American Journal of Anal yt ical Chemistry, 2011, 2, 971-978
doi:10.4236/ajac.2011.28113 Published Online December 2011 (http://www.SciRP.org/journal/ajac)
Method Validation for SPE Applied to Determination of
PAH in Petroliferous Industry Effluent Water
José Ricardo Lima Bispo, Sandro Navickiene, Haroldo Silveira Dórea*
Laboratório de Análise de Compos tos Orgânicos Poluentes, Departamento de Química,
Universidade Federal de Sergipe, São Cristóvão, Brazil
Received May 9, 2011; revised June 26, 2011; accepted July 9, 2011
Abstract
The presence of polycyclic aromatic hydrocarbons (PAH) in produced water is of environmental concern due
to their toxic properties. PAH analysis in complex samples requires pre-treatment to enrich the fraction con-
taining analytes, and eliminate matrix interferences. The objective of this work was to develop and validate
an analytical methodology for determination of PAH in produced water, using solid phase extraction (SPE)
and analysis by gas chromatography with flame ionization detection (GC-FID). Average recoveries of PAH
from produced water enriched at two concentration levels varied from 30.9% for naphthalene to 119.1% for
chrysene (RSD between 3.8% and 22.2%). The linear range was between 0.5 and 50.0 µg·mL–1, with regres-
sion coefficients better than 0.998. Detection limits were between 0.01 and 0.04 µg·L–1, and quantitation lim-
its were between 0.05 and 0.16 µg·L–1. The validated method was applied to samples of produced water
treated for disposal, in which concentrations varied from 3.5 µg·L–1 for phenanthrene to 44.3 µg·L–1 for
naphthalene ( = 177.7 μg·L–1). The method was also applied to seawater samples, in which 13 PAH
compounds were detected (
PAH

PAH

= 60.27 μg·L–1), probably derived from pyrogenic sources.
Keywords: Produced Water, Effluent, PAH, SPE
1. Introduction
One of the most significant by-products of petroleum
extraction is produced water, due to the large volumes
that are discharged or reused, as well as its chemical
composition, which can vary greatly depending on the
characteristics of the underlying geology. It is a highly
complex aqueous matrix, containing organic and inor-
ganic, soluble and insoluble, compounds derived from
petroleum fractions, with high levels of salinity (up to
250 g·L–1). Hydrocarbons predominate, with the major
groupings including alkanes, alkenes, alkynes, complex
hydrocarbons containing oxygen, nitrogen and sulphur,
volatile monoaromatic compounds, and polycyclic aro-
matic hydrocarbons (PAH) [1-4].
Although around a hundred PAH are found in the en-
vironment, only 16, possessing 2 to 6 aromatic rings, are
considered as priority compounds due to their toxicity,
mutagenicity or carcinogenicity [5]. The solubility of
these compounds in water varies greatly, and reduces as
molecular mass increases [6-8].
Liquid-liquid extraction (LLE) is the technique most
frequently used for analysis of PAH in complex matrices
including petroliferous wastewaters [3,5]. Other studies
of wastewaters have used Solid Phase Microextraction
(SPME)9 or Solid Phase Extraction (SPE) [10-12]. Direct
mode SPME is not feasible, due to attack on comer-
cially used fibres by compounds present in produced
water. Headspace mode SPME cannot efficiently extract
the 5 to 6 ring PAH. In contrast to liquid-liquid extrac-
tion, SPE does not require large volumes of organic sol-
vents, hence generating little laboratory waste, and
analysis time can be greatly reduced, particularly when
several cartridges are used simultaneously. The wide
variety of available adsorbents means that extraction
conditions can be adapted to achieve desired separation
and preconcentration goals.
While SPE is a technique that seems to offer advan-
tages compared to liquid-liquid extraction, in routine
analysis laboratories new techniques using this procedure
need to be validated so that PAH determinations in com-
plex water samples can be undertaken with confidence.

J. R. L. BISPO ET AL. 972
The objective of this work was to validate an analytic-
cal method for determination of 16 PAH, including 15 of
the 16 compounds designated as priority pollutants by
the United States Environmental Protection Agency, in
produced water, using solid phase extraction and analysis
by gas chromatography with flame ionization detection.
2.Experimental
2.1. Standards, Reagents and Solvents
Methanol, ethyl acetate, toluene, dichloromethane, ace-
tone and n-hexane were nanograde (Merck, Darmstadt,
Germany), and dichlorodimethylsilane was 99% (ACROS
Organics, New Jersey, USA). Analytical grade anhy-
drous sodium sulfate was supplied by Merck. “Prepsep”
C-18 (500 mg, 6 mL) cartridges were from Fisher Scien-
tific (Pittsburgh, PA, USA).
Certified 2000 g·mL–1 standards of the individual
PAH in dichloromethane/benzene (1:1) were purchased
from Ultra Scientific. The 16 compounds investigated
were naphthalene (Nap), acenaphthylene (Acpt), ace-
naphthene (Ace), fluorene (Flu), phenanthrene (Phe),
anthracene (Ant), fluoranthene (Flt), pyrene (Py), benz[a]
anthracene (BaA), chrysene (Chry), benzo[b]fluoran-
thene (BbF), benzo[k]fluoranthene (BkF), benzo[a]
pyrene (BaP), dibenzo[b,c]fluoranthene (DBF), dibenz
[a,h]anthracene (DBA), and benzo[g,h,i]perylene (BPe).
PAH stock solutions were prepared in dichloromethane
at 200 g·mL–1 and stored at –10˚C. Working standard
solutions were prepared by diluting the stock solutions in
dichloromethane as required.
2.2. Sampling
Produced water samples were collected from the lower
part of the treatment station tank of a petroleum produc-
tion company located in the city of Carmópolis, Sergipe,
Brazil. The water had previously been processed for re-
moval of the bulk of the oil content. For subsequent re-
cycing and reuse for injection into wells during petro-
leum extraction [13], or discharge into the sea, the pro-
duced water passes through further processes involving
filtration and chemical treatment. The samples were col-
lected in amber glass flasks, fitted with closures lined
with aluminium foil, and preserved by storing at 4˚C
prior to extraction.
2.3. Sample Preparation
5 mL aliquots of methanol were added to 100 mL sub-
samples of unfiltered produced water. The cartridges
containing the C-18 solid phase were each conditioned
using 20 mL dichloromethane, 10 mL acetone, 20 mL
methanol, and finally 20 mL ultrapure water, and then
dried for 3 minutes. The water sample was adjusted to
pH 7 with sulfuric acid (1:1), and then passed through
the C-18 cartridge. The PAH were eluted with 5 mL
acetone and 30 mL hexane. The organic extract was
concentrated in a rotary evaporator (50˚C, 60 rpm),
transferred to a glass column filled with 5 g anhydrous
sodium sulfate on silanized glass wool, and PAH were
eluted from the column using 20 mL dichloromethane.
After further concentration using the rotary evaporator,
the extract was transferred to a 1 mL volumetric flask,
and reduced under a flow of nitrogen before analysis.
2.4. Chromatographic Conditions
Quantification of PAH was performed using a Shimadzu
(Kyoto, Japan) Model 17A gas chromatograph equipped
with a flame ionization detector and split/splitless inject-
tor. The column was a Hewlett Packard (USA) fused-
silica HP-5 (30 m × 0.25 mm × 0.25 µm film thickness),
and 99.995% purity helium was used as carrier gas.
Temperatures of the injector (splitless mode, 2 min) and
detector were 250˚C and 300˚C, respectively. The col-
umn temperature was programmed as follows: 40˚C for 1
min, increasing to 160˚C at 25˚C min–1 and to 270˚C at
5˚C min–1, with a hold for 11 min. The flow rate through
the column was 1.32 mL·min-1, and the injection volume
was 1 µL.
3. Results and Discussion
Several parameters were examined at the sample prepa-
ration stage in order to optimize the performance of the
SPE technique. Method validation was undertaken after
formalization of the PAH extraction procedure, with re-
coveries determined using enriched produced water
blank samples.
3.1. Filtration of the Matrix
Possible losses by sorption onto particulate matter pre-
sent in the produced water, due to low compound solu-
bility, were examined by filtration of three samples under
vacuum through 0.45 μm cellulose acetate filters, and
comparison with three unfiltered samples. PAH concen-
trations were lower for the filtered samples, and fluorene,
phenanthrene, chrysene, benzo[k]fluoranthene, dibenzo
[b,c]fluoranthene, dibenz[a,h]anthracene and benzo[g,h,i]
perylene were not detected. With the exception of fluo-
rene and phenanthrene, the PAH investigated possess
between four and six aromatic rings. Hence, these com-
pounds are strongly adsorbed onto matrix particulates.

J. R. L. BISPO ET AL. 973
In the absence of filtration, there was partial blockage
of the C18 solid phase, which resulted in longer extrac-
tion times. This phase of the work resulted in the defini-
tion of a minimum sample volume without compromise-
ing sensitivity and the ability to quantify PAH, as a result
of which subsample volumes were fixed at 100 mL.
3.2. Organic Solvents
The solvents used for conditioning the C18 cartridges
and for subsequent elution were based on USEPA
Method 3535 [14]. Dichloromethane, n-hexane and an
n-hexane/dichloromethane mixture (70/30, v/v) were
chosen for the elution test (in triplicate) of PAH through
the C18 solid phase. Use of hexane gave the best recov-
ery, varying between 37.0% for benzo[a]pyrene and
72.2% for acenaphthylene, while recoveries using the
70/30 v/v n-hexane/dichloromethane mixture were be-
tween 18.4% for benzo[a]pyrene and 61.2% for fluoran-
thene. Dichloromethane gave the lowest recoveries. Hence,
n-hexane was selected as elution solvent.
3.3. Silanization
PAH adsorption was studied using both silanized and
standard glassware. Silanization was achieved according
to the procedure described by Doong [13], which re-
quires filling all internal volumes with a solution of 10%
dichlorodimethylsiloxane in toluene, and leaving for 8
hours. Subsequently the glassware was washed with tolu-
ene and methanol, and finally dried in a drying cabinet at
120˚C. For unsilanized glassware, the average recovery
for 14 PAH (acenaphthene and benzo[b]fluoranthene
were not extracted) was 54.0%. With silanization, the
average recovery of all 16 PAH was 70.0%.
3.4. Sample pH
Since the hydrogen ion concentration influences sample
preservation, tests were undertaken at pHs 2, 4 and 7. At
acid pH, there was a decrease in recovery of between
10% and 20% compared to neutral pH. The best recov-
eries were obtained at pH 7 (Figure 1).
3.5. Solubilization of PAH in Produced Water
Due to the low solubility of PAH in water, which de-
creases as molecular mass increases, measurement errors
can be incurred as a result of sorption of the compounds
onto the walls of the glassware used throughout the pro-
cedure [15,16]. Addition of co-solvents or organic modi-
fiers (methanol, acetonitrile or 2-propanol) is a technique
used to increase the solubility of PAH [15,17,18]. In this
work, methanol and acetonitrile were chosen, with a 5.0
mL volume of each solvent being added to the sample
containing the PAH standard. Using methanol, recover-
ies varied from 45.9% for naphthalene to 93.9% for
chrysene. Using acetonitrile, the variation was between
29.9% for naphthalene and 90.2% for dibenzo[b,c]
fluoranthene. Although both solvents showed similar
performance, slightly better results were obtained using
methanol.
3.6. Comparison of FID and PID Detectors
The photoionization detector (PID) is capable of good
0
10
20
30
40
50
60
70
80
90
100
Recuper ação (%)
HPA
Am - 1 pH 2
Am - 2 pH 4
Am - 3 pH 7
Figure 1. PAH recoveries obtained at pHs 2, 4 and 7.

J. R. L. BISPO ET AL. 974
sensitivity for detection of the volatile monoaromatic
hydrocarbons benzene, toluene, ethylbenzene, and the
xylene isomers [2]. The operating conditions of this de-
tector have been reported previously [1,2]. A test was
undertaken to measure the performance of the PID for
PAH. At a concentration of 0.4 g·mL–1, only the first
seven compounds eluting from the column were detected,
while all 16 PAH were detected at a concentration of 5
g·mL–1.
3.7. Validation of the SPE Method
Validation was based on parameters defined in standard
protocols describing chromatographic methods [15-17].
3.8. Linearity
A five-point calibration curve was constructed for each
compound over the concentration range of interest (0.5 -
50 μg·mL–1), using external standards. Calibration curve
regression coefficients (R2) were higher than 0.998 in all
cases (Table 1).
3.9. Recovery
Recoveries were measured at two enrichment levels, 10
and 100 μg·L–1 (Table 2). At the first level (10 μg·L–1),
recoveries varied from 55.0% for naphthalene to 93.1%
for anthracene. At the second level (100 μg·L–1) recover-
ies were from 30.9% for naphthalene to 119.1% for
chrysene. At these levels, the efficiency of the SPE
method was satisfactory, with recoveries lying close to
the range normally considered acceptable, between 70%
and 130% [18] (with the exception of naphthalene). At
the first level, recoveries of acenaphthylene, benzo[a]
pyrene and benzo[g,h,i]perylene were below 70%. Ta-
ble 3 lists previously reported PAH recoveries, with
some values below 30%.
3.10. Precision
Repeatability was measured using relative standard de-
viations (RSD) at two concentration levels (10 and 100
μg·L–1), and ranged from 3.8% to 22.2% (Table 2). All
RSDs were below 30%, the value considered acceptable.
[18] Confidence limits at 95% probability were also ob-
tained for each PAH.
3.11. Limits of Detection (LOD) and
Quantification (LOQ)
These were determined using the standard deviation (n =
Table 1. Analytical curves, and limits of detection and quantification, for the PAH investigated.
PAH Regression equation R2 LOD (µg·L–1) LOQ (µg·L–1)
Naphthalene y = 2362.5x – 204.2 0.99980.02 0.08
Acenaphthylene y = 2315.3x – 216.9 0.99960.02 0.07
Acenaphthene y = 2391.1x – 213.7 0.99960.01 0.05
Fluorene y = 2288.0x – 269.1 0.99950.03 0.11
Phenanthrene y = 2471.6x – 322.2 0.99920.02 0.08
Anthracene y = 2327.7x – 341.3 0.99870.03 0.11
Fluoranthene y = 2449.8x – 207.6 0.99880.03 0.12
Pyrene y = 2379.2x – 331.0 0.99860.03 0.13
Benz[a]anthracene y = 2250.8x – 293.2 0.99940.02 0.08
Chrysene y = 2468.1x – 356.9 0.99800.03 0.13
Benzo[b]fluoranthene y = 2281.6x – 268.1 0.99930.02 0.08
Benzo[k]fluoranthene y = 2312.9x – 228.2 0.99870.03 0.13
Benzo[a]pyrene y = 2067.8x – 134.3 0.99980.03 0.12
Dibenzo[b,c] fluoranthene y = 1336.3x – 269.3 1.00000.04 0.16
Dibenz[a,h]anthracene y = 1591.5x – 321.5 0.99990.04 0.15
Benzo[g,h,i]perylene y = 1579.0x – 277.4 0.99950.03 0.11
LOD: Limit of detection; LOQ: Limit of quantification.

J. R. L. BISPO ET AL.975
Table 2. Recoveries using the proposed SPE method for determination of PAH in saline samples (n = 5).
Recovery (%)
PAH Fortification (μg·L–1)
Mean ± CI1 RSD2
10 55.0 ± 10.26 15.0
Naphthalene
100 30.9  8.53 22.2
10 65.0 ± 12.37 15.3
Acenaphthylene
100 83.3  9.76 9.4
10 71.3 ± 14.57 16.4
Acenaphthene
100 88.1  14.26 13.0
10 77.2 ± 12.80 13.3
Fluorene
100 104.1  14.47 11.2
10 92.1 ± 15.51 13.5
Phenanthrene
100 115.8  11.88 8.3
10 93.1 ± 10.48 9.0
Anthracene
100 107.5  12.57 9.4
10 78.8 ± 18.29 18.7
Fluoranthene
100 111.7  9.50 6.9
10 84.1 ± 11.72 11.2
Pyrene
100 107.2  7.60 5.7
10 92.3 ± 10.33 9.0
Benz[a]anthracene
100 117.7  8.66 6.0
10 85.5 ± 10.87 10.2
Chrysene
100 119.1  5.59 3.8
10 72.3 ± 10.51 11.7
Benzo[b]fluoranthene
100 90.4  5.93 5.3
10 81.4 ± 20.13 19.9
Benzo[k]fluoranthene
100 95.0  5.12 4.3
10 58.1 ± 14.01 19.4
Benzo[a]pyrene
100 91.1  11.27 10.0
10 77.4 ± 20.77 21.6
Dibenzo[b,c]fluoranthene
100 71.8  4.33 4.9
10 68.0 ± 9.38 11.1
Dibenz[a,h]anthracene
100 69.7  3.92 4.5
10 56.9 ± 11.85 16.8
Benzo[g,h,i]perylene
100 71.8  4.27 4.8
1CI: confidence interval; 2RSD: relative standard deviation.

J. R. L. BISPO ET AL. 976
Table 3. Previously reported PAH recoveries for aqueous matrices.
Recovery (%)
PAH (SPE)1
Subsoil water
(SPE)2
Milli-Q water
(SPE)3
Sewage
(LLE)4
Seawater
(SPE)4
Seawater
(LLE)5
Seawater
(LLE)6
Seawater
1.Nap 35 99 nd nd x
2.Acpt 46 104 nd nd x
3.Ace 105 64 nd nd x
4.Flu 97 x 93 nd nd x x
5.Phe 102 72 10 52 x x
6.Ant 86 82 3 10 x x
7.Flt 113 81 13 34 x x
8.Py 112 90 13 32 x x
9.BaA 68 x 106 12 23 x
10.Chry 67 96 13 18 x x
11.BbF 86 x 91 14 25 x
12.BkF 73 x 87 13 18 x
13.BaP 61 x 77 7 10 x x
14.DBF 63 x 81 13 22 x
15.DBA 58 x 72 16 20 x
16.BPe 67 x 69 13 16 x
Range 35 - 112 95 - 104 64 - 106 3 - 16 10 - 52 95 - 112 65 - 92
nd = not detected; x = PAH analysed; 1Martinez et al. (2004); 2Garcia-Falcón et al. (2004); 3Oleszezuk and Baran (2004); 4Filipkowska et al .
(2005); 5Nemr and Abd-Allah (2003); 6Anyakora et al. (2005).
7) of the concentration at the lowest level of enrichment
and in the sample blank (LOD = t95%.s) [19]. Detection
limits varied between 0.01 and 0.04 µg·L–1 (Table 1).
Limits of quantification were calculated as ten times the
standard deviation (n = 7),and varied between 0.05 and
0.16 µg·L–1.
3.12. Comparison with Liquid-Liquid Extraction
The proposed method employing SPE was applied to the
determination of the 16 PAH in samples of produced
water. Mean concentrations varied from 3.5 μg·L–1 for
phenanthrene to 44.3 μg·L–1 for naphthalene, with a total
combined PAH concentration of 177.7 μg·L–1 (Table 4).
Previous work employing liquid-liquid extraction3 found
the following PAH concentrations in produced water:
naphthalene 26.68 μg·L–1; acenaphthylene 0.44 μg·L–1;
acenaphthene 0.34 μg·L–1; fluorene 0.01 μg·L–1; phenan-
threne 0.02 μg·L–1; anthracene 0.03 μg·L–1; fluoranthene
0.01 μg·L–1. The remaining PAH were not detected.
Seawater samples collected near Aracaju, Sergipe
State, were analyzed in order to confirm the wider appli-
cability of the SPE method. The total PAH concentration
was 60.27 μg·L–1 (Table 4), with the ratios between low
molecular weight and high molecular weight compounds,
phenanthrene/anthracene (<10) and fluoranthene/pyrene
(>1), indicating that the likely source was pyrogenic
(combustion). Furthermore, in pyrogenic material there
is a predominance of 4 to 6 ring compounds (∑PAH =
44.0 μg·L–1) relative to 2 to 3 ring compounds (∑PAH =
16.2 μg·L–1) [20,21].
4. Conclusions
An analytical method for determination of the 16 priority
polycyclic aromatic compounds in produced water using
SPE and GC-FID analysis has been validated. The pa-
rameters linearity, recovery, precision, detection limits
and quantification limits were all shown to be acceptable.
The proposed method was compared with liquid-liquid
extraction for real samples of produced water. Using SPE,
15 PAH were detected in these samples, at concentration
levels ranging from 3.5 to 44.3 μg·L–1 ( = 177.7
μg·L–1). Only seven PAH were detected using liq-
uid-liquid extraction. To confirm that the method is suit-
ble for typical saline samples, it was used for analysis of
PAH

a

J. R. L. BISPO ET AL. 977
Table 4. PAH concentrations measured in produced water and seawater (n = 2).
PAH Produced water (μg·L–1) Seawater (μg·L–1)
Naphthalene 44.30 2.64
Acenaphthylene 16.55 2.46
Acenaphthene 10.10 2.53
Fluorene 10.03 2.52
Phenanthrene 3.53 2.39
Anthracene 8.54 3.70
Fluoranthene 3.94 4.44
Pyrene 3.80 2.91
Benz[a]anthracene 8.29 12.95
Chrysene 12.10 9.53
Benzo[b]fluoranthene 6.02 9.13
Benzo[k]fluoranthene 14.44 1.97
Benzo[a]pyrene 18.58 3.10
Dibenzo[b,c]fluoranthene 7.81 nd
Dibenz[a,h]anthracene nd nd
Benzo[g,h,i]perylene 9.67 nd
∑ PAH 177.71 60.27
nd: not detected.
seawater, in which 13 PAH were detected ( =
60.27 μg·L–1).
PAH

5. Acknowledgements
The authors gratefully acknowledge the assistance of
Petrobras/UNSEAL during collection of produced water
samples. Financial support was provided by the Brazilian
agency CNPq (Process No. 461522).
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