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1
This paper describes the development and optimization, by using multivariate anal-
Instituto de Qumica,
ysis, of a GC – MS-SIM method for evaluation of the 16 polyaromatic hydrocarbons
Universidade Federal da Bahia,
Campus de Ondina, Salvador-BA, considered as priority pollutants in atmospheric particulate material by the US EPA.
Brazil In order to assure an adequate separation in the shortest analysis time, a multivari-
2
Centro Interdisciplinar em ate design was used to set the conditions of the oven temperature program. The
Energia e Ambiente – CIEnAm, optimization process was carried out using factorial fractional design and Box –
Universidade Federal da Bahia, Behnken design. The following factors were evaluated: initial temperature, temper-
Salvador-BA, Brazil ature rate #1, intermediary temperature, temperature rate #2, and final tempera-
ture. The optimized conditions were set at: 708C (2 min) fi 2008C (308C/min, 5 min)
fi 3008C (58C/min, 1.67 min). Moreover, we have also optimized the injector temper-
ature as 3108C and sampling time as 0.8 min. The total analysis time was 33 min.
Validation of GC – MS-SIM yielded satisfactory results for repetitivity of the detector
response and retention times, and linearity of calibration curves. LOD were estab-
lished as 0.13 – 0.34 ng/mL (peak area) and 0.18 – 0.72 ng/mL (peak height). The
method has been shown to be appropriate for the analysis of samples of atmo-
spheric particulate material and/or other environmental matrices.
Keywords: Atmospheric aerosols / GC – MS / Multivariate design / PAHs /
Received: November 6, 2007; revised: January 23, 2008; accepted: February 7, 2008
DOI 10.1002/jssc.200700573
have shown that average values for the 16 EPA PAHs 2 Experimental
obtained in environmental samples range between 0.04
and 15.30 ng/m3. Sample treatment is usually performed In this study, we employed 16 PAH (naphthalene, ace-
manually; hence it is tedious [19, 20] and consumes large naphthylene, acenaphthene, fluorene, phenanthrene,
amounts of many different solvents if classical PAHs anthracene, fluoranthene, pyrene, benzo[a]anthracene,
extraction methods are used [20]. chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene,
Atmospheric samples contain a large number of PAH benzo[a]pyrene, indeno[1,2,3-c,d]pyrene, dibenz[a,h]an-
compounds that differ little in structure and molecular thracene, benzo[g,h,i]perylene) standard solution (Accu-
weight; and thus require high resolution techniques for Standard Inc., USA), acetonitrile of chromatographic and
separation and analysis. Reliable data regarding the com- spectroscopic grade (J. T. Baker) for dilution of standard
position and concentration of PAHs in particulate matter solutions, a GCMS-QP2010 gas chromatograph – mass
can be obtained through different gas or liquid chroma- spectrometer system model with AOC-20i autosampler
tographic methods. The GC – MS methodology provides (Shimadzu, Japan), an Elite5MS GC column
extensive information on sample composition and ena- (30 m60.25 mm id60.25 lm film thickness) (Perkin
bles better compound identification on the basis of struc- Elmer, USA), and a NanoPure Diamond (USA) water puri-
tural information. This system records a specified ion at fying system.
a selected time and confers both better selectivity and Chromatographic analyses were carried out starting
better sensitivity [19, 20]. Some weak points of this sep- with procedures already described in the literature [28 –
aration and detection technique in PAH determination 32] as well as acquired expertise of this research group in
are mainly co-elution of isomers or structurally similar PAH analysis by GC – MS [23, 33]. Briefly, our starting col-
compounds as well as long chromatographic runs (about umn oven program, set in a univariate manner, was: (i)
45 – 90 min of total analysis time). In order to minimize oven: 608C (1 min) fi 2808C (58C/min) fi 2808C (15 min);
these undesired aspects in PAHs analysis, we propose a (ii) injector: 2708C, splitless mode; (iii) transfer line:
multivariate optimization approach to developing a 2708C, (iv) ion source: 2308C, (v) analyzer: 1508C; electron
GC – MS methodology for PAH determination, achieving impact energy: 70 eV [23, 33]. Although it is possible to
good improvements in resolution by both avoiding co- acquire reliable data for a part of the sixteen PAHs con-
elution of isomers and reducing the total time of anal- sidered using this procedure, there are some problems:
ysis. (i) it is not possible to attain completely satisfactory reso-
Optimization of the chromatographic conditions may lution for the following pairs of isomers: phenanthrene/
be carried out by a traditional univariate approach, in anthracene, benzo[a]anthracene/chrysene, benzo[b]fluor-
which each factor is studied separately, or by multivari- anthene/benzo[k]fluoranthene, and indeno[1,2,3-c,d]pyr-
ate experimental design strategy, which allows for simul- ene/dibenz[a,h]anthracene; (ii) the lower the vapor pres-
taneous variation of all evaluated factors, making it pos- sure of the PAH, the less sharp is its peak; and (iii) the pro-
sible to distinguish interactions among them that would posed temperature program takes 60 min for each sam-
not be detectable by classical univariate experimental ple to be completely eluted from column. In the face of
design [21, 22]. Multivariate optimization design also all these points, we decide to improve our experimental
allows a reduction in the number of required experi- conditions. Therefore, taking into account the chromato-
ments, without loss of information. graphic column specifications and considering the fol-
The optimization of chromatographic conditions has lowing parameters: (a) multivariate design for defining
been carried out using multivariate strategies such as of temperature programming of the oven/column; (b)
factorial design and response surface methodology type injector temperature effect; and (c) sampling time effect,
central composite design, and Box – Behnken design [23 – we have developed an optimized gas GC – MS-SIM method
27]. Moreover, since the aim of this study was to assure by multivariate strategies such as factorial design and
an adequate separation of the 16 PAHs, in the shortest response surface methodology type central composite
analysis time, a multivariate design was used to set the design, and Box – Behnken design.
conditions of the oven temperature programming (col-
umn heating). The optimization process was carried out
using both factorial fractional and Box – Behnken design.
2.1 Multivariate design
The evaluated parameters used to attain reliable chroma-
tographic conditions for separation of PAH were: initial Multivariate design was employed as a strategy to attain
temperature (8C), temperature rate #1 (8C/min), inter- higher peak resolutions in shorter total analysis time.
mediate temperature (8C), temperature rate #2 (8C/min), The following parameters related to oven temperature
and final temperature (8C), in order to attain a reliable programming were evaluated: initial temperature (8C),
improvement in the chromatographic separation of the temperature rate #1 (8C/min), intermediate temperature
priority PAH. (8C), temperature rate #2 (8C/min), and final temperature
Table 1. List of PAHs studied and selected ions (m/z) for where: Rpn is the resolution for the nth evaluated pair and
each PAH. tRn is the retention time for the second peak in each corre-
sponding pair.
PAH Abbrevi- Base Reference
ation ion ion
Design #1
Initial temperature (8C) [IT] 50 70 90
Temperature rate #1 (8C/min) [R1] 5 10 15
Intermediary temperature (8C) [MT] 140 160 180
Temperature rate #2 (8C/min) [R2] 4 8 12
Final temperature (8C) [FT] 280 300 320
Design #2
Initial temperature (8C) [IT] 60 70 80
Temperature rate #1 (8C/min) [R1] 10 20 30
Intermediary temperature (8C) [MT] 160 180 200
Temperature rate #2 (8C/min) [R2] 4 6 8
Table 3. Detailed description of the Box – Behnken design. Table 4. Critical values obtained by Box – Behnken design:
(a) central body method; (b) Derringer and Suich method
Factor [Abbreviation] Minimum Medium Maximum
level ( – ) level (0) level (+) Factor [Abbreviation] Critical value
Temperature rate #1 (8C/min) [R1] 12 24 36 (a)
Intermediary temperature (8C) [MT] 180 200 220 Temperature rate #1 (8C/min) [R1] 37.6
Temperature rate #2 (8C/min) [R2] 3 5 7 Intermediary temperature (8C) [MT] 192.7
Temperature rate #2 (8C/min) [R2] 5.6
(b)
used in the construction of the Box – Behnken design are Temperature rate #1 (8C/min) [R1] 29.1
described. Intermediary temperature (8C) [MT] 194.8
Temperature rate #2 (8C/min) [R2] 5.2
In this manner, experimental design of the Box –
Behnken type was used in association with the methodol-
ogy of the response surface for optimization of the chro-
matographic conditions, resulting in 18 experiments in the denominator of the response equation could have
with six replicates at the central point. contributed to an excessive reduction of analysis time to
The response surface (Fig. 2) enables us to conclude the detriment of analyte separations since tr is of higher
that the estimated optimum point is out of our working magnitude than the corresponding resolution. This fact
range of studied variables. This implies that it is still nec- would imply a greater weighting of analysis time, result-
essary to perform more experiments aiming to cover the ing in a poorer separation. We therefore consider that
critical point region and/or defining a response that Eq. (6) does not reach completely the desired goals and
brings both resolution improvement and analysis time should be reassessed and improved. More specifically, R1
reduction. should be fitted between 12 and 368C to have a maxi-
The chromatogram in Fig. 3 was acquired in an experi- mum in the response surface (optimum point) (Table 4a).
ment with the highest R1 (368C/min). In that chromato- In this way, we decide to construct a new equation
gram, phenanthrene (peak 5, tr = 9.41 min) and anthra- which could represent the response to be optimized by
cene (tr = 9.53 min) are too close, enabling interference using the same significant variables as are found in the
in signal integration. One possible explanation is that factorial design #2: R1, MT and R2. Thus, a new response
using the retention time (tr) of the last peak of each pair equation was developed based on the method of Der-
ringer and Suich [28, 29, 36, 37] allowing equalization of ysis time was therefore established in the range 20 –
the magnitudes of analysis time and resolution averages 50 min (50 ) 0 and 20 ) 1); the shorter the analysis
of the four pairs studied, avoiding that one of them rep- time, the closer to unity the codified analysis time value
resent an excess weight in relation to the others. would be. Resolution was defined from 1.0 – 1.6 (1.0 ) 0
The codification of both analysis time and resolution and 1.6 ) 1); the higher the resolution, the closer to
average was carried out in order to adjust the values on a unity the codified resolution would be. In this context,
scale from 0 to 1, both of them being of the same order of codification was performed in keeping with the follow-
magnitude. The employed maxima and minima values ing equations:
in the codification were based on experimental results of
resolution average between studied pairs, both for the tAcod = – (tA – 50)/(50 – 20) (4)
observed analysis time as well as for the main objective,
Rcod = (R – 1.0)/(1.6 – 1.0) (5)
i. e. best resolution in the shortest analysis time. The anal-
Figure 5. A GC – MS-SIM chromatogram of a 0.4 lg/mL PAH standard after optimization (1 – naphthalene; 2 – acenaphthylene;
3 – acenaphthene; 4 – fluorene; 5 – phenantrene; 6 – anthracene; 7 – fluoranthene; 8 – pyrene; 9 – benzo[a]anthracene; 10
– chrysene; 11 – benzo[b]fluoranthene; 12 – benzo[k]fluoranthene; 13 – benzo[a]pyrene; 14 – indeno[1,2,3-c,d]pyrene; 15 –
dibenz[a,h]anthracene; 16 – benzo[g,h,i]perylene).Chromatographic conditions: (i) injector: 3108C, (ii) injection mode: splitless;
(iii) oven: 708C (2 min) fi 2008C (308C/min) fi 2008C (5 min) fi 3008C (5.08C/min) fi 3008C (1.67 min); (iv) transfer line: 2808C;
(v) ion source: 2508C; (vi) electron impact energy: 70 eV.
Figure 6. GC – MS-SIM chromatogram of a real sample after optimization (1 – naphthalene; 2 – acenaphthylene; 3 – acenaph-
thene; 4 – fluorene; 5 – phenanthrene; 6 – anthracene; 7 – fluoranthene; 8 – pyrene; 9 – benzo[a]anthracene; 10 – chrysene;
11 – benzo[b]fluoranthene; 12 – benzo[k]fluoranthene; 13 – benzo[a]pyrene; 14 – indeno[1,2,3-c,d]pyrene; 15 – dibenz[a,h]-
anthracene; 16 – benzo[g,h,i]perylene). Chromatographic conditions: (i) injector: 3108C, (ii) injection mode: splitless; (iii) oven:
708C (2 min) fi 2008C (308C/min) fi 2008C (5 min) fi 3008C (5.08C/min) fi 3008C (1.67 min); (iv) transfer line: 2808C; (v) ion
source: 2508C; (vi) electron impact energy: 70 eV.
where tAcod is the codified analysis time; tA is the analysis 37.6 to 29.18C/min (i. e., in the established range of 12 and
time (considered as the retention time of the last peak + 368C).
2 min); Rcod is the codified resolution average; and R is According to multivariate designs, the following opti-
the average of resolution evaluated peak pairs. mized chromatographic conditions were found: temper-
After performing the codification of resolution and ature oven: (i) injector: 3108C, (ii) injection mode: splitless;
analysis time, the new response was defined as the geo- (iii) oven: 708C (2 min) fi 2008C (308C/min) fi 2008C
metric average between codified values of analysis time (5 min) fi 3008C (5.08C/min) fi 3008C (1.67 min); (iv)
and resolution: transfer line: 2808C; v) ion source: 2508C; (vi) electron impact
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi energy: 70 eV. Figure 5 shows a PAH standard chromato-
Response ¼ tAcod Rcod ð6Þ
gram, acquired under optimized conditions which per-
The response surfaces obtained by Eq. (6) allowed loca- mitted excellent separation of the 16 PAH in a total anal-
tion of maxima in the range of investigated variables ysis time of 33 min. Figure 6 depicts the chromatogram
(Fig. 4). Critical values are showed in Table 4b. of a real sample of atmospheric aerosol which shows
Comparing the new critical values (calculated with good resolution of all 16 PAHs with neither co-elutions
Eq. (6)) (Table 4b) with those from Table 4a, it is observed nor interferences. It is worthwhile mentioning that the
that the main difference is that R1 has changed from four PAHs pairs: PHE and ANT; BaA and CRY; BbF and
Table 5. Repetitivity of retention time. The analysis of detector response for each PAH showed
that there is an increment of 48.92% (CRY) to 73.61%
Peak PAH tR (min)* s CV%
(DBA) of PAH area and 25.48% (NAP) to 67.81% of PAH
1 Naphthalene 5.485 0.002 0.032 height (Fig. 9).
2 Acenaphthylene 6.918 0.001 0.019
3 Acenaphthene 7.095 0.001 0.016
4 Fluorene 7.703 0.001 0.017
5 Phenanthrene 9.410 0.002 0.022
6 Anthracene 9.534 0.002 0.024
7 Fluoranthene 13.627 0.003 0.024 3.4 Repetitivity of detector response and retention
8 Pyrene 14.583 0.003 0.019 time
9 Benzo[a]anthracene 20.299 0.002 0.012
10 Chrysene 20.453 0.003 0.014 Repetitivity of detector response was studied for both
11 Benzo[b]fluoranthene 25.101 0.003 0.012 peak area and peak height, carried out in five inter-day
12 Benzo[k]fluoranthene 25.220 0.002 0.009 replicates by injecting of 1.0 lL of each of the 10, 20, 40,
13 Benzo[a]pyrene 26.370 0.003 0.012
14 Indeno[1,2,3-c,d]pyrene 30.508 0.003 0.009 and 100 ng/mL standard solutions. The obtained results
15 Dibenz[a,h]anthracene 30.680 0.002 0.007 showed coefficients of variation (CV%) between 0.28 and
16 Benzo[g,h,i]perylene 31.338 0.003 0.009 7.55% for peak area and between 0.53 and 11.34% for
peak height.
* Refers to average of two standards, from 10 inter-day Repetitivity of retention time was evaluated in 10
determinations.
inter-day replicates with 40 and 100 ng/mL standard sol-
tR = retention time.
ution of 16 PAH. Results are listed in Table 5.
s = standard deviation.
CV = coefficient of variation.
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