluegills and
zooplankton.2
They have low solubility in water and bind very
strongly to soils, sediments and particulate matter. Benzo(a)
pyrene, a by-product of incomplete combustion or burning
of organic material, such as cigarettes, gasoline and wood, is
considered to be one of the most carcinogenic high molecular
weight PAHs.3
PAHs in surface water result from a variety of sources including
residential, industrial and commercial outlets, streets and parking
lots, and atmospheric fallout. In general, most surface waters
contain individual PAHs at levels up to 0.05 µg/L (ppb), but
highly polluted rivers can have concentrations of up to 6 ppb.
The PAH levels in groundwater and drinking water are typically
within the range of 0.0002 to 0.0018 ppb. Levels of individual
PAHs in rainwater range from 0.01 to 0.2 ppb, whereas, in highly
urban areas, levels of up to 1 ppb have been detected in snow
and fog.4
The EPA has developed ambient water quality criteria
to protect human health from the carcinogenic effects of PAH
exposure. The current recommendation is a maximum contaminant
level (MCL) of no greater than 0.2 ppb in drinking water.5
A pre-concentration step, often solid phase extraction, is
necessary to concentrate PAHs in aqueous environmental
samples to detectable levels by PDA and fluorescence. In this
application, via a spiking experiment, we explore the levels at
which PAHs in surface water can be monitored by UHPLC with
a sub-2 µm particle sized column combined with photo diode
array (PDA) and fluorescence (FL) detection.
Experimental
Hardware/Software
A PerkinElmer Altus™
UPLC®
system was used, including the
A-30 Solvent Module (quaternary pump), Sample Module
and Column Module (PerkinElmer, Shelton, CT, USA). A
PerkinElmer Brownlee™ HRes DB PAH 1.9 μm 50 x 2.1 mm
column (PerkinElmer, Shelton, CT, USA) was used for all
separations. Detection was accomplished with PerkinElmer
Altus A-30 PDA and FL detectors (PerkinElmer, Shelton, CT,
USA). All instrument control, analysis and data processing
was performed via Waters®
Empower®
3 CDS software.
Method Parameters
The HPLC method parameters are shown in Table 1.
Solvents, Standards and Samples
All solvents and diluents used were HPLC grade.
A stock PAH standard mixture was obtained from PerkinElmer
(Part # 00891543; PerkinElmer, Shelton, CT). The stock mixture
contained: naphthalene (1000 ppm), acenaphthylene (1000
ppm), 1-methylnaphthalene (1000 pm), 2-methylnaphthalene
(1000 ppm), acenaphthene (1000 ppm), fluorene (500 ppm),
phenanthrene (500 ppm), anthracene (1000 ppm), fluoranthene
(500 ppm), pyrene (500 ppm), benz(a)anthracene (500 ppm),
chrysene (500 ppm), benzo(j)fluoranthene (500 ppm), benzo(b)
fluoranthene (500 ppm), benzo(k)fluoranthene (500 ppm),
benzo(a)pyrene (500 ppm), dibenz(a,h)anthracene (500 ppm),
benzo(g,h,i)perylene (500 ppm) and ideno(1,2,3-cd)pyrene
(500 ppm) in methylene chloride.
In a 25-mL volumetric flask, a stock PAH standard solution was
prepared in methanol to equal the final standard concentration
of 1 ppm/2 ppm, depending on the analyte. The solution was
further diluted in methanol to 0.5 ppm/1 ppm for PDA detection
and to 10 ppb/20 ppb for fluorescence detection. These solutions
were used to determine linearity at ppm and ppb levels for the
PDA and FL detectors, respectively.
Surface water was collected from: 1) the Housatonic River,
downstream of a water treatment plant; 2) a suburban driveway
with sealcoating; and 3) the surface surrounding a gas station
pump area. Each water sample was spiked 1:1 with the PAH
standard solution mix. Recoveries for two PAHs, naphthalene and
benzo(a)pyrene, were calculated using both PDA and
fluorescence detection.
Prior to injection all samples were filtered through a 0.22-µm
filter to remove insoluble particles.
Results and Discussion
Figure 1 shows the peak profile of the 19 PAHs at a
concentration of 1 ppm/2 ppm. The upper chromatogram,
(a), was collected by PDA, while the lower chromatogram,
(b), was collected by fluorescence. Separation for all of the
19 PAHs was achieved in less than 4.5 minutes and most were
quantifiable down to ppb levels, as specified in Table 1. For
fluorescence detection, fluoranthene and indeno(1,2,3,-cd)
pyrene are not detected using the excitation and emission
wavelengths specified in the method.
Table1.HPLCMethodParameters.
HPLC Conditions
Column:
PerkinElmer Brownlee HRes 1.9 µm 50 x 2.1 mm
DB PAH, Part # N9303995
Mobile Phase
Gradient:
Solvent A: water
Solvent B: acetonitrile
PDA Detector: Wavelength: 254 nm
FL Detector:
Step 1: Ex 280 nm / Em 340 nm
Step 2: Ex 280 nm / Em 390 nm at 1.52 mins
Oven Temp.: 35 ºC
Injection Volume: 1 µL
Sampling (Data) Rate: 10 pts./sec
Time
(min)
Flow Rate
(mL/min)
%A %B %C %D Curve
1 Initial 0.700 50.0 50.0 0.0 0.0 Initial
2 4.00 0.700 5.0 95.0 0.0 0.0 6
3 4.50 0.700 5.0 95.0 0.0 0.0 6
4 8.00 0.700 50.0 50.0 0.0 0.0 6

3. 3
As shown in Figure 2, chromatographic repeatability was confirmed via ten injections of the 1 ppm/2 ppm PAH standard mixture,
(PDA at 254 nm).
Figure2.Chromatographic repeatability of the 19 PAH standard mixture by PDA at 254 nm.
Overlay Report
Injection Id 2468, 2464, 2480, 2455, 2492,
2484, 2476, 2472, 2488, 2460
Sample Set Id 2412
Chromatogram Overlay with Z Axis Offset
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
0.055
0.060
0.065
0.070
0.075
Minutes
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
Figure1.UHPLC chromatogram showing a standard mixture of 19 PAHs at 1 ppm/2 ppm concentrations by a) PDA at 254 nm; b) fluorescence: Ex/Em wavelengths as
specified in method.
Minutes
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
1
2 3
4
5
6
7
8
9
10
11
12
14
15
16
17
18
19
a)
b)
1. Naphthalene
2. Acenaphthylene
3. 1-Methylnaphthalene
4. 2- Methylnaphthalene
5. Acenaphthene
6. Fluorene
7. Phenanthrene
8. Anthracene
9. Fluoranthene
10. Pyrene
11. Benzo(k)antharcene
12. Chrysene
13. Benzo(j)fluoranthene
14. Benzo(b)fluoranthene
15. Benzo(k)fluoranthene
16. Benzo(a)pyrene
17. Dibenzo(a,h)anthracene
18. Benzo(g,h,i)perylene
19. Indeno(1,2,3-cd)pyrene
13

4. 4
Linearity, at ppm levels by PDA detection and ppb levels by
fluorescence, was determined for each of the 19 PAHs using a
1:1 water-diluted stock solution. Representative of the 19 PAHs,
the linearity plots for both naphthalene and benzo(a)pyrene are
shown below.
In Figure 4, linearity for benzo(a)pyrene between 0.25-2.5 ppm is shown for PDA at 254 nm. Linearity is also demonstrated from
5-50 ppb by fluorescence detection (Ex/Em wavelengths as specified in method).
In Figure 3, we demonstrate linearity for naphthalene between
0.5-5 ppm by PDA detection at 254 nm. Similarly, linearity is
demonstrated from 20-100 ppb by fluorescence detection
(Ex/Em wavelengths as specified in method).
Figure4.Linearityplotsofbenz(a)pyrenea)0.25-2.5ppmbyPDA;b)5-50ppbbyfluorescence.
CLCalReportIndivPUBLICATI
InjectionId****
SampleSetId****
ProcessingMethod: PAH_FLR_PM ProjectName: PAH
ProcessingMethodID: 3977 System: AcquitywithFLR
5030CalibrationID:ACQUITYFLRChAChannel:
Proc.Chnl.Descr.: ****
1/23/201511:53:10AMESTDateCalibrated:
CalCurveId5044
Area
0
50000
100000
150000
200000
Amount
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00
B
Benzo(a)pyrene
FL, ppb
CLCal Report Individuals
InjectionId****
SampleSet Id****
ProcessingMethod: CELPAHsppm Project Name: PAH
ProcessingMethodID: 5925 System: AcquitywithFLR
6193CalibrationID:PDACh1254nm@4.8nmChannel:
Proc. Chnl. Descr.: ****
1/30/20158:36:57AMESTDateCalibrated:
Area
-10000.0
0.0
10000.0
20000.0
30000.0
40000.0
50000.0
60000.0
Amount
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40
A
Benzo(a)pyrene
PDA, ppm
R2
= 0.99995 R2
= 0.99988
Figure3.Linearityplotsofnaphthalenea)0.5-5ppmbyPDA;b)20-100ppbbyfluorescence.
B
Naphthalene
FL, ppb
CEL Cal Curve
Project Name: PAHsPAHReported by User: System
Report Method: CEL Cal Curve Date Printed:
5361 2/2/2015Report Method ID: 5361
3:47:55 PM US/EasternPage: 1 of 1
Name: Napthalene; Processing Method: PAH_FLR_PM; Fit Type: Linear (1st Order); Cal Curve
Id: 5031; A: -1.474686e+003; B: 3.617692e+003; R^2: 0.999819; Equation Y = 3.62e+003 X -
1.47e+003; Units ppb
Area
-50000
0
50000
100000
150000
200000
250000
300000
350000
400000
Amount
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
Error Log
All Peaks Table group contains information that doesn't match the data being reported.
A
Naphthalene
PDA, ppm
R2
= 0.99988 R2
= 0.99982
Area
-2000.0
0.0
2000.0
4000.0
6000.0
8000.0
10000.0
12000.0
14000.0
16000.0
Amount
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

5. 5
Table2.PAHsandtheircalculatedLOQsandLODs,byPDAandFLdetection.
Polyaromatic
Hydrocarbon
RT (min)
PDA LOQ
(ppb)
PDA LOD
(ppb)
FL LOQ
(ppb)
FL LOD
(ppb)
Naphthalene 0.81 207 62 17 5
Acenaphthylene 0.99 343 103 ND ND
1-Methylnaphthalene 1.12 376 113 8 2
2-Methylnaphthalene 1.18 286 86 9 3
Acenaphthene 1.26 598 179 4 1
Fluorene 1.33 56 17 10 3
Phenanthrene 1.52 26 8 38 11
Anthracene 1.70 13 4 227 68
Fluoranthene 1.94 110 33 ND ND
Pyrene 2.08 146 44 6 2
Benzo(a)anthracene 2.65 52 16 2 1
Chrysene 2.75 33 10 11 3
Benzo(j)fluoranthene 3.12 134 40 11 3
Benzo(b)fluoranthene 3.24 48 14 64 19
Benzo(k)fluoranthene 3.44 74 22 45 13
Benzo(a)pyrene 3.61 57 17 15 5
Dibenzo(a,h)anthracene 3.99 232 69 7 2
Benzo(g,h,i)perylene 4.12 146 44 26 8
Indeno(1,2,3-cd)pyrene 4.25 67 20 ND ND
ND = Not Detected
As listed in Table 2, LOQ and LOD levels were established for each of the PAHs, based upon a s/n of > 10/1 for LOQ and > 3/1 for LOD.
It should be noted that for all PAHs, the LOQs established by PDA were below 1 ppm.
As can be observed in Figure 5, no quantifiable PAHs were found in any of the three unspiked, undiluted surface waters analyzed. In order
to visualize the amount of PAHs recoverable by PDA and FL detection, surface water samples were spiked with the PAH standard mixture,
as shown in Figure 6.
Figure5.Fluorescence overlay of 10 ppb/20-ppb PAH standard mixture (black) with undiluted surface water from the Housatonic River (blue), a suburban driveway with
sealcoating (green), and a gas station pump area (red).
OverlayReport FLR
ChromatogramOverlaywithZAxisOffset
SampleName: Sunocopuddlefiltered5ul; DateAcquired: 1/26/20151:17:57PMEST
SampleName: driveway filter 5ul; DateAcquired: 1/26/20151:43:21PMEST
SampleName: stratfordfilter 5ul; DateAcquired: 1/26/20151:56:04PMEST
SampleName: Std210ppb+20ppb; DateAcquired: 2/1/201510:46:50PMEST
EU
-2.00
0.00
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20.00
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24.00
26.00
28.00
30.00
Minutes
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
10 ppb/20 ppb PAH Standard Mixture by Flourescence
Housatonic River
Suburban Driveway with Sealcoating
Gas Station Pump Area

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Conclusion
This work has demonstrated the fast, effective chromatographic
separation of 19 PAHs using a PerkinElmer Altus UPLC®
system with
A-30 PDA and FL detectors. The results exhibited exceptional
linearity for each PAH over the tested concentration ranges.
Though none of the analyzed surface waters showed any
detectable amount of PAHs, the naphthalene and benzo(a)pyrene
spike recovery analysis demonstrated the ability of the A-30 PDA
and A-30 FL detectors to detect PAHs at ppb levels.
References
1. U.S. Environmental Protection Agency, Hazard Summary –
Naphthalene, 2000
2. U.S. Centers for Disease Control Agency for Toxic Substances
and Disease Registry (ATSDR) “Toxic Profile for Polyaromatic
Hydrocarbons (PAHs)”. 1995.
3. U.S. Environmental Protection Agency, “IRIS Chemical
assessment Tracking System: Benzo(a)pyrene”, 2006
4. Prabhukumar, Giridhar; et. al.; “Polycyclic Aromatic
Hydrocarbons in Urban Runoff – Sources, Sinks and Treatment:
A Review”, 2010
5. U.S. Environmental Protection Agency, Nation Primary Drinking
Water Regulations, 2015
Figure6.Fluorescence overlay of surface water from the Housatonic River (blue), a suburban driveway with sealcoating (green), and a gas station pump area (red), each spiked
1:1 with the 10 ppb/20-ppb PAH standard mixture (black).
Injection Id 1097, 1088, 1106, 1021 Sample Set Id
Chromatogram Overlay with Z Axis Offset
Sample Name: Acc Sunoco1 10+20ppb oncol; Date Acquired: 2/2/2015 4:05:20 PM EST
Sample Name: Acc Drive 1, 10+20ppb oncol; Date Acquired: 2/2/2015 3:02:23 PM EST
Sample Name: Acc Stratford 1, 10+20ppb oncol; Date Acquired: 2/2/2015 2:11:52 PM EST
Sample Name: Std1 10ppb + 20ppb; Date Acquired: 2/2/2015 12:30:58 PM EST
EU
-2.00
0.00
2.00
4.00
6.00
8.00
10.00
12.00
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18.00
20.00
22.00
24.00
26.00
28.00
30.00
Minutes
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
10 ppb/20 ppb PAH Standard Mixture by Fluorescence
Housatonic River
Suburban Driveway with Sealcoating
Gas Station Pump Area
Recoveries for naphthalene and benzo(a)pyrene, in each spiked
sample, are shown in Table 3. Recovery was determined at both
ppm and ppb levels, using both PDA and fluorescence detection.
Naphthalene and benzo(a)pyrene are reported for each water
sample, using the PDA and fluorescence detectors at ppm and
ppb levels. Recoveries were between 89-107 %.
Table3.Recoveriesofnaphthaleneandbenzo(a)pyreneinspikedsurfacewatercollected
fromtheHousatonicRiver,asuburbandrivewaywithsealcoating,andgasstationpump
area,eachdiluted1:1withmethanol.
PAH of Interest
Housatonic
River
% Recovery from
Surface Water
Suburban Driveway
with Sealcoating
Gas Station
Pump Area
PDA Detection
1-ppm benzo(a)pyrene 107 104 107
2-ppm naphthalene 102 101 98
FL Detection
10-ppb benzo(a)pyrene 102 100 102
20-ppb naphthalene 89 92 89