Fresenius J Anal Chem (2000) 368 : 697–701
O R I G I N A L PA P E R
M. N. Kayali-Sayadi · S. Rubio-Barroso ·
C. A. Díaz-Díaz · L. M. Polo-Díez
Rapid determination of PAHs in soil samples by HPLC
with fluorimetric detection following sonication extraction
Received: 9 May 2000 / Revised: 17 June 2000 / Accepted: 23 June 2000
Abstract A rapid method for the determination of PAHs
in soil samples based on their extraction with methylene
chloride by sonication and subsequent separation by HPLC
with fluorimetric detection is proposed. A Hypersil Green
PAH column was used with a gradient of acetonitrile/wa-
ter as the mobile phase, together with a program of nine
excitation and emission wavelength pairs. Recoveries
were in the range 70–98%, except for acenaphthene and
naphthalene, at concentration levels 1.08–442 µg/kg with
relative standard deviations in the range 2–15% (n = 4).
Total PAHs found in soil samples were in the range
15–282 µg/kg. The results were compared with those ob-
tained by applying the 3540 EPA method for two samples.
Introduction
Polyaromatic hydrocarbons (PAHs) are well known pollu-
tants in environmental air [1], water [2, 3], soils and sedi-
ments [4–6]. Due to their high toxicity and potential car-
cinogenity they have been the subject of intense analytical
research in environmental studies. The sources of envi-
ronmental PAHs are due to a variety of processes, both
natural and human-controlled [1].
The analytical determination of PAHs in environmen-
tal samples is carried out by gas chromatography (GC)
with flame ionization (FID) or mass spectrometry (MS)
detectors [7–9] and reverse-phase high performance liq-
uid chromatography (RP-HPLC) with photometric (UV)
or fluorimetric (FL) detectors [2, 8, 10–13].
While straightforward solid phase extraction (SPE) on
cartridges or disks is used to extract PAHs from liquid en-
vironmental samples [2, 3], the extraction of these com-
pounds from solid environmental samples is carried out by
M. N. Kayali-Sayadi · S. Rubio-Barroso () · C. A. Díaz-Díaz ·
L. M. Polo-Díez
Department of Analytical Chemistry, Faculty of Chemistry,
Complutense University of Madrid, 28040 Madrid, Spain
e-mail: kayali@eucmax.sim.ucm.es
© Springer-Verlag 2000
pressurized fluid or accelerated solvent extraction (PFE or
ASE) [4, 7, 14], sonication [5, 10], Soxhlet [4, 6], or super-
critical fluid extraction (SFE) alone or with modifiers [8].
Some of these procedures require high solvent volumes or,
and as a result, highly expensive instrumentation.
In this paper, the development of a rapid and economic
method for extraction of PAHs from soil samples is de-
scribed which is only based on solid-liquid extraction
(SLE) by means of ultrasonic agitation using low solvent
volumes. The clean-up step is not necessary. The determi-
nation of PAHs is carried out by HPLC using a program
of pairs of excitation and emission wavelength for fluori-
metric detection.
Experimental
Apparatus and materials. A chromatographic system equipped
with the following components: a Milton Roy CM 4000 high-pres-
sure gradient pump (Rivera Beach, Fl); a Perkin Elmer LS 30 lu-
minescence spectrometer (Norwalk, CT) and a Milton Roy CI
4100 integrator. A Hypersil Green PAH column (100 × 4.6 mm,
5 µm particulate size) by Sandon (England) was used for the sepa-
ration of PAHs; a P-Selecta Precisterm bath was used to maintain
the column temperature below 22 °C (Barcelona, Spain). A P-Se-
lecta ultrasonic bath was used to prepare all the PAHs solutions
and for the extraction of PAHs from samples. A Barna vacuum
pump, a Visiprep vacuum manifold system (Supelco, Bellefonte,
PA) and a P-Selecta Meditronic centrifuge able to apply up to
4800 rpm (3700 g) were also used. The mobile phase solvents
were filtered through Nylon Lida membrane filters (Kenosha, WI)
with 0.45 µm pore size; the organic extracts were also filtered
through PTFE membrane filters, with a 0.5 µm pore size (MFS,
Dublin, CA). The soil samples were screened through a sieve
(Cisa, Spain).
Chemicals. Independent stock standard solutions of PAHs were
prepared by solving solid compounds (Sigma, St. Louis, MO) in
methanol at concentration levels 35–790 mg/L. Working standard
PAH mixtures were prepared by suitable dilution of the stock stan-
dard solutions with methanol (each standard mixture solution ob-
tained independently from the individual stock solutions). HPLC
grade acetonitrile, methanol and cyclohexane (Scharlau, Barce-
lona, Spain) were used. Other chemical reagents were also of
HPLC purity. Water was purified with a Millipore Milli-Q system
(Milford, MA).
698
Procedures
1. Soil sample collection
The soil samples were collected during the 1999 winter from five
zones in Madrid city, three urbane (samples 1, 2 and 3) and two
residential urbane (samples 4 and 5), affected by different pollu-
tion levels of cars and central heating. In all the cases the 1 mm
fraction was taken. Although, the samples were initially quite het-
erogeneous, the fractions were sandy in aspect with a grey color
and a high organic content in the range 120–150 µg/kg for samples
1, 2 and 3, and low organic content 50–80 µg/kg for rest of the
samples.
The number of cars in Madrid city is about 2.5 × 106 moving
with a car frequency of about 1.6 × 105 per day through Glorieta de
Cuatro Caminos in continuous traffic jam, Paseo del Prado which
is near of the Retiro park, and Plaza del Cuzco which is a landscape
square (samples 1, 2 and 3, respectively), and of 2.7 × 104 cars per
day in Ciudad Universitaria and Dehesa de la Villa which is a quite
green zone (samples 4 and 5, respectively). Sample 6 was collected
in Almagro village (Ciudad Real), with a car frequency of about
1.0 × 103 per day.
Samples were collected as grab samples using wide mouth jars
and maintained at 0–4 °C from the time of collection until their
preparation.
using the ultrasonic bath for 5 min. The solution was filtered
through a PTFE membrane filter of 0.5 µm and analyzed by HPLC
by injecting 20 µL into the HPLC system and applying the cali-
bration procedure.
3. HPLC method and calibration
All data for quantification of the PAHs were obtained by applying
the mobile phase gradient shown in Table 1 at a flow rate of
1.0 mL/min and 22 °C. For fluorimetric detection of PAHs the pro-
gram of excitation and emission wavelengths pairs detailed in
Table 2 was used. The working standard mixtures of PAHs in the
range 1.08–885 µg/L were injected at four concentration levels to
obtain the calibration graph. The injection volume was 20 µL. The
integrated peak areas were used to quantitate the PAHs. The mo-
bile phase was degassed with helium.
Results and discussion
Optimization of experimental chromatographic
parameters

2. Soil sample preparation On the basis of previous studies [12] the mobile phase
gradient of acetonitrile/water was optimized at a flow rate
Firstly, the soil samples were screened through a sieve with 1 mm of 1 mL/min and at 22 °C; the program of excitation and
pore size and then dried at 40 °C for 24 h. Then, in a glass tube of
10 mL volume size, portions of soil (1 g), spiked or non spiked emission wavelength pairs was also optimized. Both pro-
with PAHs in the range 1.08–442 ng, were suspended in 3 mL of grams are shown in Tables 1 and 2, respectively; separa-
cyclohexane and the PAHs were extracted in an ultrasonic bath for
30 min. The mixture was centrifuged at 4000 rpm and the super-
natant liquid was decanted. Another 3 mL of cyclohexane were
added to the residue and the extraction and decant processes were
repeated. Both extracts were collected in a glass tube and a fraction
of 4 mL was evaporated for 15 min by means of the vacuum man-
ifold system; then the residue was dissolved to 0.5 mL in methanol

Table 1 Mobile phase gradient

Time, min Acetonitrile, % Water, %
0.0 45 55
5.0 45 55
22.0 73 27
30.0 100 –

Table 2 Program of excitation and emission wavelength pairs

Detected compound Time, min λex, nm λem, nm
Naphthalene, Acenaphthene, 0 280 324
Fluorene
Phenanthrene 17.2 250 365
Anthracene 19.2 254 402
Fluoranthene 20.6 285 465
Pyrene 22.5 270 390
B[a]a, Chrysene 25.0 270 384 Fig. 1 Chromatogram of a standard mixture of thirteen PAHs.
B[e]p 30.3 290 390 Conditions: Hypersil Green PAH (100 × 4.6 mm) column; temper-
B[a]p 33.3 295 405 ature, 22 °C; mobile phase, gradient of acetonitrile/water, see
Db[a,h]a, B[ghi]p 35.3 290 418 Table 1; flow-rate, 1.0 mL/min; fluorimetric detection, see Table
2; injection volume, 20 µL. Peaks: 1, Naphthalene; 2, acenaph-
B[a]a, Benzo[a]anthracene, B[e]p, Benzo[e]pyrene; B[a]p, thene; 3, fluorene; 4, phenanthrene; 5, anthracene; 6, fluoranthene;
Benzo[a]pyrene; Db[a,h]a, Dibenzo[a,h]anthracene; B[ghi]p, 7, pyrene; 8, B[a]a; 9, chrysene; 10, B[e]p; 11, B[a]p;
Benzo[ghi]perylene 12, Db[a,h]a; 13, B[ghi]p
 699

Table 3 Analytical character-

istics of the chromatographic PAH Linear rangea, µg/L RSDb, % DLc, µg/L tR, min RSDb, %
method
Naphthalene 29.5 –885 4.4 0.448 9.2 1.1
Acenaphthene 9.25–278 2.5 0.200 14.6 0.95
Fluorene 6.81–204 4.1 0.150 15.7 0.82
Phenanthrene 5.35–160 7.5 0.128 17.7 0.60
Anthracene 1.78– 53.4 2.2 0.037 19.7 0.33
Fluoranthene 1.25– 37.5 8.7 0.028 21.7 0.38
Pyrene 3.03– 90.9 7.6 0.055 23.0 0.27
B[a]a 1.71– 51.3 8.4 0.050 28.0 0.30
Chrysene 1.80– 54.0 8.2 0.046 28.9 0.29
B[e]p 10.1 –303 8.6 0.063 30.8 0.22
a Studied range B[a]p 1.08– 32.4 6.7 0.023 33.4 0.13
b Relative Standard Deviation Db[a,h]a 5.57–166 3.7 0.100 34.9 0.23
(n = 4)
c Detection Limit, DL = 3 S/N B[ghi]p 2.71– 81.3 9.0 0.055 35.6 0.36

Table 4 PAH recoveries from soil samplea

PAH c1 R1, % RSD c2 R2, % RSD c3 R3, % RSD c4 R4, % RSD
Naphthalene 29.5 – – 59.0 – – 177 – – 442 13 15
Acenaphthene 9.25 49 12 18.5 62 10 55.5 34 11 139 21 13
Fluorene 6.80 77 8 13.6 85 7 40.8 76 9 102 48 10
Phenanthrene 5.35 88 3 10.7 78 4 32.1 74 4 80.0 67 7
Anthracene 1.78 86 4 3.56 90 2 10.7 80 6 26.7 68 3
Fluoranthene 1.25 74 7 2.50 85 5 7.50 97 4 18.8 91 7
Pyrene 3.03 71 3 6.06 91 4 18.2 84 8 45.4 80 8
B[a]a 1.71 86 8 3.42 95 2 10.2 96 4 22.6 98 3
Chrysene 1.80 93 6 3.60 89 9 10.8 89 7 27.0 86 4
B[e]p 10.1 88 6 20.2 92 2 60.5 84 5 152 91 6
B[a]p 1.08 95 8 2.16 93 5 6.50 81 5 31.2 83 4
Db[a,h]a 5.55 89 9 11.1 86 2 33.3 82 3 83.0 90 3
B[ghi]p 2.71 78 2 5.42 70 7 16.2 78 8 40.6 79 7
a Soil sample number 6: 1 g; R, %: percentage recovery
c1, c2, c3 and c4 (µg/kg): PAHs concentration in spiked soil sample RSD, relative standard deviation, n = 4

tion and resolution from the baseline of 13 PAHs was at-
tained. These PAHs were selected due to their abundance
in car pollution, central heating and their toxicity. Figure 1
shows a chromatogram of a standard mixture of PAHs.
Analytical characteristics of standards
Table 3 shows the analytical characteristics obtained for
the studied PAHs. Linearity between fluorescence re-
sponse (peak area) and PAHs concentration in the range
1.08–885 µg/L was verified. The regression coefficients
were 0.9992. The relative standard deviations (RSD) at a
concentration level in the middle of the calibration range
(13–354 µg/L) for n = 4 were between 2.2–9.0% and all
the values were below 1% with respect to retention time.
Detection limits (DL) defined as three times the signal to
noise ratio (3 S/N) were lower than 0.2 µg/L except for
naphthalene (0.45 µg/L). The table also shows the reten-
tion times of the PAHs with RSD values in the range
0.1–1.1% (n = 4).
Optimization of variables for the extraction of PAHs
from soil samples by mean ultrasonication
1 g of soil sample and 2 mL of solvent were used for the
extraction of PAHs.
Two solvents, methylene chloride and cyclohexane,
were tested as PAH extractants from soil samples. Cyclo-
hexane was chosen because with methylene chloride more
interferent compounds were extracted.
Additionally, different lengths of ultrasonication cycles
(20–80 min) were applied. The highest chromatographic
peak areas were achieved for 60 min, where the mean ar-
eas were around 30% or higher.
The recoveries were a function of the number of ex-
traction stages and of the extractant volume. These pa-
rameters were thus also optimized in the ranges 2–6 mL
for cyclohexane volume and 1–3 for the number of ex-
traction stages. The best results were obtained using 3 mL
of cyclohexane twice in an ultrasonic bath for 30 min; us-
ing these conditions the mean recoveries were increased
at about 20%.
700
Fig. 2 Chromatograms of soil samples: (a) Glorieta de Cuatro
Caminos; (b) Ciudad Universitaria; (c) Almagro village. Condi-
tions: Hypersil Green PAH (100 × 4.6 mm) column; temperature,
22 °C; mobile phase, gradient of acetonitrile/water, see Table 1;
flow-rate, 1.0 mL/min; fluorimetric detection, see Table 2; injec-
tion volume, 20 µL. Peaks: 2, acenaphthene; 4, phenanthrene; 5,
anthracene; 6, fluoranthene; 7, pyrene; 8, B[a]a; 9, chrysene;
10, B[e]p; 11,B[a]p; 12, Db[a,h]a; 13, B[ghi]p

The amount of soil sample collected (0.5, 1 and 2 g)

was also evaluated. The best results were obtained with 1 g
of soil. Higher amounts gave adsorption problems of the
extractant agent, and lower amounts gave lower chroma-
tographic peak areas.
Finally, a 4 mL extract aliquot was evaporated to dry-
ness by means of the vacuum manifold system and the
residue was dissolved in 0.5 mL methanol.

Recovery studies
a
The PAHs recoveries were determined from the low pol-
luted soil sample number 6. 1.00 g of the soil was spiked
with PAHs at four concentration levels in the range
1.08–442 µg/kg, applying the experimental procedure.
The results of the analysis are shown in Table 4. In gen-
eral, recoveries were in the range 70–98% except for ace-
naphthene and naphthalene due to their volatility, spe-
cially during the concentration step of the extracts. The
relative standard deviations were in the range 2–15% for
b four replicates measurements.

Determination of PAHs in soil samples

The proposed method was applied to determine PAHs in

the six soil samples specified in the experimental section.
c Figure 2 a, b and c show the chromatograms from samples

Table 5 Determination of PAHs in soil samples (µg/kg)

PAH 1 RSD, 2 RSD, 2a RSD, 3 RSD, 3a RSD, 4 RSD, 5 RSD, 6 RSD,
% % % % % % % %
Acenaphthene 1.47 14 12.3 12 5.13 12 6.63 9 – – – – 0.59 16 – –
Fluorene – – 6.45 14 – – 7.45 12 – – – – – – – –
Phenanthrene 60.9 3 68.8 4 55.8 6 49.0 7 40.2 9 9.76 9 12.1 10 – –
Anthracene 4.85 8 1.42 3 – – 19.6 10 10.3 7 – – – – – –
Fluoranthene 10.7 7 5.26 8 4.37 9 3.64 4 6.71 3 4.65 9 6.67 5 – –
Pyrene 18.8 8 28.7 4 20.2 4 3.24 6 7.39 7 7.30 6 4.05 7 – –
B[a]a 34.2 8 9.82 10 10.2 2 6.41 4 4.58 8 21.0 9 4.34 9 – –
Chrysene 43.8 10 10.9 7 12.1 8 8.48 7 10.5 4 9.82 8 1.83 7 0.82 8
B[e]p 32.0 6 9.10 7 7.23 7 7.57 2 9.27 3 12.3 9 7.32 10 7.39 7
B[a]p 32.8 5 7.60 6 9.71 7 10.1 4 8.73 2 3.43 10 3.78 8 1.83 5
Db[a,h]a 23.4 2 8.07 8 7.24 9 13.9 2 16.1 8 4.60 2 4.18 3 3.74 2
B[ghi]p 19.3 9 6.35 8 8.58 9 6.91 3 5.21 8 4.95 7 4.42 7 1.32 10
Total PAHs 282 175 141 143 119 77.8 49.3 15.1

1–6: samples number; 1, Glorieta de Cuatro Caminos; 2, Paseo del RSD: Relative standard deviation (n = 4)
Prado; 3, Plaza del Cuzco; 4, Ciudad Universitaria; 5, Dehesa de la a Applyingthe 3540 EPA Method
Villa; from Madrid and 6, Almagro village (Ciudad Real)

number 1, 4 and 6, respectively. The results are summa-
rized in Table 5, which also shows those obtained by ap-
plying the 3540 EPA method to samples 2 and 3. In gen-
eral, the results obtained by both methods were of the
same order. However, the results for acenaphthene and
fluorene found were low when the 3540 EPA method was
used because they could be lost in the concentration step
due to the high solvent volume used for PAH extraction in
this 3540 EPA method. Naphthalene was not detected in
the samples due to its low fluorescence signal. The rela-
tive standard deviations were in the range 2–16% (n = 4).
The presence of these PAHs was also confirmed by
GC-MS. The distribution of PAHs depended on their
structure and molecular weight; the ones with higher mol-
ecular weights (4 to 6 aromatic rings) were detected in all
samples due their adsorption power on the solid particles
or surfaces. On the other hand, the PAH contents found
were in accordance with the pollution expected for the
different places where the samples were taken, as speci-
fied in experimental. Urban areas (samples 1, 2 and 3)
with high pollution level show high PAH concentrations,
which are mainly caused by traffic emission and central
heating; rural regions are generally less contaminated
(sample 6).
Conclusion
A sensitive, rapid and economic method for the determi-
nation of PAHs in soil samples is proposed. A clean-up
step is not necessary, which facilitates the determination
of low molecular weight PAHs except the naphthalene.
The amounts of PAHs in soil samples were in accordance
701
with the pollution levels from the sampling areas. PAHs
with 4–6 aromatic rings were detected in all samples.
Acknowledgements Financial support from the Spanish DGI-
CYT, project PB 96-0642, and the Servicio de Espectrometría de
Masas del Centro de Espectroscopía de la U.C.M. are gratefully
acknowledged.
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