A P P L I C AT I O N N O T E
Liquid Chromatography
Authors:
Catharine Layton
Wilhad M. Reuter
PerkinElmer, Inc.
Shelton, CT
PAHs in Surface
Water by PDA and
Fluorescence Detection
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
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),
2
Table 1. HPLC Method Parameters.
HPLC Conditions
Column:
PerkinElmer Brownlee HRes 1.9 µm 50 x 2.1 mm
DB PAH, Part # N9303995
Solvent A: water
Solvent B: acetonitrile
Mobile Phase
Gradient:
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
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
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.
7
12
8
5
6
1
2
16
19
15
4
a)
14
11
9
3
13
10
17
18
b)
0.50
1.00
1.50
2.00
1.Naphthalene
2.Acenaphthylene
3.1-Methylnaphthalene
4. 2- Methylnaphthalene
5.Acenaphthene
2.50
Minutes
6. Fluorene
7. Phenanthrene
8.Anthracene
9.Fluoranthene
10. Pyrene
3.00
11. Benzo(k)antharcene
12. Chrysene
13. Benzo(j)fluoranthene
14. Benzo(b)fluoranthene
15. Benzo(k)fluoranthene
3.50
4.00
4.50
16. Benzo(a)pyrene
17. Dibenzo(a,h)anthracene
18. Benzo(g,h,i)perylene
19. Indeno(1,2,3-cd)pyrene
Overlay Report
Figure 1. 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.
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).
Chrom atogram Overlay with Z Axis Offs et
0.075
0.070
0.065
0.060
0.055
0.050
0.045
AU
0.040
0.035
0.030
0.025
0.020
0.015
0.010
0.005
0.000
0.50
1.00
1.50
2.00
2.50
Minutes
3.00
3.50
4.00
4.50
Figure 2. Chromatographic repeatability of the 19 PAH standard mixture by PDA at 254 nm.
Inj ecti on Id 2468, 2464, 2480, 2455, 2492,
2484, 2476, 2472, 2488, 2460
Sampl e Set Id 2412
3
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 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).
CEL Cal Curve
16000.0
A
400000
B
14000.0
Naphthalene
PDA, ppm
350000
Naphthalene
FL, ppb
12000.0
300000
200000
Area
250000
8000.0
Area
10000.0
6000.0
150000
4000.0
100000
2000.0
50000
0.0
0
R2 = 0.99988
-2000.0
0.00
0.50
1.00
1.50
2.00
2.50
Amount
3.00
3.50
4.00
4.50
5.00
0.00
CEL PAHs ppm
5925
Project Name:
System:
10.00
20.00
30.00
40.00
50.00
Amount
60.00
70.00
80.00
90.00
100.00
CLName:
Cal Report
Individuals
Napthalene;
Processing Method: PAH_FLR_PM;
Fit Type: Linear (1st Order); Cal Curve
5031; A: PAH_FLR_PM
-1.474686e+003; B: 3.617692e+003;
R^2: 0.999819;
ProcessingId:
Method:
Project Name:
PAH Equation Y = 3.62e+003 X Units
Processing1.47e+003;
Method ID:
3977 ppb
System:
Acquity with FLR
Figure 3. Linearity plots of naphthalene a) 0.5-5 ppm by PDA; b) 20-100 ppb by fluorescence.
Processing Method:
Processing Method ID:
CLCalReportIndivPUBLICATI
R2 = 0.99982
-50000
Channel:
Proc. Chnl. Descr.:
PAH
Acquity with FLR
ACQUITY FLRChA
****
Calibration ID:
5030
PDA Ch1for
254nm@
4.8nm
Cbetween
alibration ID:
6193
InChannel:
Figure 4, linearity
benzo(a)pyrene
0.25-2.5
ppm is shown Date
for
PDA at 254
nm.AMLinearity
is also demonstrated from
Calibrated:
1/23/2015 11:53:10
EST
Proc. Chnl. Descr.:
****
D
ate
C
alibrated:
1/30/2015
8:36:57
AM
EST
5-50 ppb by fluorescence detection (Ex/Em wavelengths as specified in method).
Cal Curve Id 5044
60000.0
Error Log
All Peaks Table group contains information that doesn't match the data being reported.
B
A
200000
Benzo(a)pyrene
PDA, ppm
50000.0
40000.0
Benzo(a)pyrene
FL, ppb
150000
Area
30000.0
Area
100000
20000.0
50000
Reported by User: System
Report Method: CEL Cal Curve
Report Method ID:5361
5361
0
Page: 1 of 1
10000.0
0.0
R2 = 0.99995
-10000.0
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
Amount
1.60
1.80
2.00
2.20
0.00
2.40
Figure 4. Linearity plots of benz(a)pyrene a) 0.25-2.5 ppm by PDA; b) 5-50 ppb by fluorescence.
Injection Id ****
Sample Set Id ****
Injection Id ****
Sample Set Id ****
4
5.00
10.00
Project Name: PAHs\PAH
Date Printed:
2/2/2015
3:47:55 PM US/Eastern
R2 = 0.99988
15.00
20.00
25.00
Amount
30.00
35.00
40.00
45.00
50.00
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.
Table 2. PAHs and their calculated LOQs and LODs, by PDA and FL detection.
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 can be observed in Figure 5, no quantifiable PAHs were found in any of the three unspiked, undiluted surface waters analyzed. In order
Overla
ythe
RePAH
port
FLRmixture,
to visualize the amount of PAHs recoverable by PDA and FL detection, surface water samples were spiked
with
standard
as shown in Figure 6.
ChromatogramOverlaywithZAxis Offset
30.00
28.00
26.00
24.00
10 ppb/20 ppb PAH Standard Mixture by Flourescence
Housatonic River
Suburban Driveway with Sealcoating
Gas Station Pump Area
22.00
20.00
18.00
EU
16.00
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
-2.00
0.50
1.00
1.50
2.00
2.50
M inutes
3.00
3.50
4.00
4.50
Sample Name: Sunoco puddle filtered 5ul; Date Acquired: 1/26/2015 1:17:57 PMEST
mplestandard
Name: d
riveway(black)
filter 5ul;
Da
te Acquire
d: 1/26
/2015from
1:43:2
1P
MEST
Figure 5. Fluorescence overlay of 10 ppb/20-ppbSa
PAH
mixture
with
undiluted
surface
water
the
Housatonic
River (blue), a suburban driveway with
mple Name: stratford filter 5ul; Date Acquired: 1/26/2015 1:56:04 PMEST
sealcoating (green), and a gas station pump areaSa
(red).
Sample Name: Std2 10ppb + 20ppb; Date Acquired: 2/1/2015 10:46:50 PMEST
5
Chrom atogram Overlay with Z Axis Offs et
30.00
28.00
26.00
24.00
10 ppb/20 ppb PAH Standard Mixture by Fluorescence
Housatonic River
Suburban Driveway with Sealcoating
Gas Station Pump Area
22.00
20.00
18.00
EU
16.00
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
-2.00
0.50
1.00
1.50
2.00
2.50
Minutes
Sampl e Name: Acc Sunoco1 10+20ppb oncol ;
3.00
3.50
4.00
4.50
Date Acqui red: 2/2/2015 4:05:20 PM EST
Figure 6. Fluorescence overlay
of surface
water from
theDri
Housatonic
River (blue), aoncol;
suburban driveway
with sealcoating
(green), and
a gas station
pump
area (red), each spiked
Sampl
e Name:
Acc
ve 1, 10+20ppb
Date Acquired:
2/2/2015
3:02:23
PM
EST
1:1 with the 10 ppb/20-ppb
PAH standard
mixture
(black).
Sampl
e Name:
Acc
Stratford 1, 10+20ppb oncol ; Date Acquired: 2/2/2015 2:11:52 PM EST
Sampl e Name: Std1 10ppb + 20ppb;
Date Acqui red: 2/2/2015 12:30:58 PM EST
Recoveries for naphthalene and benzo(a)pyrene, in each spiked
sample, are shown in Table 3. Recovery was determined at both
ppm andInj
ppb
both
PDA1106,
and fluorescence
detection.
ectilevels,
on Idusing
1097,
1088,
1021
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 %.
Table 3. Recoveries of naphthalene and benzo(a)pyrene in spiked surface water collected
from the Housatonic River, a suburban driveway with sealcoating, and gas station pump
area, each diluted 1:1 with methanol.
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
Conclusion
This work has demonstrated the fast, effective chromatographic
Sampl
Id
separation of 19 PAHs
usingeaSet
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
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