biomonitoring of polycyclic aromatic hydrocarbons and ... · biomonitoring of polycyclic aromatic...
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Faculty of Bioscience Engineering
Academic year 2014 – 2015
Biomonitoring of polycyclic aromatic hydrocarbons
and derivatives in Belgium
Zhimiao Zuo
Promotor: Prof. dr. ir. Herman Van Langenhove
dr. ir. Christophe Walgraeve
Master’s dissertation submitted in partial fulfillment of the
requirements for the degree of
Master of Environmental Sanitation
I
Acknowledgement
The dissertation is almost finished, which means the two-year study in the University of
Ghent and the living in Belgium are going to the end as well. The experience of studying
abroad is one of the most important parts of my life. The people I met here will not be
forgotten ever. At the end of my student period, lots of people should be showed of my
gratitude.
Professor Herman Van Langenhove is the promoter of my thesis. He is modest and gentle.
Besides the thesis, several lectures were given by him. The erudite of knowledge, the precise
on research, the diligent on work, the humorous on speech and the optimistic on living
impressed me a lot. I learnt not only knowledge from him, but also knew better on how to
behave correctly. An old saying in Chinese ‘先生之风,山高水长’ describes Professor Herman
Van Langenhove perfectly. The meaning of the sentence is: ‘The prestige of the master will
be spread and remembered forever’.
My tutors Christophe Walgraeve and Dohai Duc guided me pretty well while the thesis
proceeded. Christophe worked with me from the very beginning of the thesis. He was so
scrupulous and conscientious. Even though the experiments always went late to dark, he was
there in the lab until the last moment to give help and guidance. While dissertation
compiling, Christophe was so nice that he gave up part of his summer vacation to read and
modify the text.
My gratitude goes to Professor Kristof Demeestere as well, who always listened to my report
and gave some constructive suggestions kindly. His professional opinion enlightened me to
avoid the detour while thesis designing. He was so responsible to all the students in the lab,
and tried to help the best all the time.
The staff from The Department of Sustainable Organic Chemistry and Technology, such as
Wouter De Soete, Joren Bruneel and so on, although I hardly talked to all of them, the smile
and warm greeting they gave encouraged me every time I arrived at the lab. Especially, my
special thanks to Patrick De Wispelaere, who supported me on GC-MS operation.
Professor Peter Goethals, the coordinators of Master of Science in Environmental Sanitation
(IMENVI) Sylvie Bauwens and Veerle Lambert. Thank you so much for approving my
application and giving such a wonderful and rare opportunity to study in the best university
around the world, and to work with so many amiable and brilliant brains.
I want to show my great thanks to my lovely classmates, we came together from different
continents. Every one of you gave me lots of help and encouragement while I was
disoriented. Daniel Paul Odhiambo Ombaka, Audisny Apristiaramitha Teddy and Workineh
Mengesha Fereja who were working together in the same lab for thesis, gave me lots of
II
suggestion and introduction about lab work as well.
My parents and grandfather are the inner pillar supported and motivated me to finish this
program straight. My friends in Beijing are the ones released my pressure in the two-year.
My girlfriend is the one consoled and accompanied me while I was frustrated. Without them,
I would never success.
At last, the great thanks to the committee. Thank you for the time and patience to read my
dissertation.
III
List of Abbreviations
ACN Acetonitrile SEC Size Exclusion Chromatography
AC Adsorption Chromatography SPE Solid Phase Extraction
ASPEC Automated Solid-Phase Extraction Clean-up USEPA U.S. Environmental Protection Agency
DCM Dichloromethane VOCs Volatile Organic Compounds
DNA Deoxyribonucleic Acid WHO World Health Organization
d.w. Dry Weight
F-PAHs Mono-fluorinated PAHs
FW Fresh Weight
GC Gas Chromatography
EI Electron Ionization
GMF Glass Microfiber Filter
GPC Gel Permeation Chromatography
HEX Hexane
HPLC High Performance Liquid Chromatography
IARC International Agency for Research on Cancer
IS Internal standard
LRAT Long-range Atmospheric Transport
MS Mass Spectrometry
NF Not Found
NPAHs Nitro-polycyclic Aromatic Hydrocarbons
oxy-PAHs Oxygenated Polycyclic Aromatic Hydrocarbons
PACs Polycyclic Aromatic Compounds
PAHs Polycyclic Aromatic Hydrocarbon
PCBs Polychlorinated Biphenyls
PLE(ASE)
Pressurized Liquid Extraction (Acceleration
Solvent Extraction)
PM Particulate Matter
POPs Persistent Organic Pollutants
PTFE Polytetrafluoroethylene
RPLC Reversed Phase Liquid Chromatography
RPA Relative Peak Area
RSRF Relative Sample Response Factor
S_B Spiked Blank
S_SAM_A Sample Spiked After
S_SAM_B Sample Spiked Before
SAM Sample
IV
Abstract
The aim of this study was to develop a bio-monitoring method to analyze polycyclic aromatic
hydrocarbons (PAHs) and oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs). Taxus
baccata (European Yew) was chosen as the bio-monitor species in this thesis. This is the first
time Taxus baccata applied on PAHs and oxy-PAHs researches. The 16 PAHs mentioned in
this thesis are the 16 U.S. Environmental Protection Agency (USEPA) priority PAHs. The
oxy-PAHs and PAHs in samples were analyzed by Gas Chromatography Mass Spectrometry
(GC-MS). The approach of sample preparation, extraction solvent choice, and solid phase
extraction (SPE) clean-up were optimized and developed. PAHs and oxy-PAHs were
quantified by internal standards calibration with deuterated PAHs. The recovery and matrix
effects of PAHs and oxy-PAHs were obtained. The concentration of target compounds in
sample was obtained and expressed as in mass of leaf dry weight.
Air pollution, PAHs, oxy-PAHs, vegetation, Taxus, bio-monitoring.
V
CONTENT
Acknowledgement ............................................................................................................................. I
List of Abbreviations ......................................................................................................................... III
Abstract ........................................................................................................................................... IV
CHAPTER 1 Introduction................................................................................................................- 1 -
CHAPTER 2 Literature Review .......................................................................................................- 9 -
2.1 Sampling ..........................................................................................................................- 9 -
2.2 Sample Preparation & Extraction ................................................................................. - 13 -
2.3 Clean-up ....................................................................................................................... - 19 -
2.4 PAHs and oxy-PAHs Analysis ......................................................................................... - 24 -
2.5 Concentration Levels .................................................................................................... - 28 -
CHAPTER 3 Chemical Materials .................................................................................................. - 32 -
3.1 Chemicals & Reagents .................................................................................................. - 32 -
3.2 Standards Solution ....................................................................................................... - 33 -
CHAPTER 4 Results & Discussion ................................................................................................ - 34 -
4.1 Selection of Plant Species ............................................................................................ - 35 -
4.2 Dry Matter Content ...................................................................................................... - 36 -
4.3 Solvent Selection .......................................................................................................... - 36 -
4.4 Size of the Sample Determination ................................................................................ - 37 -
4.5 Extraction ..................................................................................................................... - 39 -
4.6 Clean-up & Concentration ............................................................................................ - 40 -
4.6.1 Clean-up & Concentration Procedure ............................................................... - 40 -
4.6.2 Determination of Eluent Volume ...................................................................... - 41 -
4.7 GC-MS Analysis ............................................................................................................. - 47 -
4.8 Recovery and Matrix Effects ......................................................................................... - 47 -
4.8.1 Experiment Design ............................................................................................ - 48 -
4.8.2 Data Processing Method ................................................................................... - 49 -
4.8.3 Results of Analysis ............................................................................................. - 50 -
4.9 Discussion ..................................................................................................................... - 53 -
CHAPTER 5 Conclusions & Prospect ........................................................................................... - 55 -
5.1 Conclusions .................................................................................................................. - 55 -
5.2 Prospect ....................................................................................................................... - 55 -
REFERENCES ............................................................................................................................... - 56 -
- 1 -
CHAPTER 1 Introduction
In industrialized and densely populated areas, air pollution has been regarded as one of the
most important environmental issues, especially in recent decades (Rodriguez et al., 2010).
Reviewing the top 10 of the pollution incidents in past century, half of them were related to
air pollution, which are listed in Table 1-1 (Zuo, 2010). With the evolution of human
technology, increasing pollutants were produced due to the increasing natural resource
consumption (Agrillo et al., 2013; Yu, 2014). Moreover, human health and air quality are
associated intimately (Esposito et al., 2014; Matus et al., 2012). Epidemiological studies,
which carried out to investigate the relationship between health risks and air pollution,
indicated that ambient air pollution have a high possibility to be responsible for the
increasing rates of lung cancer and respiratory system disease (Du Four et al., 2004; Goldberg
et al., 2001). Therefore, the researches on air quality, air pollution and air purification were
highlighted and focused by more and more scientists.
Air pollutants can be classified in different ways. Firstly, by formalism, air pollutants can be
divided into two groups, primary pollutants and secondary pollutants. Primary pollutants are
the ones emitted from the pollution sources directly, for instance carbon monoxide (CO),
sulfur dioxide (SO2) and nitric oxide (NO) etc. Secondary pollutants are the ones produced by
the chemical or photochemical reactions of primary pollutants, for instance, ozone (O3),
H2SO4, aerosol, etc. Secondly, by physical property, air pollutants can also be divided into two
groups, gaseous pollutants and particulates. A large proportion of air pollution is caused by
gaseous pollutants, for instance, SOx, NOX, chlorofluorocarbon (CFCs), etc. Thirdly, by
chemical property, air pollutants can be divided into two groups, organic pollutants and
inorganic pollutants. (Hao, 2010; Kallenborn et al., 2012; Xia, 2003).
Previous reports have studied the major air contaminants, for instance, O3, NOx, SOx, COx,
volatile organic compounds (VOCs), Particulate Matter (PM), heavy metals, etc. (Cheng et al.,
2008a; Cheng et al., 2008b; Sujaritpong et al., 2014). Various organic compounds are known
to be present as airborne particles. Of primary concern are the polycyclic aromatic
hydrocarbons (PAHs) and their derivatives, for instance nitro-polycyclic aromatic
hydrocarbons (NPAHs) and oxygen containing polycyclic aromatic hydrocarbons (oxy-PAHs).
They are known as polycyclic aromatic compounds (PACs) collectively (Du Four et al., 2004).
PAHs and oxy-PAHs have drawn a lot of attention recently. With the speedy development of
modern industry and transportation, more fuel is required and consumed, thus more air
pollutants are produced nowadays (Srogi 2007; ötvös et al., 2004). Among the air pollutants,
PAHs and their derivatives oxy-PAHs are reported to be one of the biggest environmental
risks (Nocun and Schantz, 2013; Wang et al., 2006). The properties of high carcinogenic
possibility and toxicity to the environment are concerning people (Niu et al., 2003;
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Pongpiachan, 2013). Therefore, PAHs and oxy-PAHs are chosen as the main object of this
study.
Table 1-1 the Air Pollution Incidents among the Top 10 Pollution Incidents in the 20th
Century
PAHs is the name of a large group constituted of more than 100 organic compounds having
Year Subject Region Inducement Details
1930 The 1930
Meuse Valley
Fog
Belgium,
Industrial
Temperature
Inversion
The first public nuisance recorded in the
20th century. It was induced by the
temperature inversion. The valley was full
of heavy industries. 60 people were killed
by the trapped gaseous emission in one
week.
1943 Los Angeles
Photochemical
Smog Episode
U.S., City Heavy Traffic The hydrocarbon, emitted by petrol
burning, catalyzed by ultraviolet
irradiation, resulted in the production of
‘blue smog’, which was known as
photochemical smog later. Two similar
incidents were reported in 1955 and
1970, more than 75% population of the
city was recorded disease.
1948 The 1948
Donora Smog
Donora,
Pennsylvania,
U.S., Industrial
Temperature
Inversion
The city of Donora was well-known by lots
of large-scale iron works, smelters and
sulfuric acid plants. The foggy morning in
1948, controlled by the anticyclone and
temperature inversion, the plants’
emission was trapped in lower
atmosphere, which brought disaster to
the city. More than 20,000 people were
affected.
1952 The London
Smogs
Britain, City Coal Burning 12 severe smog incidents were recorded
in London since 1952. All of them were
induced by the emission of sulfur dioxide
and particulate matter while coal burning.
Reported, more than 12,000 people dead
in the smog of 1952.
1984 Bhopal
Disaster
Bhopal, India,
City
Explosion The city of Bhopal was attacked by 45
tons highly toxic methyl isocyanate smog
which was produced by the explosion of
the farm chemical plant. 20,000 casualty,
50,000 blindness were reported after the
disaster. More than 200,000 people were
affected.
- 3 -
two or more fused aromatic rings (Qiu et al., 2013). The properties were described as
relatively low solubility in water, but highly lipophilic (Srogi, 2007). Vapor phase and the
condensed (aerosol) phases are the two main present states of PAHs in the atmosphere
(Inomata et al., 2013). To specify, low molecular weight PAHs have the tendency to be more
concentrated in the vapor phase. The ones often associated with particulates are higher
molecular weight PAHs (Inomata et al., 2013; Ravindra et al., 2006).
PAHs can be arisen either naturally or as the result of anthropogenic activities (Węgrzyn et al.,
2006; Zhan et al., 2013). Naturally, PAHs can be the products of thermal decomposition; can
be formed during incomplete combustion of organic materials (Niu et al., 2003; Ratola et al.,
2006) and geochemical formation of fossil fuels (Foan and Simon, 2012). On the other hand,
domestic heating, power plants, industrial processes, waste incineration, and most
importantly, the emissions of motor vehicles can be the major anthropogenic sources of
PAHs (Mu et al., 2013; Qiu et al., 2013; Ravindra et al., 2006).
The 16 individual PAHs, which are defined as priority by US Environmental Protection Agency
(USEPA) (Ma et al., 2010), involved in the present studies are naphthalene, acenaphthylene,
acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene (PYR),
benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene,
dibenz[a,h]anthracene, benzo[g,h,i]perylene, and indeno[1,2,3-cd]pyrene (Ziegenhals et
al.,2008; Srogi, 2007). The basic physical properties and structure of the 16 selected PAHs
are described in Table 1-2 (Working Group on Polycyclic Aromatic Hydrocarbons, 2001).
Previous studies have proved that PAHs, as persistent organic pollutants (POPs) could affect
human health by bio-accumulation through food chain (Mu et al., 2013). Also, adverse
characters of PAHs, such as toxicity, slow rates of degradation and the potential for either
bioaccumulation in living organisms or long-range transport were observed recently (Foan
and Simon, 2012). Furthermore, PAHs were proved to be responsible for carcinogenic,
mutagenic (Hubert et al., 2003; Navarro-Ortega et al., 2012; Węgrzyn et al., 2006),
immunotoxic (Rodriguez et al., 2012; Sanz-Landaluze et al., 2010), endocrine disruption,
reproductive and developmental toxicity (Pongpiachan, 2013). Besides, some of the PAHs
may also act as co-carcinogens or tumor promoters (Rodriguez et al., 2010), which are
detrimental to human health and non-human organisms (Zhan et al., 2013).
Among them, the best known carcinogenic PAHs compound is benzo[a]pyrene which has
been used as a leading substance, due to its reasonably well-known emission inventory and
toxicological significance (Pongpiachan, 2013; Ziegenhals et al., 2008; Van Jaarsveld et al.,
1997). Base on the classification of the International Agency for Research on Cancer (IARC),
some of the PAHs compounds are classified as probable or possible to human carcinogens
(Du Four et al., 2005). The details of the PAHs compounds classification about carcinogens of
IARC can be found also in Table 1-2.
- 4 -
Table 1-2 the Properties and Structure of the 16 EPA PAHs
PAHs Formula Boiling Point/℃ Melting Point/℃ Structure IARC Classification
Naphthalene C10H8 217.9 80.5
n.e.*
Acenaphthylene C12H8 280 93
n.e.
Acenaphthene C12H10 279 95 CH2 CH2
n.e.
Fluorene C13H10 295 116 CH2
3**
Phenanthrene C14H10 340 100.5
3
Anthracene C14H10 342 216.4
3
Fluoranthene C16H10 375 108.8
3
Pyrene C16H10 150.4 393
3
Benzo[a]anthracene C18H12 400 160.7
2A***
Chrysene C18H12 448 253.8
NA
Benzo[b]fluoranthene C20H12 481 168.3
2B****
Benzo[k]fluoranthene C20H12 480 215.7
2B
Benzo[a]pyrene C20H12 496 178.1
2A
Dibenzo[a,h]anthracene C24H14 524 266.6
2B
Benzo[g,h,i]perylene C22H12 NA 277
3
Indeno[1,2,3-c,d]pyrene C22H12 536 163.6
2A
* n.e.: not evaluated
** 3: not classifiable
*** 2A: probably carcinogenic
**** 2B: possibly carcinogenic
- 5 -
Oxy-PAH is a group of compounds having the structure of aromatic rings attached with
oxygen atom(s). Besides oxygen, other chemical groups are possible to attach (O’Connell et
al., 2013). Normally, oxy-PAHs were commonly observed in highly polluted areas, especially
the industrial areas with PAHs emission (Lundstedt et al., 2007).
Oxy-PAHs can be both primary pollutants and secondary pollutants. There are several
sources of oxy-PAHs formation, for instance, petrogenic process, pyrogenic process,
biological transformation, photo-oxidation and chemical oxidation (Kojima et al., 2010;
Lundstedt et al., 2007; O’Connell et al., 2013). It is notable that, the emission sources of
oxy-PAHs were reported as same as PAHs’. Incomplete combustion was regarded as one of
the major sources of oxy-PAHs. It was also indicated that the gas-phase reactions between
PAHs and ozone or between PAHs and different radicals (such as hydroxyl radicals, nitrate
radicals), were contributed to the oxygenated derivatives formation (Nocun and Schantz,
2013). Besides, oxy-PAHs were found persistent in the environment (O’Connell et al., 2013).
Therefore, oxy-PAHs are also worthy to be studied carefully.
Recent researches showed that the derivatives of PAHs, such as oxy-PAHs, are much more
toxic to both human and environment than the un-substituted PAHs (Lundstedt et al., 2014;
Nocun and Schantz, 2013). Diseases induced by allergy can be caused by exposure to
oxy-PAHs (Chung et al., 2007). Mutagenic was reported as one of the most adverse effects
can be caused by several oxy-PAHs, for instance, polycyclic aromatic ketones, quinones and
anhydrides (Kojima et al., 2010; Wei et al., 2012).
Oxy-PAHs have been noticed by more and more scientists, but the regulations and standards
of oxy-PAHs were not established completely (Wei et al., 2012), different oxy-PAHs were
analyzed in different researches. 9-Fluorenone, 9,10-anthraquinone, 1,8-naphthalic
anhydride and benzanthrone were analyzed by Kojima et al., (2010). 24 compounds of
oxy-PAHs were analyzed by O’Connell et al., (2013), which are shown in Table 1-3. Only 3
compounds, benzanthrone, 9,10-anthraquinone, and 9-fluorenone were identified and
quantified by Souza et al., (2014). Some of the important oxy-PAHs are listed in Table 1-3.
It comes to be a top priority task to monitor the concentration of PAHs and its derivatives in
all programs for evaluating environmental hazards and the human health risk. Traditionally,
PAHs and oxy-PAHs had been monitored in soils, water, air and sediments etc. (Cochran et al.,
2012; Lang et al. 2008; Navarro-Ortega et al., 2012). The result of multi-year systematic air
sampling has revealed the seasonality in PAHs concentrations, the levels were observed
highest during the colder winter months (Halsall et al., 2001).
- 6 -
Table 1-3 Some of the Important oxy-PAHs Species
Oxy-PAHs Structure Oxy-PAHs Structure
1,4-Benzoquinone O
O
Chromone
O
O
1,2-Naphthoquinone O
O
1,4-Naphthoquinone O
O
Perinaphthenone O
9-Fluorenone O
1,2-Acenaphthenequinone O O
Xanthone
O
O
9,10-Phenanthrenequinone O
O
1,4-Anthraquinone O
O
9,10-Anrhraquinone O
O
Phenanthrene-1,4-dione O
O
2-Ethyl-9,10-Anthraquinone O
O
4H-Cyciopenta[d,e,f]phenanthrenone
O
1,9-Benz-10-Anthrone
O
Benzo[a]-11-fluorenone
O
Pyrene-4,5-dione
O
O
Aceanthrenequinone O O
5,12-Naphthacenequinone O
O
Benzo[c]phenanthrene-1,4-quinone O O
6H-Benzo[c,d]pyrenone O
Benz[a]anthracene-7,12-dione O
O Benzo[a]pyrene-7,8-dione
O
O
Benzo[a]pyrene-1,6-dione O
O
A range of different passive sampling devices have been used to evaluate the air quality
(Seethapathy et al., 2008). The relevant information was provided by the existence of
worldwide natural bio-monitoring matrices (Ratola et al., 2012). Two main limitations of
PAHs and oxy-PAHs urban monitoring have been presented to the researches. Firstly, the
- 7 -
wide variation of PAHs and oxy-PAHs concentration bothered a lot. Secondly, the monitoring
in urban is an intensive task in labor, equipment and time (Noth et al., 2013). Therefore,
plant bio-monitors are commonly used, and regarded as the complementary to automatic
monitoring devices (Rodriguez et al., 2012; Wang et al., 2008). Time-integrated information
of a large number of persistent pollutants on atmospheric depositions is provided by plant
bio-monitors, furthermore, the pollutants deposition maps at different scales are permitted
while bio-monitoring (De Nicola et al., 2013). Therefore, considering the large scale of
landscape plants distribution in modern cities, bio-monitors are chosen to discuss in this
study.
Some articles reported a direct relationship between PAHs and oxy-PAHs concentrations in
soil and plants. They believed a strong absorption of PAHs in soil was conducted by root cells
(Fismes et al, 2002; Lundstedt et al., 2014; Srogi, 2007; Zhan et al., 2013). But the primary
pathway PAHs transport into the leaves is air which has been verified by both field and
chamber studies recently. According to the articles, “most PAHs (80%) were accumulated in
the wax fraction, and most of the remainder (17%) penetrated the inner tissues of the leaves”
(Murakami et al., 2012). This conclusion makes vegetation bio-monitoring a feasible way to
examine the spatial distribution and composition of PAHs and oxy-PAHs (Noth et al., 2013).
Various groups of plants (e.g. moss, aquatic plants, grasses, vegetable sand trees) have been
recommended as bio-monitors for PAHs by researchers (De Nicola et al., 2005; De Nicola et
al., 2013; Noth et al., 2013; Rinaldi et al., 2012; Rodriguez et al., 2012; Sanz-Landaluze et al.,
2010). They are able to accumulate in vegetation after deposition. Some articles indicated
that leaves which possess a high surface area can be contaminated by PAHs more seriously
(Ziegenhals et al., 2008).
Pine needles can be applied in the PAHs monitoring because of the strong tendency to
accumulate airborne contaminants with the cover of lipidic-waxy, also its perennial character
enable the monitor in the high-pollutant-concentration-winter (Ratola et al., 2012; Ratola et
al., 2006; Navarro-Ortega et al., 2012). For these reasons, pine needles may provide
complementary monitoring information. In some regions, pine needles have already been
used as passive samplers to monitor the concentration, the sources and the spatial
distribution of PAHs (Navarro-Ortega et al., 2012). Specifically, spruce needles are highlighted
in the study of Niu et al., (2003), because its leaf surface is rich with wax components, which
can accumulate many kinds of lipophilic organic compounds. On the other hand, the
bio-monitor species for oxy-PAHs were hardly reported.
Gas chromatography mass spectrometry (GC-MS) is the technique used most widely to
analyze PAHs and oxy-PAHs (Bamford et al., 2003; Forsberg et al., 2014; Zhang et al., 2011).
Before GC-MS analysis, purification was recommended to minimize the influence of the
biological material. Solid phase extraction was recommended as the purification method
(Augusto et al., 2009; Cochran et al., 2012; Ratola et al., 2012).
The advantages on economic aspect of using bio-monitor could be seen easily as well. Due to
- 8 -
the costly price of technical equipment used in conventional atmospheric pollutants
measurement for PAHs, the use of bio-monitors is important and helpful in developing
countries. (Wannaz et al., 2012).
This thesis aims to develop the existing bio-monitoring method for PAHs quantificational
determination; to introduce the new species Taxus baccata for PAHs and oxy-PAHs
bio-monitoring; to innovate the method for oxy-PAHs bio-monitor.
- 9 -
CHAPTER 2 Literature Review
2.1 Sampling
Needle-shape leaf species and broad leaf species were applied widely as biological material
for analyzing PAHs and the derivatives. For instance, Pinus pinea, the representative of
needle species and Quercus ilex, the representative of broad species have been used
commonly. Besides, moss is another species which attracted scientists a lot (Augusto et al.,
2013; Martins et al., 2014). However, the species of moss used in different researches were
not consistent.
As for the sampling strategy, some studies pointed that the leaves should be picked from all
parts of the tree and mixed homogeneously (Rodriguez et al., 2012). While others indicated
that only the leaves from outer part or from the canopy should be collected, to ensure the
accuracy of the result (De Nicola et al., 2005; Sun et al., 2010). Consistently, nearly all of the
experiments made the choice to collect leaves growing at the height of 2-4 meter from the
ground, because the comparison between the leaves from bottom branches and the leaves
from higher branches showed that the bottom branches leaves provided less information
than the higher ones (Ratola et al., 2006).
The age of leaf was also mentioned by many researches. Based on different species and
location information, leaf aged ranged from 5 month to 3 years was mostly used. In some
particular cases, the age of leaf was defined as the period of exposure, for example, 1 week
after the fire disaster (Rey-Salgueiro et al., 2008) or 12 weeks after transplanting from
cultivating field to monitoring point (Rinaldi et al., 2012).
There was a procedure of sampling recommended by most of the authors. Briefly, 1) the
leaves were picked by hand, tried to minimize the contact with leaf surface, gloves were
highly recommended; 2) the leaves were wrapped by aluminum foil; 3) the aluminum foil
packages were sealed in plastic bags and stored in cool box while transport; 4) if the tests
didn’t proceed at the day of sampling, the plastic bags with leaves would be stored in freezer
under -20℃ in the lab until analyzing. More information can be found in Table 2-1.
- 10 -
Table 2-1 Literature Review of Sampling
Location Species and Type Plant Part Amount Age Sample method Reference
Spain, Barcelona Needle: Pinus pinea L. Randomly from single
tree
NA*
NA NA Ratola et al.,
2006
Portugal (rural/seaside),
Porto; Spain, Barcelona
Bottom branches NA NA NA
Fresno, California,
consider population and
traffic, 91 locations
totally
Needle: Jeffrey pine trees (Pinus
jeffreyi)
The 2 branches from
different sides of one
tree.
99 samples All in same old Wrapped by solvent-washed
aluminum foil, samples were
labeled and tape sealed,
transported with dry ice
Noth et al.,
2013
Spain, the Ebro River
basin, urban, industrial,
and agricultural area
from source to mouth
Needle: Pinus halepensis, Pinus
pinea and Pinus nigra
NA 30 samples NA NA Navarro-Ortega
et al., 2012
Greater Cologne
Conurbation, Industry
and residence combined
Needle: Pine trees NA 3 trees for each
sites
Different ages
collected
separately
Sampled twice in winter and
summer
Lehndorff &
Schwark, 2009
Industrial site Needle: Masson pine (Pinus
massoniana L.)
Outer part of the
middle canopy
5 similar mature
trees/site
Distinct
different ages
Wrapped and sealed by
polyethylene bags, stored in a
homemade cryogenic storage
container
Sun et al., 2010
Canada, Ottawa,
municipal sanitary landfill
(high way and agriculture
land nearby)
Needle: Norway spruce (Picea abies) NA NA NA Wrapped by solvent-cleaned
aluminum foil, stored under -80℃
St-Amand et al.,
2008
Portugal, Spain, and
Greece, 4 urban and 5
non-urban areas in each
country
Needle: Pinus pinea L. Outer branches about
2m height
NA 1-year old
needles
Sampled from the bottom of each
needle, wrapped by aluminum foil
and plastic bag, frozen to transport
& store
Ratola et al.,
2012
- 11 -
Table 2-1 continue
Location Species and Type Plant Part Amount Age Sample method Reference
Argentina, coastal area Needle: Pinus radiata;
Broad leaf: Populus hybridus;
Long slim leaf: Eucalyptus rostrata
Randomly from single
tree
150-200 g
plant/site
1-year old
needles
Collected a distance between 2.5-3
m in each direction from each plant.
Rodriguez et
al., 2012
Germany, Stuttgart,
Urban (traffic,
background), suburban
(background, rural,
traffic)
Needle: Tillandsia capillaris;
Long slim leaf: Lolium multiflorum
(both are cultivated, not natural)
NA NA 12 weeks Wrapped by labeled paper
bags and kept in a cooling box
Rodriguez et
al., 2010
Brazil, Cubatao, 12km to
costal, surround by
mountain
Needle: Lolium multiflorum;
Broad leaf: P. guajava (tropical
biomonitors)
(both are cultivated, not natural)
Randomly from single
tree
NA NA Wrapped by aluminum foil-freezer Rinaldi et al.,
2012
Germany, Leipzig–Halle
region
Needle: Pinus sylvestris L.;
Broad leaf: Maple leaves (Acer
campestre)
NA 5 trees/site, 300g
needle,10g leaves
1) 5 month
leaves; 2)
years needles
Stored under -25℃ Hubert et al.,
2003
Mexico, Coatzacoalcos
Veracruz, industrial area
Broad leaf: white mangrove
(Laguncularia racemosa), red
mangrove (Rhizophora mangle),
medlar (Eriobotrya japonica)
Randomly from single
tree
NA NA Wrapped by Aluminum foil, plastic
bag and cold boxes successively,
one single tree from one site
Sanz-Landaluze
et al., 2010
Italy, Naples Broad leaf: Quercus ilex L.
(Mediterranean evergreen oak)
Outer part of canopies,
2-4m height
40 leaves/tree, 8
trees/site
1-year old Minimize contact with the leaf
surface while sampling, stored
under -20℃ in polyethylene bags
De Nicola et al.,
2005
China, Beijing, urban
roadsides and inside a
university campus
Broad leaf: Gingkgo (Ginkgo biloba),
peach (Prunus persica), Japanese
pagodatree (Sophora japonica),
purple leaf plum (Prunus cerasifera),
Peking lilac (Syringa pekinensis) and
green spire (Euonymus japonicus).
1.5 m height 50 leaves from 4-6
trees, each site
each species
Annual fresh
leaves
NA Wang et al.,
2008
- 12 -
Table 2-1 continue
Location Species and Type Plant Part Amount Age Sample method Reference
Italy, Naples and Salerno
in Campania region 19
sites in urban, 2 sites in
the remote
Broad leaf: Quercus ilex L. Outer part of the
canopies, about 2m
height
NA One-, two-
and
three-year-old
leaves
Leaves picked by hand, minimize
contact with the leaf surface,
transported and stored under -20℃
in polyethylene bags, avoid light
De Nicola et
al., 2011
Estuary, towns and
industry area
Broad leaf: Cabbage, maize, grape,
‘‘Padron-type” pepper and tomato
NA NA 1 week after
fire
Stored in a room under 0-4℃ Rey-Salgueiro
et al., 2008
Italy, Campania &
Tuscany, urban,
periurban (5km away) &
extraurban (70-100km)
area
Broad leaf: Quercus ilex;
Moss: leptodon smithii (epiphytic
moss)
1) leaves: randomly
from 15-20 trees,
canopy 3-4 m height; 2)
moss: from same tree
as leaves, 1-2m height
NA 1) leaves:
1-year old; 2)
moss: 2-3
years
Sample stored under -20℃, without
washing the leaves
De Nicola et
al., 2013
Portugal highly
industrialized coast
Moss: Parmotrema hypoleucinum
(Steiner) Hale
Collected from
branches and trunks of
Quercus suber L. &
Pinus pinea L
34 samples NA Leaves were put in brown glass
bottle after picking, stored under
4℃
Augusto et al.,
2009
Spain, Navarra, Bertiz
Nature Reserve
Moss: Isothecium myosuroides Brid.
and Hypnum cupressiforme Hedw
Brown part are not
included
9 samples of Brid,
3 of Hedw
Less than 3
years,
according to
the duration
of research
NA Foan et al.,
2012
*NA: Not mentioned in the articles.
- 13 -
2.2 Sample Preparation & Extraction
First of all, the dry weight of the sample was determined. The temperature of oven was
suggested to be set at 80 - 85℃ (Hubert et al., 2003; Ratola et al., 2012), while others did
not mention clearly. Secondly, to make the sample prepared, reduce the size of leaf was
applied by most of the literature. Two mainstreams of particle size were adopted: cut into 1
mm sections and grinded into powder under 0.05 mm. In most cases, pulverization was
performed after liquid nitrogen freezing.
Secondly, to separate the target compound from the sample leaves, extraction procedure
was taken. The conventional methods are ultra-sonication and Soxhlet extraction, which
have been widely applied on biological materials extraction. The results of the traditional
ones were considered reliable (De Nicola et al., 2011; Rodriguez et al., 2012; Sun et al., 2010).
However, shortages were reported as well, for instance, cost of time and solvent (Augusto et
al., 2009; Hubert et al., 2003), loss of volatile compounds and filtration is required
mandatorily (Wang et al., 2008), etc. The extraction temperature and power of
ultra-sonication were not clearly stated in literature. The time of extraction normally should
be set at 10 minutes and repeated 3 times (Ratola et al., 2012; Ziegenhals et al., 2008). Little
information about Soxhlet extraction was introduced. The extract time of 24 hours was
suggested by some researches (Rinaldi et al., 2012).
The advanced extraction methods pressurized liquid extraction (PLE) was suggested by
recent researches (Foan et al., 2012; Lehndorff & Schwark, 2009). The accelerated solvent
extractor (ASE 200 and ASE 300) manufactured by Dionex were applied in these researches.
The configuration of the device was found similar after comparing different literature. The
temperature was around 100℃, the pressure was around 100bar, the flush volume was 60%,
the time was 10min, 2-3 static cycles were set and the purge time was 120s.
Once, saponification was also applied for extraction. The details of the extraction were not
introduced. 30mL of cyclohexane was applied as extraction solvent (Ziegenhals et al., 2008).
Lots of solvents were applied as extraction solvents. The mixture hexane:dichloromethane
(1:1, v/v), dichloromethane and hexane were the most commonly used extraction solvents.
Besides, varies choices of solvents were reported, for instance, acetone and the mixture
dichloromethane:acetone (1:1,v/v), etc. The amount of solvent was not clearly explained.
Anhydrous sodium sulfate was applied as desiccant and was suggested to be added while
concentrating (De Nicola et al., 2013; Sun et al., 2010). Filters with 0.22μm or 0.45μm pore
diameter were used for the extracted solvent filtration. The filter composited by Na2SO4 and
celite (70:30, w/w) applied by Ziegenhals et al (2008) only.
To concentrate the extract, nitrogen flow performed by TurboVap and rotary evaporator
were suggested mostly. The temperature, vapor pressure and rotate speed were not
described clearly. The application of combining the TurboVap and the rotary evaporator was
- 14 -
recommended several times. The solvent was evaporated down to 4-5mL by rotary
evaporator and then blow down by nitrogen flow until the extract dryness (De Nicola et al.,
2011; De Nicola et al., 2013; Rinaldi et al., 2012). The endpoint of concentration was
introduced lower than 1mL by lots of authors.
The comparison between various extract methods was made previously. The articles with
comparison were bold framed in the following table. The difference between PLE and
Soxhlet was studied by Hubert et al., (2003) and Foan and Simon (2012). The recovery (about
70%) and total PAHs concentration (200ng/g) were found similarly when PLE and Soxhlet
were performed, however, the advantages of less consumption of solvent and time were
found, it was 30mL for PLE against 100mL for Soxhlet and 24 samples in 8 hours for PLE
against 2 samples in 3.5 hours for Soxhlet, therefore, PLE extraction was recommended
(Foan and Simon, 2012). The difference between PLE, ultra-sonication and Saponification
was studied by Ziegenhals et al., (2008). PLE was recommended after comparing the
efficiency of extraction. The difference between PLE, Soxhlet and ultra-sonication was
studied by Ratola et al., (2006). The recoveries and relative standard deviation (RSD) of the 3
different extraction technologies were 65-102% with RSD <7.5% (Soxhlet), 72-100% with RSD
<8% (ultra-sonication), 70-137% <11% (PLE), therefore, ultra-sonication was recommended
considering accessibility, lower cost and less time (Ratola et al., 2006).
Two different technologies PLE and non-device extraction was applied on different parts of
the sample leaf by Wang et al., (2008). PLE was applied on inner part of the leaf, while
non-device extraction was applied on the cuticle. Therefore, the differences between the
two technologies were not stated clearly in the article. More information can be found in
Table 2-2.
- 15 -
Table 2-2 Literature Review of Sample Preparation & Extract
Sample Preparation Extract method & description Solvent information Extra information Evaporation after
extraction
Filter after
extraction
Reference
No Ultra-sonication 2mL of homogenized
n-hexane:acetone (1:1, v/v)
NA Nitrogen used to blow
down solvent
0.22μm Rodriguez et al.,
2012
No Ultra-sonication n-hexane NA Nitrogen used to blow
down solvent
NA Rey-Salgueiro et
al., 2008
No Ultra-sonication dichloromethane:acetone (1:1,
v/v)
5g leaves, 3g moss,
anhydrous sodium
sulfate (=mass of
sample)
Rotary evaporator
dried to 4 mL, then
blew down to dryness
by nitrogen
NA De Nicola et al.,
2013
FW/DW determined Ultra-sonication: repeated 3 times 100mL of
dichloromethane:acetone (1:1,
v:v)
5 g sample Rotary evaporator
dried to 5 mL, then
blew down to dryness
yes De Nicola et al.,
2011
Dried in room
temperature for 1 week,
grilled and sieved by
63цm, stored under 4℃
until analysis
Ultra-sonication: 30s, 20% amplitude,
25℃
Acetone, dichloromethane,
n-hexane, methanol, iso-octane
and n-hexane:acetone (1:1, v/v)
NA Nitrogen used to blow
down solvent
0.22μm Sanz-Landaluze et
al., 2010
Defrosted in room
temperature, FW/DW
determined under 80℃
Ultra-sonication: 10min, repeated 3
times
30mL of hexane:dichloromethane
(1:1, v/v)
5g sample Rotary evaporator to
dryness
NA Ratola et al., 2012
Samples divided to 2
groups, one for PAHs,
one for FW/DW
Soxhlet extraction: 24h 250mL of dichloromethane and
50mL of hexane
0.2g sodium
sulfate
1) rotary evaporator to
10ml; 2) add Na2SO4
then centrifugal to
separate; 3) evaporate
to dryness
0.45μm Rinaldi et al.,
2012
No Soxhlet extraction: 24h 200mL of acetonitrile 2g sample Rotary evaporator NA Augusto et al.,
2009
- 16 -
Table 2-2 continue
Sample Preparation Extract method & description Solvent information Extra information Evaporation after
extraction
Filter after
extraction
Reference
Samples were separated
into two groups, one
washed with deionized
water, another left
original, while testing,
cut into 1-cm sections,
freeze-dried
Soxhlet extraction: 48h dichloromethane 5g sample, Na2SO4 NA NA Sun et al., 2010
Dried spruce needles PLE*: ASE
** 200, 140℃, 1000psi
(≈69bar)***
, 10min, 2 static cycles,
purged time 120 s
hexane:dichloromethane (1:1, v/v) NA Concentrated using a
TurboVap
NA St-Amand et al.,
2008
FW/DW determined
under 50℃
PLE: 120℃, 75bar hexane:dichloromethane (99:1,
v/v)
Centrifugation to
remove waxes
NA NA Lehndorff &
Schwark, 2009
1) FW/DW determined
under 85℃; 2)
ultra-sonication with
100mL of DCM for
10min, the wax layer was
filtered; 3) inner
substance shredded
PLE: ASE 200, two steps at 40 and
120℃, 15MPa (≈150bar)****
, 10min, 3
static cycles
n-hexane NA NA NA Hubert et al.,
2003
No Soxhlet extraction: 20h n-hexane 100g sample NA NA
Freeze-dried
recommended, grinded
to <0.05mm
PLE: ASE 200, 80℃, 150bar, warm up
for 5min, 2 static cycles, purged with
purified nitrogen for 120s
n-hexane 1.5g sample, 0.75g
Na2SO4
Nitrogen used to blow
down to 1mL
NA Foan et al.,
2012
No Soxhlet extraction: Soxtec System
HT2, immersed in boiling solvent 2h,
rinsed for 1h
100mL of n-hexane 1.5g sample, 50ng
surrogate standard,
0.75g Na2SO4
Nitrogen used to blow
down to 1mL
NA
- 17 -
Table 2-2 continue
Sample Preparation Extract method & description Solvent information Extra information Evaporation after
extraction
Filter after
extraction
Reference
Homogenized, levigate
with acrylic acid
PLE: ASE 200, 100℃, 100 bar, 10 min,
4-cycle, flush volume 60%, 2 static
cycles, purge time 120s
n-hexane, n-hexane:acetone (1:1,
v/v)
1.5–2 g tea NA Glass
microfiber
filters
Ziegenhals et
al., 2008
Samples were heated
with 30 mL methanolic
(KOH) under reflux 1 h,
filter by glass wool
Saponification: after extraction,
washed 3 times with 30mL portions of
distilled water
30mL of cyclohexane 1.5–2 g tea Rotary evaporator Na2SO4/Celite
(70:30, w/w)
No Ultra-sonication: repeated 3 times. dichloromethane:acetone (1:1,
v/v)
1.5-2g sample Rotary evaporator Na2SO4/Celite
(70:30, w/w)
Cut into 1cm bits,
analysis immediately
after sampled
Soxhlet extraction: 24 h 100mL of
hexane:dichloromethane (1:1, v/v)
10g sample Rotary evaporator to
0.5mL
NA Ratola et al.,
2006
Cut into 1cm bits,
analysis immediately
after sampled
Ultra-sonication: 360W Selecta
ultrasonic bath, 10min, repeated 3
times.
30mL of hexane:dichloromethane
(1:1, v/v)
10g sample Rotary evaporator to
dryness
NA
Grind and fill to the top
with hydro matrix
PLE: ASE 200, 150℃, 1500psi
(≈103bar), warm up for 7min, run for
10min, 2 static cycles, purge time 90s
hexane:dichloromethane (1:1, v/v) NA TurboVap LV
evaporator with
Nitrogen to 0.5mL
NA
- 18 -
Table 2-2 continue
Sample Preparation Extract method & description Solvent information Extra information Evaporation after
extraction
Filter after
extraction
Reference
Fresh leaves were
air-dried (25℃) for 1 h
NA 150mL of dichloromethane 1-6g sample 50mL extraction dried
by nitrogen, weighted
as cuticle wax
0.45цm Wang et al.,
2008
Step1: Fresh leaves were
air-dried (25℃) for 1h;
Step 2: (extracted leaves)
freeze-dried and
pulverized for 40-mesh
sieve
PLE: ASE 300 dichloromethane:acetone (1:1,
v/v)
0.5-2g sample NA NA
* PLE: Pressurized Liquid Extraction
** ASE: Accelerated Solvent Extractor
*** 1bar ≈ 14.5psi
**** 1bar ≈ 0.1MPa
- 19 -
2.3 Clean-up
The co-extraction and co-elution of plant lipids and pollutants can lead to great interference
on GC–MS analysis (Hubert et al., 2003). Therefore, chromatographic columns packed with
Silica/Alumina/Florisil cartridges (Solid Phase Extract-SPE cartridges) were applied to remove
the influence of polar compound and gel permeation chromatography (GPC) was applied to
eliminate the influence caused by lipids (Sun et al., 2010).
According to the literature, the procedure of SPE clean-up can be concluded as following: 1)
the devices and tools were washed by distilled water; 2) the devices and tools were
conditioned by solvent; 3) the solvent after extraction was added into the SPE columns
followed by the eluents; 4) every drop of the liquid was collected into one tube; 5) collected
solvent was concentrated to almost dryness by rotary evaporator or TurboVap; 6) the
residue after concentration was reconstituted by 1mL of solvent; 7) the prepared sample
was transferred to vials and stored in freezer waiting for further analysis (Ratola et al., 2006;
Ratola et al., 2012; Ziegenhals et al., 2008). Dichloromethane, cyclohexane and the mixture
hexane:dichloromethane (1:1, v/v) were the eluents which widely used for SPE (Ratola et al.,
2006; Ratola et al., 2012; Ziegenhals et al., 2008). The eluent volume was not explained
clearly in the articles.
GPC was recommended by some articles. Briefly, the residue from extraction was eluted and
filtered, and then the liquid was transferred for GPC clean up. The product of GPC was
concentrated to 0.5mL or to dryness by rotary evaporator. Further purification maybe
performed depends on different situation. Cyclohexane:ethylacetate (1:1 v/v) was used as
the eluent for GPC (Rodriguez et al., 2010).
Other clean up method were introduced, for instance, size exclusion chromatography (SEC),
medium pressure liquid chromatography etc., some researchers also suggested it is not
necessary for cleaning up (Rodriguez et al., 2012; Sanz-Landaluze et al., 2010).
The comparison between various extraction methods was made previously. The literature
with comparison was bold framed in the following table. The difference of Florisil, alumina
and silica between cartridges format and glass chromatography was studied by Ratola et al.,
(2006). The similar results were obtained by glass chromatography and by alumina and silica
in cartridges format. The recovery was 100% for most of the 16 PAHs with RSD <13%, except
for the ones less volatile which the recovery was around 50%. Inconsistence and higher high
molecular weight PAHs recovery were obtained by Florisil SPE cartridge. ’Due to the more
time-consuming set-up and operation of this approach, it was decided to consider alumina
cartridges as the best and most selective clean-up method.’ (Ratola et al., 2006).
The difference between SEC and conventional method was studied by Hubert et al., (2003).
The concentration of each PAHs compound was improved while comparing SEC clean up to
the conventional clean up. The RSDs for SEC (10%) were found lower than the conventional
- 20 -
(20-30%). Therefore, SEC was recommended for clean-up (Hubert et al., 2003). Both GPC
and SPE were applied by Ziegenhals et al., (2008), but the difference was not clearly stated.
More information can be found in Table 2-3.
- 21 -
Table 2-3 Literature Review of Clean-up
Clean-up method Extra information Program Reference
No clean up NA NA Rodriguez et al.,
2012
No clean up NA NA Sanz-Landaluze et
al., 2010
Centrifuge NA 3500rpm, -2℃, 15min, twice Rinaldi et al., 2012
GPC* NA 1) residue from extraction eluted with 10mL of cyclohexane:ethylacetate (1:1 v/v); 2) 5mL of above used
in GPC and concentrate to 0.5mL in 40℃ water bath with rotary evaporator; 3) further purify with
adsorption chromatography (AC) with silica gel of petrolether:dichloromethane (4:1, v/v); 5) mix with
propan-2-ol for final purification on Sephadex LH-20 column; 6) the rest was re-constituted by 1mL of
hexane.
Rodriguez et al.,
2010
SPE**
(Polypropylene) 5g, 25mL of Alumina
cartridges
1) 50mL of hexane:dichloromethane (1:1, v/v) to condition; 2) extract add to column, elute with 50mL of
hexane:dichloromethane (1:1, v/v) and 50mL of dichloromethane; 3) pre-concentrate to 0.5mL with
rotary evaporator; 4) re-constituted by 1mL of hexane.
Ratola et al., 2012
SPE cartridges of Florisil 30mL of acetonitrile
as eluting solvent
NA Augusto et al., 2009
SPE cartridges of silica NA NA Rey-Salgueiro et al.,
2008
Silica column chromatography column information:
10mm i.d. × 350mm
length, 10g of silica
gel and 20mm length
of Na2SO4
1) concentrated using rotary evaporator; 2) transferred with cyclohexane and eluted with 25mL of
n-hexane, followed by 50mL of pentane:dichloromethane (3:2, v/v) at the rate of 2mL/min; 3) evaporated
to dryness by rotary evaporator; 4) rinsed 3 times in Kudema-Danish concentrator with n-hexane; 5)
blown down to 1mL by nitrogen
Wang et al., 2008
- 22 -
(Table 2-3 continue)
Clean-up method Extra information Program Reference
Small-deactivated silica column NA 1) eluted with hexane; 2) concentrated to approximately 200μl using a TurboVap St-Amand et al.,
2008
Medium pressure liquid
chromatography
NA NA Lehndorff &
Schwark, 2009
GPC NA 1) 4.5mL of cyclohexane:ethyl acetate (1:1, v/v); 2) polytetrafluoroethylene (PTFE) filter (pore size 1mm)
with flow rate of 5mL/min, waste time 0–36min, collect time 36–65min; 3) rotary evaporator used to
remove solvent, then blew down to dryness by nitrogen
Ziegenhals et al.,
2008
SPE: ASPEC***
Xli Before clean-up, 1)
1g silica dried for 12h
under 550℃; 2)
deactivate with 15%
distilled water into
8mL column
1) condition column with 3mL of cyclohexane; 2) 10mL of cyclohexane added to the sample as solvent
SPE cartridges of Florisil 5g 1) conditioned with 50mL of hexane:dichloromethane (1:1, v/v); 2) sample added and eluted with 50mL
of hexane:dichloromethane (1:1, v/v), followed by 50mL of dichloromethane; 2) Rotary evaporator dried
to 0.5mL, then blew down to dryness by nitrogen; 3) reconstitute in 1mL of hexane.
Ratola et al., 2006
SPE cartridges of silica 5g
SPE cartridges of alumina 5g
Glass column with Florisil, silica
and alumina
5g Florisil / Silica /
Alumina and 1cm
high of Na2SO4
Activated at 400℃ for 12h and deactivated with 1.2% ultrapure water
- 23 -
(Table 2-3 continue)
Clean-up method Extra information Program Reference
SEC****
The extracted solvent
was concentrated to
dryness first
1) 600μL THF was used to dissolve the solvent; 2) SEC condition: flow rate 5mL/min, run for 15min; 3) the
solvent collected between 0-9.2min was discarded; 4) the solvent between 9.2-12min was collected and
concentrated to dryness by rotary evaporator.
Hubert et al., 2003
Conventional The extracted solvent
was concentrated to
2mL first
1) extracted solvent was mixed with 15g deactivated Florisil; 2) eluted with 160mL
n-hexane:dicholoromethane (1:1, v/v); 3) the first 60mL eluate were mixed with 3.5g deactivated Florisil
and eluted with 60mL same eluent; 4) concentrate to dryness.
* GPC: Gel Permeation Chromatography
** SPE: Solid Phase Extraction
*** ASPEC: Automated Solid-Phase Extraction Clean-up
**** SEC: Size Exclusion Chromatography
- 24 -
2.4 PAHs and oxy-PAHs Analysis
Two technologies were applied to analyze PAHs and oxy-PAHs, High Performance Liquid
Chromatography (HPLC) and GC-MS. GC-MS was applied more widely than HPLC.
Apparently, GC-MS is combined by two parts. Gas chromatography (GC) is the first part
which is applied to analyze the chemical species existed in the sample. Firstly, the sample
was heated into gaseous phase. The gaseous sample was carried by inert gases, such as
helium, and was transported into the chromatographic column. The absorption ability of
absorbent to the components existed in the sample was different, resulting in that the speed
of various components was different in GC. The components that have the weakest
absorption ability to the adsorbent were discarded the earliest. (Wiersma, 2004; Zeng, 2010).
Mass spectrometry is one of the quantitative approaches based on the measurement of
mass-to-charge ratio. The components are ionized into positive ions with different
mass-to-charge ratio first. (Wiersma, 2004; Zeng, 2010).
HP-5 MS capillary column with 30m × 0.25mm i.d. × 0.25μm film thickness GC-MS column
was applied in most of the experiments. TR-50MS 10m × 0.1mm i.d. × 0.1μm film thickness
GC-MS column was applied by Ziegenhals et al., (2008). Fused-silica capillary column with
15m × 0.25mm i.d. × 0.25μm film thickness GC-MS column was applied by St-Amand et al.,
(2008). Vydac 201TP C18, 250mm × 2.1mm (length i.d.), 5μm particle size column was
applied by Rodriguez et al., (2012). SupelcosilTM LC-PAH C18 column with 25m × 0.32mm i.d.
× 0.52μm film thickness HPLC column was applied by Foan et al., (2012). More information
can be found in Table 2-4.
- 25 -
Table 2-4 Literature Review of Detection & Separation
Equipment Re-constitution Column Flow Program Reference
GC-MS*
NA HP-5MS capillary column (30m ×
0.25mm i.d. with a 0.25μm film
thickness)
NA 1) 33℃, 1min; 2) 20℃/min to 280 ℃; 3) keep for
15min
De Nicola et
al., 2005
GC-MS NA TR-50MS column (10m × 0.1mm ×
0.1μm)
NA 1) 50℃, 1min ; 2) 25℃/min to 280℃; 3) 1℃/min to
330℃; 4) hold 30min
Ziegenhals et
al., 2008
GC-MS NA HP ultra 2 capillary column (25m ×
0.32mm i.d. × 0.52μm film thickness)
NA 1) 60℃, 1min; 2) 10℃/min to 260℃; 3) keep 1min Hubert et al.,
2003
GC-MS NA HP-5 MS capillary column (30m ×
0.25mm i.d. × 0.25μm film thickness)
1mL/min,
helium
Start at 60℃, raise to 280℃ in 6 minutes Wang et al.,
2008
GC-MS The solvent after clean-up was
concentrated and re-dissolved in
4mL of cyclohexane
HP-5MS capillary column (30m ×
0.25mm i.d. × 0.25μm film thickness)
1.11mL/min,
helium
1) started at 70℃; 2) increased by 20℃/min, to
280℃; 3) held for 24min.
De Nicola et
al., 2011
GC-MS 1) The solvent after clean-up was
dried to 1mL by rotary evaporated;
2) further concentrated to 0.2mL; 3)
1μl hexamethyl-Benzene was added
NA 1.2 mL/min,
helium, linear
velocity
25.4cm/sec at
300°C
1) start at 60℃ for 3 min; 2) raised to 200℃ by
20°C/min; 3) to 260℃ by 4℃/min; 4) to 270℃ by
2℃/min; 5) 10 min hold time
Sun et al.,
2010
GC-MS NA DB-5 column (30m × 0.25mm i.d. ×
0.25μm film thickness) coated with 5%
diphenyl-olydimethylsiloxane
Helium 1) 60℃, 1min; 2) 6℃/min to 175℃, hold 4min; 3)
3℃/min to 235℃; 4) 8℃/min to 300℃, hold 5min
Ratola et al.,
2006
GC-MS The solvent after clean-up was
concentrated and reconstituted to
4mL by 0.4mL of internal standard
and cyclohexane
HP-5MS capillary column (30m ×
0.25mm i.d. × 0.25μm film thickness)
1.11mL/min,
helium
1) started with 70℃; 2) up by 20℃/min to 280℃; 3)
keep 24min
De Nicola et
al., 2013
- 26 -
(Table 2-4 continue)
Equipment Preparation Column Flow Program Reference
GC-MS The solvent after clean-up was
concentrated and re-dissolved in
1mL of hexane
VF-5MS (30m × 0.25mm i.d. × 0.25μm
film thickness)
1mL/min helium 1) 60℃ for 1min; 2) up to 175 by 6℃/min hold 4min;
3) up to 235℃ by 3℃/min; 4) up to 300℃ by 8℃
/min; 5) keep to total time of 60min
Ratola et al.,
2012
GC-MS The solvent after clean-up was
concentrated and re-dissolved in
1mL of hexane
HP-5 MS (30 m × 0.25 mm i.d.,
0.25μm) fused-silica capillary column
1mL/min helium 1) 90℃ for 4 min; 2) raise to 100 by 10℃/min; 3)
raise to 290℃ by 3℃/min; 4) keep 22 min
Rodriguez et
al., 2010
GC–EIMS**
NA Fused-silica capillary column (15m ×
0.25mm) coated with 0.25μm
chemically bonded HP-5MS phase (5%
phenyl methyl siloxane)
NA 1) Initial 150℃ for 2 min; 2) 10℃/min to 240 ℃; 3)
5℃/min to 300℃; 4) held for 5 min.
St-Amand et
al., 2008
HPLC***
The solvent after clean-up was
concentrated and re-dissolved in
2mL of acetonitrile
25cm × 4.6mm (length—i.d.) 5μm
particle Supelcosil LC-PAH coupled
with a guard column
NA NA Rinaldi et al.,
2012
HPLC The solvent after clean-up was
concentrated and re-dissolved in
200μl acetonitrile:water (6:4, v/v)
Vydac 201TP C18, 250mm × 2.1mm;
5μm
0.3 ml/min 1) ACN:water (60:40, v/v); 2) 18min, 100% CAN; 3)
21min back to step one; 4) total time 28min
Rodriguez et
al., 2012
HPLC NA Supelcosil LC-PAH C18 column (250
mm × 4.6mm i.d. 5μm particle size)
and a precolumn (20mm × 4.6mm i.d.
5μm particle size)
NA NA Foan et al.,
2012
HPLC The solvent after clean-up was
concentrated and filled up to a final
volume of 0.5mL with ACN
Supelcosil LC-PAH (25cm × 4.6mm
(length × i.d.), 5μm particle size), kept
constant at 33℃.
Inject volume:
50μl by flow
rate of 1mL/min
Mobile phases were acetonitrile (ACN) and water. 1)
the gradient was: 80:20 ACN/H2O; 2) changed to 95:5
ACN/H2O in 40 min; 3) changed to 80:20 ACN/H2O in 1
min; 4) hold for 10 min giving an analysis time of
51min.
Rey-Salgueiro
et al., 2008
- 27 -
* GC-MS: Gas Chromatography Mass Spectrometry
** GC-EIMS: Gas Chromatography - Electron Ionization Mass Spectrometry
*** HPLC: High Performance Liquid Chromatography
- 28 -
2.5 Concentration Levels
The highest total PAHs concentration in industry area was reported in Guangdong Province, China.
PAHs concentration was recorded 1927.2ng/g (d.w.) around a ceramic manufactures (Sun et al.,
2010). On the other hand, the lowest was reported in Argentina, which is 49ng/g (d.w.) monitored
around an aluminum industry (Rodriguez et al., 2012).
The total PAHs concentration in cities was the most common topic which the researches focused on.
The difference between the urban area and suburban area was also usually compared. The highest
PAHs concentration of 8500 ng/g (d.w.) was found by De Nicola et al., (2011) in an urban area of
Campania, Italy. The result was not convinced enough to be the representative to describe the
situation of ‘Urban area’. The average city PAHs concentration was reported range from 154.2 to
4420ng/g (d.w.) depending on different traffic condition, city scale and climates. The highest PAHs
concentration was found in January (De Nicola et al., 2005). The difference age of leaf was verified
having no effect on the accumulation of PAHs (De Nicola et al., 2011). The concentration of oxy-PAHs
in leave samples or other biological samples was rarely found in previous researches. More
information can be found in Table 2-5.
According to the previous studies, phenanthrene was found has the highest concentration in natural
environment, while, indeno[1,2,3-c,d]pyrene and benzo[b]fluoranthene was found the lowest
(Ratola et al., 2006; Navarro-Ortega et al., 2012). Fluoranthene, phenanthrene and pyrene were
found have higher concentration than other PAHs compounds, relating to different traffic condition
in cities (Rodriguez et al., 2012). Naphthalene, dibenzo[a,h]anthracene, and indeno[1,2,3-c,d]pyrene
were found the lowest in cities (Rodriguez et al., 2012). Naphthalene, phenanthrene, pyrene,
chrysene and fluoranthene were found have higher concentration than others relating to different
industrial area (Sun et al., 2010). More information can be found in Table 2-6.
- 29 -
Table 2-5 Literature Review of Current Results in Sum of PAHs
Total PAHs Concentration in Leaf (ng/g, d.w.)
(Details of the Location Information)
Type of Location Reference
97/111/111.2 (Fresh leaves) 3 different polluted sites in Germany Hubert et al., 2003
290 Along the whole river in Spain Navarro-Ortega et al., 2012
41 Urban area Noth et al., 2013
1927 (ceramic manufactures)
1565 (steel industry)
1144 (petrochemical)
1045 (blank, no industry)
Industry (Guangdong, China) Sun et al., 2010
1100-2076 (May, 2001)
1349-1930 (September, 2001)
1798-4420 (January, 2002)
1038-1962 (May, 2002)
300-500 (control site)
Urban area of Naples De Nicola et al., 2005
1216 (1-year-old leaves)
1126 (2-year-old leaves)
1102 (3-year-old leaves)
500-2000 (urban site mean)
8500 (highest urban value)
Urban area (Campania) De Nicola et al., 2011
382 (moss); 714 (leaves) Urban area (Campania) De Nicola et al., 2013
154 (moss); 365 (leaves) Urban area (Tuscany)
326 (moss); 218 (leaves) Periurban (Campania)
72 (moss); 172 (leaves) Periurban (Tuscany)
125 (moss); 426 (leaves) Extraurban (Campania)
111 (moss); 141 (leaves) Extraurban (Tuscany)
400 Industry (Mexico) Sanz-Landaluze et al., 2010
2080 (heavy traffic)
1031 (moderate traffic)
423 (low traffic)
Urban area (Stuttgard) Rodriguez et al., 2010
- 30 -
(Table 2-5 continue)
Total PAHs Concentration in Leaf (ng/g, d.w.)
(Details of the Location Information)
Type of Location Reference
49-1830 Aluminum industry (Argentina) Rodriguez et al., 2012
200 Bertiz Nature Reserve (Spain) Foan and Simon, 2012
22 (Costa Nova)
59 (Cerveira)
75 (Barcelona)
339 (Porto)
Natural polluted Ratola et al., 2006
807 (Porto)
454 (Setubal)
309 (Evora)
348 (Faro)
Urban area (Portugal) Ratola et al., 2012
350 (Miranda de Ebro)
620 (El Prat)
791 (Barcelona)
346 (Vic)
Urban area (Spain)
563 (Chania)
713 (Rethymno)
566 (Malia)
344 (Ierapetra)
Urban area (Greece)
121 (Antua)
135 (Quintas)
155(Souselas)
78(Sines)
105(Alcoutim)
Sub-urban area (Portugal)
111 (Villodas)
84 (Monteagudo)
128 (Movera)
83 (Alcolea de Cinca)
135 (Torres de Segre)
Sub-urban area (Spain)
180 (Elafonisi)
98 (Paleochora)
124 (Festos)
87 (Analipsi)
39 (Moni Toplou)
Sub-urban area (Greece)
- 31 -
Table 2-6 Literature Review of Current Findings in Individual PAHs
Reference Ratola et al., 2006 Rodriguez et al., 2012 Sun et al., 2010 Navarro-Ortega
et al., 2012
Site information Costa Nova
(Natural)
Cerveira
(Natural)
Barcelona
(Natural)
Porto
(Natural)
High
Traffic
Moderate
Traffic
Low Traffic Ceramic Steel Petrochemical No Industry Natural
PAHs (±SD) ng/g d.w.
Naphthalene 4.8 2.3 2.9 14.8 1.9±0.9 2.5±2.2 0.5±0.3 141.0±34.4 116.1±21.6 198.9±77.5 320.1±30.3 12.3±8.6
Acenaphthylene NA 0.9 2.8 7.0 11.9±2.2 6.0±2.2 3.9±0.8 21.9±0.2 9.2±1.8 31.8±3.3 33.1±3.6 4.9±3.2
Acenaphthene NA 6.2 1.8 5.5 67.9±25.4 29.9±15.0 18.6±10.1 46.8±7.9 50.8±0.5 49.8±11.4 55.4±6.5 6.8±8.6
Fluorene 0.9 2.9 5.2 28.9 50.2±17.4 20.9±6.2 8.5±4.3 143±82 69.4±8.3 45.9±24.7 36.6±8.4 52.4±47.7
Phenanthrene 7.3 11.6 20.4 102.9 595±293 263±122 98.6±29.5 467±246 252±29.9 157.5±59.2 203.9±32.83 124±111
Anthracene 0.6 1.4 1.9 7.6 21.8±5.8 10.2±4.4 6.3±1.6 70.2±35.9 76.9±8.9 35.1±11.1 49.1±11.4 4.1±3.5
Fluoranthene 1.3 3.8 8.4 42.9 614±149 335±258 128±43 207±100 198.5±62.3 134.7±70.3 79.3±18.4 20.7±19.8
Pyrene 0.9 4.0 9.1 37.1 311±47 154±107 58.2±17.9 366±172 182.2±15.5 266±115 94.7±27.4 30.8±27.5
Benzo[a]anthracene 0.5 2.3 2.4 10.1 29.8±11.2 12.5±5.1 6.6±0.8 93.3±49.0 150.1±30.4 116.1±76.5 81.1±30.3 7.5±7
Chrysene 2.7 6.2 8.9 45.9 199±81 91.2±34.1 52.4±11.4 287±152 182.6±54.3 61.5±33.2 57.2±21.5 16.9±9
Benzo[b]fluoranthene 0.3 2.5 3.0 7.2 50.8±15.6 22.5±6.4 16.4±1.6 111.3±3.7 93.7±17.5 83.8±28.8 25.0±6.1 3.8±3.1
Benzo[k]fluoranthene 0.5 2.3 3.0 7.9 16.6±2.1 9.3±2.8 9.0±3.3 69.5±13.1 79.0±7.6 73.7±9.6 33.9±7.0 3.7±1.8
Benzo[a]pyrene NA 3.5 1.8 6.8 22.4±8.6 10.8±4.4 7.4±2.4 72.6±22.6 53.5±12.1 44.0±10.1 21.2±11.8 1.4±0.9
Dibenzo[a,h]anthracene 0.7 2.8 1.3 5.7 65.2±8.1 NA NA 47.6±43.6 40.9±10.8 NA 10.9±3.5 0.4±0.5
Benzo[g,h,i]perylene 0.7 3.1 1.2 4.9 12.7±6.3 36.3±21.0 8.4±3.7 126.9±20.1 NA NA NA 0.6±0.4
Indeno[1,2,3-c,d]pyrene 0.5 3.4 0.8 4.3 10.3±1.1 6.5±4.6 NA 61.4±4.4 37.7±9.7 NA 39.3±114.8 0.4±0.3
- 32 -
CHAPTER 3 Chemical Materials
3.1 Chemicals & Reagents
The chemicals and reagents used in this thesis are listed in Table 3-1. Except
dichloromethane is purchased from ACROS, others reagents are purchased from SIGMA –
ALDRICH.
Table 3-1 Chemicals & Reagents
Subject Manufacture Description
cyclohexane SIGMA - ALDRICH ACS reagent ≥99%
hexane SIGMA - ALDRICH CHROMASOLV® Plus, for HPLC, ≥95%
dichloromethane ACROS 99.9% for residual analysis
SPE Tube SIGMA - ALDRICH LC-Florisil®, 1g, 6mL
Sodium Sulfate SIGMA - ALDRICH ACS reagent ≥90%, anhydrous, powder
Two internal standards were applied in this thesis, which are mono-fluorinated PAHs and
deuterated PAHs. The relationship between the 16 kinds of PAHs and the internal standards
(IS) were shown in the following table. The internal standards were chosen follow the rules
of minimum difference of molecule weight between the analytes and the internal standards.
The details can be found in Table 3-2 and Table 3-3.
Table 3-2 Internal Standards Information (1)
Internal standards Concentration
ng/mL
Analytes
Deuterated PAHs Naphthalene-d8 196 Naphthalene
Biphenyl-d10 199 Acenaphthene
Acenaphthylene
Phenanthrene-d10 198 Anthracene
Phenanthrene
Fluorene
Pyrene-d10 199 Pyrene
Fluoranthene
Benz[a]anthracene-d12 197 Benz[a]anthracene
Chrysene
Benzo[a]pyrene-d12 197 Benzo[b]fluoranthene
Benzo[k]fluoranthene
Benzo[a]pyrene
Benzo[g,h,i]perylene-d12 197 Dibenz[a,h]anthracene
Indeno[1,2,3-cd]pyrene
Benzo[g,h,i]perylene
- 33 -
Table 3-3 Internal Standards Information (2)
Internal standards Concentration
ng/mL
Analytes
Mono-fluorinated
PAHs
1-Fluoronaphthalene 200 NA
4-Fluorobiphenyl 201 NA
3-Fluorophenanthrene 201 NA
1-Fluoropyrene 199 NA
3-Fluorochrysene 199 NA
9-Fluorobenzo[k]fluoranthene 100 NA
According to Lundstedt et al., (2014), Kojima et al., (2010), Cochran et al., (2012) and O’
Connell et al., (2013), pyrene-d10, was chosen as the single IS for oxy-PAHs analysis.
3.2 Standards Solution
The stock solution was diluted 7 times which were named standard 1 to standard 7. In this
thesis, standard 1 and standard 4 were chosen to be the standard solutions. Standard 1 was
prepared by blending 50μL of PAHs stock solution, 50μL of oxy-PAHs stock solution, 900μL of
dichloromethane, 1μL of internal standard mono-fluorinated PAHs and 1μL of internal
standard deuterated PAHs. Standard 4 was prepared by blending 20μL of standard 1, 980μL
of dichloromethane, 1μL of internal standard mono-fluorinated PAHs and 1μL of internal
standard deuterated PAHs. The two standards were prepared by the PAHs and oxy-PAHs
stock purchased from SIGMA – ALDRICH, in the lab of the Department of Environmental
Organic Chemistry and Technology. The details of the stocks are shown in Table 3-4.
Table 3-4 Details of PAHs and oxy-PAHs Stock Solution
PAHs Conc. in Toluene µg/ml oxy-PAHs Conc. in DCM µg/ml
Naphthalene 100 Napthalene-1-carboxaldehyde 100
Acenaphthylene 100 Napthalene-1,4-dione 105.2
Acenaphthene 100 Fluorene-9-one 130
Fluorene 100 Fluorene-2-carboxaldehyde 107.6
Phenanthrene 100 Anthracene-9,10-dione 114.8
Anthracene 100 1,8-napthalic anhydride 99.6
Fluoranthene 100 Phenanthrene-9-carboxaldehyde 108
Pyrene 100 Phenanthrene-9,10-dione 106.8
Benz[a]anthracene 100 7H-benzo[d,e]anthracene-7-one 102.4
Chrysene 100 Pyrene-1-carboxaldehyde 115.6
Benzo[b]fluoranthene 100 Benz[a]anthracene-7,12-dione 104.4
Benzo[k]fluoranthene 100 Napthacene-5,12-dione 105.6
Benzo[a]pyrene 100 Anthracene-9-one 114.4
Dibenz[a,h]anthracene 100
Benzo[g,h,i]perylene 100
Indeno[1,2,3-cd]pyrene 100
- 34 -
CHAPTER 4 Results & Discussion
The design of this thesis contains four main parts, which are sample preparation, extraction,
SPE clean-up and concentration. The experiment begins with the preparation of sample. The
size of leaves was tested first. Three leaf shapes, ‘intact leaf’, ‘1mm leaf particle’ and ‘N2
freeze-dry grinded leaf’ were selected. Samples consisting of 5g leaves of each size, 1g of
Na2SO4, 9mL extraction solvent and internal standard were prepared and extracted by
ultra-sonication.
Secondly, the extraction solvent was determined. Three alternatives were chosen according
to the literature, which were the mixture hexane:dichloromethane (1:1, v/v), hexane and
dichloromethane. The prepared samples were extracted in 10mL BD PlastipakTM syringe by
CPX2800H-E BRANSONIC ultra-sonication bath, and were filtered by 0.45μm GMF WHATMAN
syringe filter. The rest was concentrated to 0.5mL by nitrogen on Caliper TurboVap®LV
concentration evaporator workstation.
Florisil solid phase extraction cartridge supplied by Supelco (LC-Florisil) was used to perform
clean up. The device was cleaned by acetone first. Then the cartridge and the device were
conditioned by the extraction solvent. The eluent was the mixture
dichloromethane:cyclohexane (1:1, v/v). The amount of eluent was determined during the
experiments. The clean-up samples were concentrated by TurboVap to the last drop. The
samples were reconstituted using 1mL of dichloromethane, then transferred into CleanPack®
vials and were stored in freezer for further GC-MS analysis.
Figure 4-1 is given to draw a clear image of the procedure of PAHs and oxy-PAHs analysis.
- 35 -
Figure 4-1 Flow Chart of the Design
4.1 Selection of Plant Species
Taxus baccata, which is shown in Figure 4-2, is an evergreen species growing widely in
western, central and southern Europe, northwest Africa and southwest Asia. It is also known
as ‘yew’ or ‘European yew’ which can be found easily in Ghent, Belgium. It can be seen in
most of the urban landscape designs, for instance parks, lawns, belts between both way
- 36 -
motor roads etc. Taxus baccata is a medium size species which can reach 10-20m tall. The
leaves are green or dark green and flat. The size of the leaves can be 1-4cm long and 2-3mm
broad. Taxus baccata is considered as one of the most longevity species around the world.
Figure 4-2 Taxus baccata
4.2 Dry Matter Content
5g of leaf was taken and was left in oven under 110℃ for 24 hours. Triplicates were
performed. The water content of Taxus baccata was calculated as 55.11% in average, which
was applied in further experiments. The water content and dry weight can be calculated by
Equation (1) and Equation (2).
𝑴𝒂𝒔𝒔 𝒐𝒇 𝑫𝒓𝒊𝒆𝒅 𝑳𝒆𝒂𝒗𝒆𝒔 = 𝑩𝒆𝒂𝒌𝒆𝒓 𝒘𝒊𝒕𝒉 𝑭𝒓𝒆𝒔𝒉 𝑳𝒆𝒂𝒇 − 𝑩𝒆𝒂𝒌𝒆𝒓 𝒘𝒊𝒕𝒉 𝑫𝒓𝒚 𝑳𝒆𝒂𝒇 (𝟏)
𝑫𝒓𝒚 𝑴𝒂𝒕𝒕𝒆𝒓 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 = (𝑴𝒂𝒔𝒔 𝒐𝒇 𝑫𝒓𝒊𝒆𝒅 𝑳𝒆𝒂𝒗𝒆𝒔
𝑴𝒂𝒔𝒔 𝒐𝒇 𝑭𝒓𝒆𝒔𝒉 𝑳𝒆𝒂𝒗𝒆𝒔) × 𝟏𝟎𝟎% (𝟐)
4.3 Solvent Selection
According to the literature, hexane, dichloromethane and the mixture of
dichloromethane:hexane (1:1 v/v) were nominated as extraction solvent. To determine
which solvent is the most suitable for Taxus baccata, the following experiment was designed.
Three piles of 5g intact leaf were put into tubes which filled with 50mL of three different
solvents. The tubes were soaked in ultra-sonication bath to extract. The extracts were
- 37 -
transferred into 50mL BD PlastipakTM syringes. The syringes were equipped with 0.45μm
syringe filter and filled with 2g of Na2SO4. The filtered solvents were sunk in BUCHI Heating
Bath B-490 water bath kettle and were rotary evaporated by BUCHI Rotavapor R-200 rotary
evaporator. Triplicates were performed for each solvent. The results are shown in Figure 4-3.
Figure 4-3 the Result of Solvent Test.
Visual test was performed. Figure 4-3 shows the turbidity of each extract after evaporation.
From left to right they were the mixture of hexane:dichloromethane (1:1, v/v),
dichloromethane and hexane. The highest turbidity was given by the mixture. The highest
clarity was given by dichloromethane. Therefore, dichloromethane and hexane were feasible
in this case. The following experiments performed base on these two solvents. The efficiency
and recovery of the two were compared.
4.4 Size of the Sample Determination
3 different sample preparations were compared.
1) Intact leaf (intact)
1g fresh leaves and 1g Sodium Sulfate were weighed and transferred into a syringe. The
plunger of syringe was removed in advance. The syringe was blocked by a stopper at the
mouth. 9mL solvent, dichloromethane or hexane, was filled in next. Afterwards, the syringe
was sealed by the syringe plunger. The prepared syringe was shown in Figure 4-4.
2) Cut into 1mm cube leaf particles (cut 1mm)
The fresh leaves were sliced down to 1mm parts by Swann Morto Ltd. surgical blades on
‘everyday’ 15 micrometer, 30cm × 30m Aluminum foil. The following operation was as same
procedure as 1).
The Mixture
- 38 -
3) Liquid nitrogen dried and grinded (N2)
The whole Taxus baccata branches were left under room temperature for 1 week to dry.
Leaves were picked from the branches and collected in a mortar. Liquid nitrogen was applied
to freeze. The frozen leaves were grinded into particles smaller than 1mm. Then following
operation was as same procedure as 1).
Figure 4-4 the Prepared Syringe
To make the results easy to be interpreted, the ratio of peak area was calculated. The results
of ‘intact’ were regarded as reference, and were compared with the results of ‘cut 1mm’ and
‘N2’. The interpreted results are shown in Table 4-1 and Table 4-2. The best results extracted
by dichloromethane are highlighted in green, while the best ones extracted by hexane are
highlighted in blue.
Table 4-1 Result of Sample Size Selection: PAHs
Preparation
PAHs
DCM HEXANE
Intact Cut 1mm N2 Intact Cut 1mm N2
Naphthalene 1.00 1.26 1.42 1.00 1.16 1.42
Acenaphthylene 1.00 1.53 1.00 1.00 0.86 0.60
Acenaphthene 1.00 1.32 0.53 1.00 0.84 0.61
Fluorene 1.00 1.13 0.66 1.00 1.48 0.88
Phenanthrene 1.00 0.86 0.88 1.00 1.53 1.49
Anthracene 1.00 0.86 NF 1.00 1.36 NF
Fluoranthene 1.00 1.33 1.32 1.00 1.45 1.50
Pyrene 1.00 1.21 1.17 1.00 1.32 1.44
Benzo[a]anthracene 1.00 1.19 1.21 1.00 0.95 0.95
Chrysene 1.00 1.15 1.15 1.00 1.26 1.19
Benzo[b]fluoranthene 1.00 1.30 1.14 1.00 1.14 0.87
Benzo[k]fluoranthene 1.00 1.18 1.39 1.00 0.99 1.45
Benzo[a]pyrene 1.00 1.34 1.54 1.00 0.74 1.20
Dibenzo[a,h]anthracene NF NF NF NF NF NF
Benzo[g,h,i]perylene NF NF NF NF NF NF
Indeno[1,2,3-c,d]pyrene 1.00 1.69 2.04 1.00 0.95 0.96
- 39 -
Table 4-2 Result of Sample Size Selection: oxy-PAHs
Preparation
oxy-PAHs
DCM HEXANE
Intact Cut 1mm N2 Intact Cut 1mm N2
Napthalene-1,4-dione 1.00 0.89 0.03 1.00 0.96 NF
Fluorene-9-one 1.00 0.89 0.59 1.00 1.13 0.87
Anthracene-9,10-dione 1.00 1.21 0.91 1.00 2.04 2.23
1,8-napthalic anhydride NF NF NF NF NF NF
7H-benzo[d,e]anthracene-7-one 1.00 0.91 0.67 1.00 2.09 1.36
Benz[a]nthracene-7,12-dione 1.00 0.77 0.76 1.00 1.33 NF
Napthacene-5,12-dione NF NF NF NF NF NF
According to the tables, ‘cut 1mm’ and ‘N2’ gave better extraction results than ‘intact’. The
difference between ‘cut 1mm’ and ‘N2’ was not clear. Different behaviors were shown by
different compounds. Therefore, to avoid the unexpected influence, nitrogen freezing was
performed on fresh leaves in this thesis.
4.5 Extraction
Extraction was performed by ultra-sonication bath under 28℃, 4 lever power (maximum) for
20 minutes. The sealed syringes were hold by tube stand and sunk into ultra-sonication bath.
The bath container was filled to 10mL mark line of syringe by deionized water. The syringes
were taken out and shaken for 2-3 seconds every 2-3 minutes. This is aimed to mix the
samples and solvent homogeneously to increase the efficiency of extract.
The stoppers were replaced by 0.8 × 50mm BD MicrolanceTM syringe needles. The needles
were bended manually into a ‘U’ shape conduit. The syringes and needles were connected by
a 0.45μm syringe filter. The design is shown in Figure 4-5. The syringes were held upside
down and squeezed manually until no continuous liquid flow can be observed. The detail can
be seen in Figure 4-6. Air was sucked in aiming to squeeze again. All syringes were squeezed
3 times. The needles were removed, and the syringes were squeezed harshly and directly
from the mouth of syringe filter to tube until the last drop. The extract was concentrated
down to around 0.5mL by TurboVap. Nitrogen was applied as flowing gas under 30℃, 10psi.
Figure 4-5 The Manually Bended ‘U’ Shape Conduit.
- 40 -
Figure 4-6 the Process of Extract Transfer
4.6 Clean-up & Concentration
4.6.1 Clean-up & Concentration Procedure
Florisil solid phase extraction cartridge was applied to remove the influence made by polar
compounds. The device and configuration were shown in Figure 4-7. The device was washed
by acetone. Then the columns were connected and conditioned by 5mL of dichloromethane
or hexane slowly. The mixture of dichloromethane:cyclohexane (1:1 v/v) was prepared as
eluent. The switches of the columns were switched off at the beginning. The solvents were
transferred into the cartridge by the VWR 230mm disposable glass Pasteur pipettes. The
solvent was slowly collected drop by drop into the VWR 100 × 16.00 × (0.8-1.0) mm tubes.
The switches were switched off immediately the liquid in the cartridge tubes sunk into the
solid phase. Afterwards, the cartridges were filled by eluent and the same operation was
performed.
The collected solvent was concentrated to almost dryness by TurboVap nitrogen gas flow,
under the condition of 30℃ and 10psi. The concentrated clean-up solvent was re-dissolved
in 1mL of dichloromethane. To retrieve all compounds, the re-dissolved tube was sunk into
ultra-sonication bath for 5 seconds under the condition of 25℃, 4 lever power (maximum).
The re-dissolved sample extract was transferred into vials and stored in freezer until analyze.
- 41 -
Figure 4-7 the Clean-up Device
4.6.2 Determination of Eluent Volume
To decide how much eluent is sufficient for elution, the following experiment was designed.
1mL sample were prepared by 995μL extraction solvent and 5μL standard 1 solutions. Both
hexane and dichloromethane were applied for extraction in this design. To brief the
procedure, 16mL eluent was prepared for elution, 1mL sample was added first, then the
eluent. The eluent was added milliliter by milliliter. The solvent were collected in 16 tubes
separately.
Most of PAHs compounds behaved similarly, most of the compounds were recovered before
the 5th milliliter. The recovery of naphthalene was found differently, but the recovery pattern
was similar to others. Four oxy-PAHs compounds were recovered. They performed similarly
comparing to the PAHs compounds, the recovery peak can be observed about the 6th
milliliter of eluent, except the DCM extraction of 7H-benzo[d,e]anthracene-7-on. The details
of the elution results of each PAHs compound and oxy-PAHs compound are shown in Figure
4-8 to Figure 4-27.
- 42 -
Figure 4-8 Eluent Volume Test Result: Naphthalene Figure 4-9 Eluent Volume Test Result: Acenaphthylene
Figure 4-10 Eluent Volume Test Result: Acenaphthene Figure 4-11 Eluent Volume Test Result: Fluorene
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Naphthalene
Hexane
DCM
0
10
20
30
40
50
60
70
80
90
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Acenaphthylene
Hexane
DCM
0
10
20
30
40
50
60
70
80
90
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Acenaphthene
Hexane
DCM
0
10
20
30
40
50
60
70
80
90
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Fluorene
Hexane
DCM
- 43 -
Figure 4-12 Eluent Volume Test Result: Phenanthrene Figure 4-13 Eluent Volume Test Result: Anthracene
Figure 4-14 Eluent Volume Test Result: Fluoranthene Figure 4-15 Eluent Volume Test Result: Pyrene
0
10
20
30
40
50
60
70
80
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Phenanthrene
Hexane
DCM
0
10
20
30
40
50
60
70
80
90
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Anthracene
Hexane
DCM
0
10
20
30
40
50
60
70
80
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Fluoranthene
Hexane
DCM
0
10
20
30
40
50
60
70
80
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Pyrene
Hexane
DCM
- 44 -
Figure 4-16 Eluent Volume Test Result: Benz[a]anthracene Figure 4-17 Eluent Volume Test Result: Chrysene
Figure 4-18 Eluent Volume Test Result: Benzo[b]fluoranthene Figure 4-19 Eluent Volume Test Result: Benzo[k]fluoranthene
0
10
20
30
40
50
60
70
80
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Benz[a]anthracene
Hexane
DCM
0
10
20
30
40
50
60
70
80
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Chrysene
Hexane
DCM
0
10
20
30
40
50
60
70
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Benzo[b]fluoranthene
Hexane
DCM
0
10
20
30
40
50
60
70
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Benzo[k]fluoranthene
Hexane
DCM
- 45 -
Figure 4-20 Eluent Volume Test Result: Benzo[a]pyrene Figure 4-21 Eluent Volume Test Result: Dibenz[a,h]anthracene
Figure 4-22 Eluent Volume Test Result: Benzo[g,h,i]perylene Figure 4-23 Eluent Volume Test Result: Indeno[1,2,3-cd]pyrene
0
10
20
30
40
50
60
70
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Benzo[a]pyrene
Hexane
DCM
0
10
20
30
40
50
60
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Dibenz[a,h]anthracene
Hexane
DCM
0
10
20
30
40
50
60
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Benzo[g,h,i]perylene
Hexane
DCM
0
10
20
30
40
50
60
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% m
ass
in f
ract
ion
Fraction nr (+/- 1ml)
Indeno[1,2,3-c,d]pyrene
Hexane
DCM
- 46 -
Figure 4-24 Eluent Volume Test Result: Benz[a]anthracene-7,12-dion Figure 4-25 Eluent Volume Test Result: Anthracene-9,10-dionen
Figure 4-26 Eluent Volume Test Result: Fluorene-9-one Figure 4-27 Eluent Volume Test Result: 7H-benzo[d,e]anthracene-7-on
0
5
10
15
20
25
30
35
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Benz[a]anthracene-7,12-dion
Hexane
DCM
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Anthracene-9,10-dione
Hexane
DCM
0
5
10
15
20
25
30
35
40
45
50
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Fluorene-9-one
Hexane
DCM
0
2
4
6
8
10
12
14
16
18
20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
7H-benzo[d,e]anthracene-7-on
Hexane
DCM
- 47 -
The details of clean-up and concentration procedure applied in further experiments are
described step by step: 1) 16mL eluent were prepared for elution; 2) sample added into the
cartridge; 3) eluent was added into the cartridge following this program: 1mL, 1mL, 1mL,
1mL, 1mL, 2mL, 2mL, 2mL, 2mL, and 3mL; 4) the first and second milliliter of eluent was used
to wash the residual sticking on the wall of tube; 5) the 16mL eluent were collected in two
tubes, the first tube were replaced by the second tube right after the 9th milliliter eluent
added; 6) the first tube was concentrated by nitrogen gas flow under 30℃, 10psi; 7) the
solvent collected in the second tube was transferred into the first tube when more than half
of the solvent in the first tube had been evaporated; 8) the second tube was washed by 1mL
of dichloromethane and the washing liquor was transferred into the first tubes as well.
4.7 GC-MS Analysis
To brief, the concentrated extract was injected in splitless mode via a BEST programmed
temperature vaporizer (PTV) injector (Interscience, Louvain-la-Neuve, Belgium). One μl was
injected. Chromatographic separation was performed on a Restek Rxi-17Sil MS column (30 m;
0.25 mm ID; 0.25 μm; Interscience, Louvain-la-Neuve, Belgium). To protect the analytical
column, a 50 cm guard column was connected in front of the analytical column (deactivated,
0.25 mm ID). The GC (Trace GC Ultra) oven temperature was initially set at 70°C (2.5 min),
and then heated to 320°C at a heating rate of 10°C/min. The final temperature was held for
15 min. The MS-transfer line was heated to 240°C.
After separation, compounds were subjected to Electron Ionization (EI; 70eV). A
perfluorokerosene mix was continuously introduced (1 to 2 μl per 24h of analysis) into the
source via a heated (150°C) capillary leak. The mass fragments of the perfluorokerosene
were used as internal reference ions enabling accurate mass measurements. The mass
spectrometer was run in multiple ion detection (MID) mode with a mass resolution above 10
000 (10% valley definition).
4.8 Recovery and Matrix Effects
The following 5 experiments were designed to obtain the recovery and matrix effects of PAHs
and oxy-PAHs. Five experiments were conducted: 1) ‘Blank’, 2) ‘Standard tests’, 3) ‘Leaves’, 4)
‘Spiked before’ and 5) ‘Spiked after’. Each experiment was carried out with two solvents
dichloromethane and hexane. Each experiment was conducted in three fold. Therefore, 30
tests were carried out in total. Figure 4-28 is given to draw a clear image of the tests. The
details are described in following section.
- 48 -
Figure 4-28 Flow Chart of the Recovery Determination
4.8.1 Experiment Design
The experiment was carried out without leaves. 9mL solvent and 1g anhydrous sodium
sulfate were added into syringe. The extraction, clean-up and analysis were performed next
as described in previous sections.
The experiment was carried out without leaves. 9mL solvent, 1g anhydrous sodium sulfate,
20μL standard-1 and internal standards (1μL F-PAHs) were added into the syringe. The
extraction, clean-up and analysis were performed next as described in previous sections. 1μL
deuterated-PAHs were added into the reconstituted vials before GC-MS analysis.
The experiment was carried out with leaves. The leaves were prepared as described in
previous sections. 1g leaf, 9mL solvent, 1g anhydrous sodium sulfate and internal standards
(1μL F-PAHs) were added into the syringe. The extraction, clean-up and analysis were
performed next as described in previous sections. 1μL deuterated-PAHs were added into the
reconstituted vials before GC-MS analysis.
The experiment was carried out with leaves. The leaves were prepared as described in
previous sections. 1g leaf, 9mL solvent, 1g anhydrous sodium sulfate, 20μL standard-1 and
internal standards (1μL F-PAHs) were added into the syringe. Standard-1 was dropped onto
the leaves first. The syringe was shaken to make the leaves and Standard-1 mix well. F-PAHs
were dropped onto the leaves when the Standard-1 and leaves were well mixed. The solvent
was added next. The extraction, clean-up and analysis were performed next as described in
previous sections. 1μL deuterated-PAHs were added into the reconstituted vials before
GC-MS analysis.
- 49 -
The experiment was carried out with leaves. The leaves were prepared as described in
previous sections. 1g leaf, 9mL solvent, 1g anhydrous sodium sulfate and internal standards
(1μL F-PAHs) were added into the syringe. F-PAHs were dropped onto the leaves before the
solvent added. The extraction, clean-up and analysis were performed next as described in
previous sections. 20μl Standard-1 and 1μL deuterated-PAHs were added into the
reconstituted vials before GC-MS analysis.
4.8.2 Data Processing Method
PAHs and oxy-PAHs analysis were carried out by GC-MS. The mass of the compounds were
calculated as following procedure:
𝑹𝑷𝑨∗ =𝑻𝒂𝒓𝒈𝒆𝒕 𝑪𝒐𝒎𝒑𝒐𝒖𝒏𝒅𝒔 𝑷𝒆𝒂𝒌 𝑨𝒓𝒆𝒂
𝑰𝑺∗∗ 𝑷𝒆𝒂𝒌 𝑨𝒓𝒆𝒂 (𝟑)
*RPA: Relative Peak Area
**IS: internal standard
𝑹𝑺𝑹𝑭∗𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅 = 𝑹𝑷𝑨 ×
𝑰𝑺 𝑴𝒂𝒔𝒔 (𝒏𝒈)
𝑴𝒂𝒔𝒔 𝒐𝒇 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅(𝒏𝒈) (𝟒)
*RSRF: Relative Sample Response Factor
𝑴𝒂𝒔𝒔 𝒐𝒇 𝑻𝒂𝒓𝒈𝒆𝒕 𝑪𝒐𝒎𝒑𝒐𝒖𝒏𝒅 (𝒏𝒈) = 𝑹𝑷𝑨 ×𝑰𝑺 𝑴𝒂𝒔𝒔
𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝒐𝒇 𝑹𝑺𝑹𝑭 𝒐𝒇 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 (𝟓)
𝑹𝒆𝒄𝒐𝒗𝒆𝒓𝒚 =𝑹𝑷𝑨𝒔𝒑𝒊𝒌𝒆 𝒃𝒆𝒇𝒐𝒓𝒆 −
𝑹𝑷𝑨𝒍𝒆𝒂𝒗𝒆𝒔
𝑴𝒂𝒔𝒔𝒍𝒆𝒂𝒗𝒆𝒔× 𝑴𝒂𝒔𝒔𝒔𝒑𝒊𝒌𝒆 𝒃𝒆𝒇𝒐𝒓𝒆
𝑹𝑷𝑨𝒔𝒑𝒊𝒌𝒆 𝒂𝒇𝒕𝒆𝒓 −𝑹𝑷𝑨𝒍𝒆𝒂𝒗𝒆𝒔
𝑴𝒂𝒔𝒔𝒍𝒆𝒂𝒗𝒆𝒔× 𝑴𝒂𝒔𝒔𝒔𝒑𝒊𝒌𝒆 𝒂𝒇𝒕𝒆𝒓
× 𝟏𝟎𝟎 (𝟔)
𝑴𝒂𝒕𝒓𝒊𝒙 𝒆𝒇𝒇𝒆𝒄𝒕𝒔 =𝑹𝑷𝑨𝒔𝒑𝒊𝒌𝒆 𝒂𝒇𝒕𝒆𝒓 −
𝑹𝑷𝑨𝒍𝒆𝒂𝒗𝒆𝒔
𝑴𝒂𝒔𝒔𝒍𝒆𝒂𝒗𝒆𝒔× 𝑴𝒂𝒔𝒔𝒔𝒑𝒊𝒌𝒆 𝒂𝒇𝒕𝒆𝒓
𝑹𝑷𝑨𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅
× 𝟏𝟎𝟎 (𝟕)
- 50 -
𝑳𝒆𝒂𝒇 𝑴𝒂𝒔𝒔 𝑪𝒐𝒏𝒕𝒆𝒏𝒕 =𝑴𝒂𝒔𝒔 𝒐𝒇 𝑻𝒂𝒓𝒈𝒆𝒕 𝑪𝒐𝒎𝒑𝒐𝒖𝒏𝒅
𝑹𝒆𝒄𝒐𝒗𝒆𝒓𝒚 × 𝑴𝒂𝒕𝒓𝒊𝒙 𝒅𝒊𝒔𝒕𝒖𝒓𝒃𝒂𝒏𝒄𝒆 (𝒏𝒈) (𝟖)
𝑫𝒓𝒚 𝑾𝒆𝒊𝒈𝒉𝒕 𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 =𝑳𝒆𝒂𝒇 𝑴𝒂𝒔𝒔 𝑪𝒐𝒏𝒕𝒆𝒏𝒕
𝑳𝒆𝒂𝒗𝒆𝒔 𝑫𝒓𝒚 𝑾𝒆𝒊𝒈𝒉𝒕 (𝒏𝒈/𝒈) (𝟗)
4.8.3 Results of Analysis
The recovery, matrix effects and compounds dry weight on leaves of dichloromethane
extraction are shown in Table 4-3 and Table 4-4.
Table 4-3 DCM extraction results: PAHs
Results
PAHs R
1 (%) SD
2 RSD
3 Matrix
4 SD RSD d.w.
5 (ng/g)
Naphthalene 24.7 0.22 90 0.98 0.39 40 1519
Acenaphthylene 66.0 0.05 8 1.23 0.03 2 3.4
Acenaphthene 73.6 0.07 9 1.06 0.06 5 8.7
Fluorene 66.2 0.08 12 1.20 0.02 1 13.8
Phenanthrene 63.6 0.06 10 1.22 0.01 1 60.6
Anthracene 72.1 0.07 10 1.17 0.06 5 NF
Fluoranthene 69.9 0.06 8 1.37 0.02 2 59.4
Pyrene 64.9 0.07 11 1.28 0.04 3 41.2
Benzo[a]anthracene 69.2 0.07 10 1.20 0.04 3 17.9
Chrysene 70.2 0.07 11 1.22 0.11 9 67.4
Benzo[b]fluoranthene 66.6 0.05 7 1.18 0.02 2 25.8
Benzo[k]fluoranthene 64.1 0.05 8 1.08 0.01 1 14.6
Benzo[a]pyrene 70.1 0.07 10 1.14 0.07 6 4.8
Dibenzo[a,h]anthracene 68.0 0.06 9 1.33 0.00 0 NF
Benzo[g,h,i]perylene 66.1 0.06 9 1.12 0.02 2 5.6
Indeno[1,2,3-c,d]pyrene 67.0 0.05 8 1.25 0.02 2 4.3
Sum 1847
Sum without naphthalene 327
1. R: Recovery 2. SD: Standard deviation
3. RSD: Relative standard deviation 4. Matrix: Matrix effects
5. d.w.: Dry weight concentration on leaf
- 51 -
Table 4-4 DCM extraction results: oxy-PAHs
Results
oxy-PAHs R (%) SD RSD Matrix SD RSD d.w. (ng/g)
Napthalene-1,4-dione 45.0 0.03 6 1.62 0.06 4 0.7
Fluorene-9-one 67.3 0.04 6 1.89 0.07 4 10.5
Anthracene-9,10-dione 66.5 0.06 9 2.22 0.11 5 5.6
1,8-napthalic anhydride NF NF NF NF NF NF NF
7H-benzo[d,e]anthracene-7-one 58.6 0.06 10 3.26 0.04 1 5.8
Benz[a]nthracene-7,12-dione 63.2 0.05 7 1.98 0.04 2 4.17
Napthacene-5,12-dione 13.7 0.01 11 2.44 0.02 1 NF
Sum 26.7
As for PAHs, the result of naphthalene was found higher than others, while the recovery was
found lower. The concentration of naphthalene was found 10-100 times higher than others
in blank experiments. Therefore, the sum results were shown in two ways, with naphthalene
and without. The recovery of PAHs compounds ranged between 63-74%. The matrix effects
coefficient ranged between 0.9-1.3, which means the influence given by solvents was not
significant. The sum concentration of PAHs in leaf samples was 327ng/g (d.w.). The
concentration of chrysene, phenanthrene and fluoranthene were found higher than others,
which were 67.4ng/g (d.w.), 60.4ng/g (d.w.) and 59.4ng/g (d.w.) respectively. Anthracene and
dibenzo[a,h]anthracene were not found in the sample leaf.
AS for oxy-PAHs, 1,8-napthalic anhydride was not found in most of the tests, except ‘spiked
after’. It was rarely found in the natural background, and it could be absorbed to the SPE
cartridges while cleaning up. Therefore, 1,8-napthalic anhydride is not discussed. The
recovery of oxy-PAHs ranged between 58-67%, except naphthalene-1,4-dione was 45% and
napthacene-5,12-dione was 14%. The matrix effects coefficient was ranged between 1.6-2.4,
except 7H-benzo[d,e]anthracene-7-one was 3.3. The concentration of oxy-PAHs in leaf
samples was 26.7ng/g (d.w.). Fluorene-9-one was found having the highest concentration
which was 10.5ng/g (d.w.). Napthacene-5,12-dione was not found in the sample leaf.
The recovery, matrix effects and compounds dry weight on leaves of hexane extraction are
shown in Table 4-5 and Table 4-6.
- 52 -
Table 4-6 hexane extraction results: PAHs
Results
PAHs R (%) SD RSD Matrix SD RSD d.w. (ng/g)
Naphthalene 33.2 0.30 92 0.99 0.10 11 925
Acenaphthylene 55.7 0.07 12 1.31 0.08 6 4.3
Acenaphthene 59.8 0.08 14 1.14 0.07 6 13.8
Fluorene 60.5 0.08 14 1.13 0.08 8 14.7
Phenanthrene 57.9 0.09 15 1.06 0.10 9 68.6
Anthracene 70.5 0.09 12 1.07 0.05 5 NF
Fluoranthene 59.2 0.09 15 1.50 0.13 8 42.3
Pyrene 57.3 0.08 14 1.39 0.10 7 26.8
Benzo[a]anthracene 60.7 0.06 10 1.05 0.06 6 9.3
Chrysene 56.9 0.08 14 1.08 0.10 9 37.6
Benzo[b]fluoranthene 58.6 0.08 13 1.10 0.06 5 12.6
Benzo[k]fluoranthene 60.8 0.09 14 0.98 0.04 4 9.1
Benzo[a]pyrene 65.2 0.09 14 0.99 0.06 6 3.9
Dibenzo[a,h]anthracene 57.7 0.07 12 1.28 0.06 5 NF
Benzo[g,h,i]perylene 57.9 0.07 12 1.05 0.06 6 3.5
Indeno[1,2,3-c,d]pyrene 57.7 0.07 13 1.20 0.07 5 2.5
Sum 1174
Sum without naphthalene 249
Table 4-5 hexane extraction results: oxy-PAHs
Results
PAHs R (%) SD RSD Matrix SD RSD d.w. (ng/g)
Napthalene-1,4-dione 35.6 0.04 11 1.51 0.09 6 0.0
Fluorene-9-one 59.2 0.07 11 1.77 0.08 4 13.4
Anthracene-9,10-dione 66.1 0.07 10 2.04 0.08 4 5.3
1,8-napthalic anhydride NF NF NF NF NF NF NF
7H-benzo[d,e]anthracene-7-one 52.8 0.06 11 2.93 0.10 4 2.9
Benz[a]nthracene-7,12-dione 59.6 0.07 12 1.89 0.06 3 0.0
Napthacene-5,12-dione 4.8 0.01 11 2.33 0.13 6 NF
Sum 21.6
The higher d.w. concentration and lower recovery of naphthalene than others, and NF of
1,8-napthalic anhydride were found when extracted by hexane as well. Therefore, neither of
the two was discussed in the sum result. Anthracene, dibenzo[a,h]anthracene, and
napthacene-5,12-dione were not found when hexane was applied as the extraction solvent.
As for PAHs, the recovery ranged between 56-61%, except anthracene was about 71%. The
matrix effects coefficient ranged between 0.9-1.3, except fluoranthene was 1.5 and pyrene
was 1.39. The concentration of PAHs in leaf samples was 249ng/g (d.w.). Different to DCM
extraction, the concentration of phenanthrene was the highest which was 68.6ng/g (d.w.),
and was followed by fluoranthene (42.3ng/g (d.w.)) and chrysene (37.6ng/g (d.w.)).
- 53 -
As for oxy-PAHs, the recovery of oxy-PAHs ranged between 53-67%, except
Napthalene-1,4-dione was 36% and napthacene-5,12-dione was 5%. The matrix effects
coefficient was ranged between 1.5-2.3, except 7H-benzo[d,e]anthracene-7-one which was
2.93. The concentration of oxy-PAHs in leaf samples was 21.6ng/g (d.w.). Fluorene-9-one was
found having the highest concentration which was 13.4ng/g (d.w.), while
benz[a]nthracene-7,12-dione and n apthalene-1,4-dione was not found.
Comparing the results between two solvents, higher recovery and dry weight concentration
were obtained using dichloromethane. Therefore dichloromethane was recommended to be
the extraction solvent for Taxus baccata.
4.9 Discussion
This thesis is designed to develop a method using Taxus baccata to bio-monitor PAHs and
oxy-PAHs in atmosphere. Existed results were found rarely about oxy-PAHs bio-monitoring
and Taxus baccata PAHs bio-monitoring. To reduce the size of sample leaves, fresh leaves
nitrogen freezing followed by pulverizing was applied because of the better extraction results.
Nitrogen freezing followed by pulverizing provides smaller particles and bigger specific
surface area than other methods. Therefore, more contact area between sample and
extraction solvent was given. Furthermore, freezing and pulverizing broke the lipid layer
covered on leaves and broke the cells to some extent, which made it possible to extract the
PAHs and oxy-PAHs stored in inner parts of the leaves.
Due to the good recovery, 16mL eluent was applied to clean up the extracted solution.
Compared to other PAHs compounds, different pattern of recovery was showed by
naphthalene. Naphthalene is a widely used household substance (Praharaj and Kongasseri,
2012) and chemical intermediate in industries of dye, o-phthalic anhydride, pesticide, etc.
(Gerd et al., 2003; Sava et al., 2014; Zhang and Zhao, 2008). Volatility is one of the important
properties of naphthalene and one of the major reasons that naphthalene can be found
more easily than other PAHs compounds in atmosphere. Another important reason of the
different recovery pattern can be deduced as the adsorption of the Florisil cartridge to
naphthalene, which needs to be studied further. Oxy-PAHs are the derivatives of PAHs, the
recovery and behavior in biological samples were not studied clearly in previous researches.
Further studies are required to explain the different recovery pattern between PAHs and
oxy-PAHs.
Based on visual test, recovery test and the PAHs/oxy-PAHs concentration extracted from
leaves, dichloromethane was chosen as the best extraction solvent for Taxus baccata.
Turbidity was observed reproducibly when the mixture of dichloromethane and hexane was
applied as extraction solvent. Some chemical reactions between the extraction solvent and
part(s) of the leaves may be accelerated by the mixture, which can be deduced as one of the
reasonable explanation. Further studies are required to figure out the exact reasons.
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Comparing to the previous studies, considering the traffic flow and industrial polluted
scenario of Ghent, the total PAHs results of this thesis are higher than the suburban area of
Campania (218ng/g (d.w.)) and Tuscany (172ng/g (d.w.)) (De Nicola et al., 2013), and also
higher than the suburban area of Portugal (mean 118ng/g (d.w.)), Spain (mean 108ng/g
(d.w.)) and Greece (105ng/g (d.w.)) (Ratola et al., 2012).
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CHAPTER 5 Conclusions & Prospect
5.1 Conclusions
Taxus baccata was chosen as the bio-monitoring species for PAHs and oxy-PAHs. The leaves
were taken in the city area of Ghent, Belgium. The methods were optimized in the extraction
step and clean up step. Liquid nitrogen freeze was proved better than other methods for
Taxus baccata. The ‘U’ shape conduit was developed for extraction and filtration. The 16mL
eluent program was innovated for SPE clean-up. Dichloromethane was selected as the most
suitable extraction solvent for Taxus baccata. The recovery of PAHs compounds ranged
63-74%, matrix disturbance ranged 0.9-1.3, and the sum of dry weight concentration was
327ng/g. Oxy-PAHs was first tested by bio-monitoring. The recovery of oxy-PAHs compounds
ranged 58-67%, matrix disturbance was ranged between 1.5-2.4, and the sum of dry weight
concentration was 26.7ng/g.
5.2 Prospect
As for pollutants information, NPAHs are another important and toxic group of PAHs
derivatives. The existence and concentration are recommended to be studied in other
researches. As for extraction efficiency, the temperature, frequency and extraction time of
ultra-sonication bath are expected to be studied further. As for clean-up, automatic
operation is expecting to be applied. At last, the difference between the results of
bio-monitors and conventional chemical methods in the same regions are expected to be
compared.
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