Upload
others
View
9
Download
0
Embed Size (px)
Citation preview
UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)
UvA-DARE (Digital Academic Repository)
Fullerene nanoparticles in soil: Analysis, occurrence and fate
Carboni, A.
Link to publication
Citation for published version (APA):Carboni, A. (2016). Fullerene nanoparticles in soil: Analysis, occurrence and fate.
General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, statingyour reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Askthe Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,The Netherlands. You will be contacted as soon as possible.
Download date: 27 Jul 2020
1
Fullerene Nanoparticles in Soil:
Analysis, Occurrence and Fate
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus
prof. dr. ir. K. I. J. Maex
ten overstaan van een door het College voor Promoties ingestelde
commissie, in het openbaar te verdedigen in de Agnietenkapel
op dinsdag 18 oktober, te 14:00 uur
door
Andrea Carboni
geboren te Narni, Italië
2
Promotiecommissie:
Promotors: Prof. Dr. W.P. de Voogt - Universiteit van Amsterdam
Prof. Dr. K. Kalbitz - Universiteit van Amsterdam /
Technische Universität Dresden
Copromotor: Dr. J. R. Parsons - Universiteit van Amsterdam
Overige leden: Prof. Dr. Ir. P. J. Schoenmakers - Universiteit van Amsterdam
Prof. Dr. B. de Bruin - Universiteit van Amsterdam
Prof. Dr. A.P. van Wezel - Universiteit Utrecht
Dr. W. Th. Kok - Universiteit van Amsterdam
Dr. Ir. N.W. van den Brink - Wageningen UR
Faculteit der Natuurwetenschappen, Wiskunde en Informatica
Cover by Alessandro Cavalletti, Archinit
Printed by Leoni Tipografia Grafiche s.n.c. - Amelia, Italy
Fullerene nanoparticles in soil: analysis, occurrence and fate by Andrea Carboni
Proefschrift Universiteit van Amsterdam, FNWI, IBED, 2016
ISBN 978-94-91407-36-9 / Copyright © 2016
This work was funded by NanoNextNL, a micro and nanotechnology consortium of the Government of The Netherlands and 130 partners
3
“Pollution is nothing but the resources we are not harvesting. We allow
them to disperse because we've been ignorant of their value.”
R. Buckminster Fuller
4
5
Table of contents
Chapter 1. General Introduction ................................................................. 7
Chapter 2. Optimization of the extraction procedure for fullerenes into
soil ...............................................................................................................17
Chapter 3. A HPLC-UV method for the analysis of fullerenes in soils ........31
Chapter 4. An UHPLC-HRMS method for the analysis of fullerenes in
soils ..............................................................................................................53
Chapter 5. Analysis of fullerenes in soils from The Netherlands ...............75
Chapter 6. Incubation of solid state C60 fullerene under environmentally
relevant conditions .....................................................................................91
Chapter 7. Synthesis..................................................................................109
Appendices ...............................................................................................119
Summary ...................................................................................................143
Samenvatting ............................................................................................145
References ................................................................................................147
List of papers used in this thesis ..............................................................165
Curiosities .................................................................................................166
Acknowledgements ..................................................................................169
6
7
Chapter 1
Introduction
This introduction gives an overview of the terminology and classification of
nanomaterials and nanotechnology (1.1) before proceeding to focus on the
fullerenes (1.2), their sources and fate in the environment (1.3) and the
analysis of fullerenes in the environment (1.4). Finally the justification and
objectives of the thesis (1.5) are discussed.
1.1 Engineered Nanomaterials and Nanotechnology
Nanomaterials (NMs) consist of a group of chemical substances defined by
their external size between 1 and 100 nm in at least one dimension, and can
be considered a bridge between atomic or molecular structures and bulk
materials (EU Commission, 2011; Henglein et al., 1993). This is a
heterogeneous group of chemicals that can be further classified depending
on their composition (e.g. metallic, organic) as well as on the basis of their
origin (e.g. natural or anthropogenic). Although particles in the nano-size
range have likely been always present on Earth (Nowack et al., 2007), only
recently they have attracted a lot of attention due to their innovative
properties. In particular, due to the small size, NMs display a larger surface
to volume ratio in comparison with bulk materials that determines changes
in the physico-chemical properties related to electrical conductivity, colour,
solubility, mechanical strength, and catalytic activity (Reed, 1993; Ebbesen
et al., 1996; Pal et al., 1997; Powell et al., 1998; Pan et al., 2012). This makes
them appealing for novel applications and uses and in the last decades,
8
large efforts were dedicated to the synthesis of engineered nanomaterials
(ENMs) aimed to enhance technological and industrialized processes. In this
context, nanotechnology refers to the characterization, production and
application of NMs that can be achieved by manipulating their composition
and arrangement. This field is very broad, covering a wide range of different
techniques, scientific and commercial applications and products (RS & RAE,
2004). According to some, nanotechnology is expected to determine the
“next industrial revolution” and current and future applications could hold
societal benefits including economic development and employment and
advancements in material, environmental and medical sciences (RS & RAE,
2004; Roco et al., 2005). Nowadays, nanotechnology is estimated to be
worth $2.6 trillion in manufactured goods (Lux Research 2006) and the
production of engineered nanomaterials is expected to be 58,000 tons in
the 2011-2020 period (Maynard, 2006). The increasing production and use
(Wijnhoven et al., 2010; Hendren et al., 2011; Maynard et al., 2012) will
inevitably result in a larger release of these chemicals into the environment.
However, the implications of a larger occurrence and exposure to ENMs are
not fully understood and several studies raised concern about their fate,
transport, and potential adverse effects for the environment and human
health (Christian et al., 2008; Peralta-Videa et al., 2011). Nonetheless it is
difficult for regulatory agencies to develop standards on the usage,
manufacture and regulation because NMs may display a different
behaviour in comparison with “traditional” materials (Bhatt et al., 2011).
For instance, in toxicity test particles number and/or surface area may have
a more important role than concentration, raising questions about the
validity of current dose-metric methodologies and risk assessment in
general (Pakaninen, 2013). This is further complicated by the fact that not
only the size but also the shape and surface properties are important in
affecting the behaviour of nanostructured matter (Yadav et al., 2008). In
this context, key limitations include the lack of data on the environmental
fate and behaviour of engineered nanomaterials and of direct
measurements of their occurrence in aquatic or terrestrial systems (Klaine
et al., 2008; Gottschalk et al., 2010). These are in turn determined by the
lack of analytical methodologies capable of quantifying trace
concentrations of NPs in environmental matrices (Hasselhov et al., 2008;
Mueller et al., 2008; Gottschalk et al., 2009; Pan et al., 2012).
9
1.2 Fullerenes
The synthesis of fullerene in 1985 by Kroto et al., represented a milestone
in the development of nanotechnology and signed the beginning of one of
its most prominent fields, the carbon nanotechnology. Fullerenes, Cn, are
an allotrope of carbon. However, unlike other forms of carbon such as
diamond and graphite, which consist of the repetition of atomic structures,
fullerenes are discrete molecules with a defined number of atoms (Fig. 1.1).
Fig. 1.1. Structures of carbon allotropes. A) C60 fullerene, B) diamond, C) graphene and D) graphite (layers of graphene).
They display a closed-cage molecular shape where the carbon atoms are
interconnected in five- and six- membered rings. Depending on the number
of hexagonal rings that combine with 12 pentagonal rings, fullerenes can
present a theoretically unlimited number of structures. Among these, C60
was the first to be discovered and consists of 12 pentagonal and 20
hexagonal rings arranged in a football-like structure of 0.72 nm in diameter.
C60 is the most stable fullerene because of energetic reasons (Zhang et al.,
1992) and by far the most studied to date. All the carbon atoms are
equivalent in the molecule and present a sp2 hybridization, with lengths of
the bonds of 1.38 Å and 1.45 Å, for six-six and five-six bonds, respectively.
Due to the π-electrons in the rings, fullerenes could be described as three-
dimensional aromatic molecules. However, since double bonds do not
locate in the pentagonal rings, resulting in poor electrons delocalisation,
they are not “super aromatic” as initially proposed (Buhl et al., 2001; Yadav
10
et al., 2008). Fullerenes are more electronegative than most hydrocarbons,
behave like electron deficient alkenes and react readily with electron rich
species (Hirsch et al., 2005). In general, these compounds are extremely
versatile and can undergo a large number of reactions including redox,
nucleophilic attack, addition, cycloaddition and photochemical reaction as
photocycloaddiction (Kroto et al., 1994).
These reactions also give the possibility to derivatize the closed-cage
structure with consequent production of novel fullerenic materials with
modified physico-chemical properties that provide new features and
chemical characteristics (e.g. higher water solubility or conductivity). In
particular, the derivatization of fullerenes can be achieved with the
inclusion of atoms other than carbon within the structure (heterohedral
fullerenes), the inclusion of chemical species (e.g. metals, water) in the
inner space (endohedral fullerenes) and / or the functionalization of the
outer space (exohedral fullerenes) (Chai et al., 1991; Hummelen et al., 1995)
(Fig. 1.2). The unique molecular nano-size structure, combined with the
possibly unlimited number of species that may be derived, make fullerenes
appealing for a large number of possible applications. According to the
Nanotechnology Consumer Products Inventory, as of March 2006 carbon
nanomaterials (fullerenes and carbon nanotubes) were the most widely
used nanoparticles with regard to the number of products on the market.
Current applications include personal care products (e.g. as antioxidants in
cosmetics) and photovoltaics (as electron acceptors) as well as electronics
and optics (Tagmatarchis et al., 2001; Guldi et al., 2002; Burangulov et al.,
2005; Kim et al.,2006; Benn et al., 2011; Li et al., 2012). Fullerenes are also
expected to play a role in novel medical and environmental strategies
(Bakry et al., 2007; Cantrill, 2011).
11
Fig. 1.2. Examples of fullerenes derivatives structures. Left side: endohedral fullerenes displaying A) a water molecule (H2O@C60) and B) a potassium atom (K@C60) included into the C60 structure. Right side: exohedral fullerenes, C) [6,6]-Phenyl-C61-butyric acid methyl ester ([60]PCBM) and D) poly-hydroxylated fullerenes also known as fullerol or fullerenol (e.g. C60(OH)n).
1.3 Sources and environmental fate of fullerenes
The understanding of fullerenes occurrence and fate in the environment is
limited and complicated by the multiple sources from which they may
derive. Similar to any industrially produced chemical, the increasing
production and application will inevitably mean a release of these
compounds in the environment at larger concentration and in a wider
variety of ecosystem than is currently the case (Nowack et al., 2007; Tiwari
et al., 2014). In addition, fullerenes can be formed naturally during wildfire,
lightening, meteor impact and combustion processes in general where
carbonaceous materials are consumed (Daly et al., 1993; Heyman et al.,
12
1994; Tiwari et al., 2016). Thus, unintentional emissions of anthropogenic
origin may be expected due to industrial processes and transportation that
involve coal, fuel and organic matrices (e.g. as by-product of combustion,
oil refinery; Sanchis et al., 2013). A precise picture of the current worldwide
production of fullerenes is hindered by the lack of data concerning their
production and a complete inventory of the products they are included in.
However, manufacturing of fullerenes is currently estimated to be in the
range of tens of tons per year and natural/incidental contributions could
exceed that of engineered nanomaterials (ENMs) (Hendren et al., 2011;
Tiwari et al., 2016).
Once in the environment, little is known about the environmental fate of
fullerenes. The assessment of their behaviour is further complicated by
their dualistic character in water solubility. Indeed, although extremely non
polar, fullerenes are well known to create stable aqueous suspensions with
the formation of nanometer-sized agglomerates that are negatively
charged and whose water solubility is much higher than that of the pristine
structures (Deguchi et al., 2001; Javfert et al., 2008). Although it is not clear
yet to which extent these colloidal structures will form in the environment,
such phenomena of homo-aggregation, as well as the hetero-aggregation
with natural components (e.g. humic and fulvic acids), will likely determine
mobility in water and soil matrices (Wang et al., 2012; Haftka et al., 2015).
The colloidal stability can be further affected by other environmental
conditions such as ionic strength and type of ions present in the water and
the zeta potential of the fullerenes (Haftka et al., 2015). Upon release,
fullerenes will presumably interact with solar radiation, water, natural
materials (e.g. organic matter), soil, air and biota and may be transformed
or degraded as a consequence (e.g. Avanasi et al., 2014; Tiwari et al., 2014).
In this context, some studies suggest that fullerenes will be functionalized
upon interaction with ozone, water and light, and will undergo oxidative
pathways that could lead to mineralization (Hou et al., 2009; Lee et al.,
2009; Hwang et al., 2010; Tiwari et al., 2014). However, other studies
highlighted the stability of C60 in soil matrices that may determine their
accumulation in the environment (Jehlicka et al., 2005; Parthasarathy et al.,
2008). Thus, soil may act as a sink (Gottschalk et al., 2009) receiving
fullerenes through several pathways such as the direct release of ENMs, the
deposition of incidental species from the atmosphere, water transport and
13
use of biosolids (Utsunomoya et al., 2002; Sanchis et al., 2012; Navarro et
al., 2013).
1.4 Environmental analysis of fullerenes
Understanding the occurrence and fate of nanoparticles necessarily relies
on the collection of empirical data. At the time this thesis project started,
several studies had highlighted the lack of analytical methodology able to
detect and quantify fullerenes in environmental samples (Hasselhov et al.,
2008; Gottschalk et al., 2009; Isaacson et al., 2009), a task which is
complicated by the low concentrations expected and the difficulties in the
extraction from environmental matrices (Jehlicka et al., 2005). The existing
techniques presented several limitations. For instance, the majority of the
studies addressed the analysis of C60 only, whereas other pristine fullerenes
(e.g. C70 and to a lesser extent larger fullerenes such as C84) and
functionalized structures were seldom included (e.g. Bouchard et al., 2008).
Furthermore, most of the studies were focussed on water and wastewater
media, whereas soil and sediments, where these chemicals are expected to
accumulate to a larger extent, received little or no attention. Eventually,
although several methods were developed for the extraction and analysis
of fullerenes in soil and sediment matrices, these were not suited for the
extraction of samples presenting high organic carbon content, the detection
at environmentally relevant concentrations and were not able to distinguish
between particles of engineered or natural origin (Jehlicka et al., 2005; Vitek
et al., 2009; Shareef et al., 2010). These issues likely hindered the analysis
of fullerenes in several studies and made the reproducibility of positive
results a difficult task. For instance, the concentration detected in samples
from the Sudbury impact ranged from part per mil to nothing, in three
different studies on the same material (Becker et al., 1994; Heymann et al.,
1999; Elsila et al., 2005).
14
In the last few years several efforts have been made to overcome these
issues and analytical methodologies are now available that allow the
determination of fullerenes and functionalized structures in environmental
samples.
In detail, fullerenes are extremely non-polar chemicals and their analysis
typically makes use of organic solvents, such as toluene, where they are
more soluble (Ruoff et al., 1993). Robust methods for their extraction
include liquid-liquid extraction and solid-phase extraction (for water
samples, e.g. Bouchard et al., 2008; van Wezel et al., 2011) as well as
soxhlet, ultrasonication, microwave assisted extraction (MAE) and
accelerated solvent extraction (ASE) (for solid samples, e.g. Jehlicka et al.,
2005). With regard to the analysis, although separation techniques such as
electrophoresis, size exclusion chromatography and field flow fractionation
were tested, the general opinion is that the combination of liquid
chromatography with ultraviolet-visible and mass spectrometric detection
offers the greatest potential for routine analysis (Isaacson et al., 2009). This
usually consisted of non-aqueous reverse phase methodologies employing
non-polar mobile phases (e.g. toluene or toluene-acetonitrile mixtures)
(e.g. Bouchard et al., 2008, Shareef et al., 2010). The separation of the
fullerenes has been achieved with both standard octadecyl silica as well as
functionalized silica columns and several stationary phases such as 5PBB
and pyrenylpropyl silica that were specifically designed for the analysis of
fullerenes (e.g. Hou et al., 2009).
Concerning the detection methodologies, fullerenes possess strong light
absorptivity in the UV range (specifically at circa 330 nm) and UV detection
has been employed in their analysis in water, sediments, soil and biological
samples (Moussa et al., 1997; Xia et al., 2006; Bouchard et al., 2008; Shareef
et al., 2010; Wang et al, 2011). However, it must be noted that, although
close to some mass spectrometric techniques in terms of sensitivity (Wang
et al., 2010), UV detection lacks the specificity needed for an unambiguous
identification, especially in complex matrices where it can suffer from the
presence of co-extractants. Mass spectrometry can overcome these issues
due to the higher selectivity provided by the m/z signals and the larger
sensitivity, especially in the analysis of exohedral fullerenes (i.e.
functionalized structures that undergo fragmentation in MS/MS) (e.g. van
Wezel et al., 2011; Kolkman et al., 2013; Astefanei et al., 2014b). Although
15
ionization is usually achieved with electrospray interfaces operating in
negative mode, other techniques, such as heated electrospray (H-ESI) and
atmospheric pressure chemical and photo ionization systems (APCI and
APPI, respectively), have been tested and can improve the ionization
efficiency (Nunez et al., 2012; Astefanei et al, 2014b; Emke et al., 2015;
Sanchis et al., 2015). Eventually, identifications of fullerenes in
environmental matrices at concentrations in the part per billion range were
achieved with high resolution MS instruments such as Orbitrap and FTICR
that can provide better identification due to higher mass and isotopic
cluster distribution accuracies (Astefanei et al., 2014b; Emke et al., 2015;
Sanchis et al., 2015).
1.4 Justification and objectives of the thesis
The last two decades have been characterized by the rise of
nanotechnology. However, the interest toward the novel and possible
applications of nanomaterials has been accompanied by the concern about
the implications that these may have for humans and the environment. In
this context, the NanoNextNL project was started with the aim of studying
several aspects of micro- and nano-materials, from engineering to
environmental and life science as well as regulatory and societal aspects.
Within this framework, research was started at the University of
Amsterdam (UvA) and Watercycle Research Institute (KWR) and focussed
on a class of carbon-based nanomaterials, the fullerenes, whose
characterization in the environment was still largely unknown and limited
by the lack of analytical methodologies.
Thus, the general objective of the present thesis was to study the
occurrence and fate of fullerenes in the terrestrial environment, which is
done by first developing analytical methods that allow their analysis in soil
16
samples. First, the extraction of several fullerenes and functionalized
structures was tested from soil samples differing in properties such as the
texture and composition. To this end a non-selective UV detection method
was developed that enables one to determine the totality of fullerenes
species in the samples with an extraction methodology that is potentially
applicable in routine analysis (Chapter 2 and 3). Next, a more sensitive and
selective methodology was developed employing mass spectrometric
detection, in order to fulfil identification criteria and provide unambiguous
determination of the fullerenes in soil samples. This included the
optimization of the method for the analysis of fullerenes at environmentally
relevant concentrations and in complex soil matrices (Chapter 4). These
methodologies were then applied for the study of fullerenes occurrence
and fate in the environment. First, an environmental survey was carried out
for the detection of fullerenes in soils collected in the Netherlands. This also
allowed a partial understanding of their sources and transformation in the
environment (Chapter 5). Following, in Chapter 6 incubation studies were
carried out in order to shed light on the fullerenes fate upon release in the
environment. This included the irradiation with UVA light at
environmentally relevant conditions and the characterization of
transformation products that may derive from their degradation. Chapter 7
finally provides a synthesis of the results obtained.
17
Chapter 2
Optimization of the extraction
procedures for fullerenes in soil
18
Abstract
Two extraction techniques, accelerated solvent extraction (ASE) and a
combination of ultra-sonication and shaking extractions (SSh) were
compared for the recovery of fullerenes spiked into sandy soil matrices.
Consecutive steps of extractions were tested at different concentrations and
the use of polar solvents was tested for the enhancement of the
performance. The results indicate that average recoveries were in the range
of 70% or higher with both techniques at all the concentrations tested.
Furthermore, although SSh delivered higher recoveries at larger
concentrations, when fullerenes and functionalized structures were spiked
at the concentration of 5 μg/kg, both techniques had similar performance.
Eventually, more polar solvents were not applicable in the extraction or in
clean-up of the samples. In general, although ASE is an automatize process
and requires one cycle of extraction, a procedure involving two cycles of SSh
is recommended as more feasible for the routine analysis of a large number
of samples.
19
2.1 Introduction
The presence of fullerenes in natural samples reflects very specific
conditions and natural events in which these chemicals are formed (Buseck
et al., 1992; Becker et al., 1995). However, their increasing manufacturing
and application in emerging nanotechnology is expected to lead to a higher
occurrence in the environment and soil could act as a sink for their
accumulation (Piccinno et al., 2012; Gotthschalk et al., 2009). The lack of
proper analytical methodologies likely hindered both the analysis of these
nanoparticles in several studies (Vitek et al., 2009) and the reproducibility
of positive results (Decker et al., 1994; Heymann et al., 1999; Elsila et al.,
2005). In particular, extraction methodologies are needed for monitoring
studies and routine analysis, which are critical for the understanding of
fullerenes occurrence and fate in the environment. In this context,
extraction techniques such as Soxhlet and supercritical CO2 extraction can
recover fullerenes from solid matrices (soil, carbon soot) but can be
expensive and / or time-consuming and are not suitable for routine analysis
or screening of a large number of samples in general (Issacson et al., 2009).
Thus, techniques such as ultrasonication and accelerated solvent extraction
can provide faster analysis with similar or better performance. In particular,
ultrasonication is a robust method whose performance for C60 extraction
from soil samples was already investigated by Jehlicka et al. (2005) and
Vitek et al. (2009) who reported acceptable recoveries of extraction (>
75%). On the other hand, accelerated solvent extraction (ASE) presents the
advantage of being automated and was recently employed by Shareef et al.
(2010), who reported good recoveries of extraction (>80%) in several soil
matrices. However, when these techniques were compared for the
extraction of fullerenes from carbonaceous matrices (e.g. bituminous coal,
shungite), both techniques presented low recoveries (usually < 5%) (Jehlicka
et al., 2005). In these studies a comparison of the two methodologies was
not performed for soil samples that may be representative of the matrices
in which fullerenes are present in the environment. Furthermore, only C60
was investigated and no other fullerenes and functionalized structures were
included. Thus, in the present work the extraction of sandy soil samples
spiked with fullerenes was performed with both ASE and a combination of
ultra-sonication and shaking extraction. A first experiment was aimed to
20
compare these techniques for the extraction of C60 and C70 at different
concentrations and to assess the contribution of consecutive steps of
extraction to the overall recovery. Then, a second experiment compared
the techniques for the extraction of fullerenes and functionalized fullerenes
at lower concentrations. In this case, the effect of a pre-extraction and pre-
washing with polar solvents was tested.
2.2 Materials and methods
2.2.1 Reagents and chemicals
Toluene, methanol and acetonitrile (Biosolve, Dieuze, France) were all
analytical grade. C60 fullerene (purity >99.9%) was obtained from MER
Corporation. (Tuscon, Arizona, United States). C70 fullerene (purity >99%),
was purchased from Sigma-Aldrich (Steinheim, Germany). [6,6]-Phenyl-C61-
butyric acid methyl ester ([60]PCBM) (purity >99) and [6,6]-Phenyl C71
butyric acid methyl ester ([70]PCBM) (purity >99%) were purchased from
Solenne B.V. (Groningen the Netherlands). Stock solutions of the individual
fullerenes were prepared in toluene at a concentration of 500 mg/L
according to the method described by Kolkman et al. (2013). The solutions
were placed in the dark overnight on a rotary shaker to achieve complete
dissolution of the fullerenes. Diluted solutions for the individual fullerenes
and their mixture were obtained by diluting aliquots from the individual
stock solutions. The solutions were stored at 4°C in the dark and sonicated
for 2 min before use. Sandy soil was collected in the Flevopark area,
Amsterdam, the Netherlands (52°21'55.09"N, 4°57'3.88"E) and consisted in
a top-soil A horizons (top 10-15 cm from the surface) whose texture was
assessed according to WRB 2006. The sample was placed in a freezer at -
20°C overnight and lyophilized with a Scanvac Coolsafe freeze-dryer
(Labogene, Lynge, Denmark) in order to remove traces of water. The dried
sample was finely ground with an agate mortar and sieved. Amendment of
the soil was achieved as follows: three samples (200 g each) of soil were
placed into glass jars and fullerenes were added by spiking a C60 and C70
21
stock solution in toluene to obtain a final concentration of 80 µg/kg, 15
µg/kg and 5 µg/kg each. Following, a sample was made with the same
procedure but spiking both fullerenes and functionalized fullerenes to
obtain a final concentration of 5 µg/kg. The soils were then homogenized
by stirring and left in the dark for 24 h to allow the solvent to evaporate.
2.2.2 Extraction procedures
Two sets of experiments were carried out to compare ASE with a
combination of ultrasonic and shaking extraction. In the first, sandy soil was
spiked at three different levels: 80, 15 and 5 µg/kg, respectively and toluene
was used as the extraction solvent. For ultrasonication and shaking
extraction, 15 g of soil were weighed and placed into a glass centrifuge tube,
15 ml of toluene were added and the samples were placed open into a
Bransonic 12 ultrasonic bath (Branson, Danbury CT, United states)
operating at 50 kHz for 30 min. Then, the tubes were closed with a glass
stopper and shaking extraction was performed with an orbital shaker at 160
rpm for 90 min. Subsequently, the samples were centrifuged at 2000 rpm
and the toluene supernatant was filtered through a 4-7 µm pore size
prepleated paper filter (Whatman, Maidstone, United Kingdom) into 60 ml
amber glass vials. The filter was rinsed with 3 ml of toluene and the
extraction was repeated a second time with the same procedure. For the
first experiment, the extracts were collected separately.
For accelerated solvent extraction (ASE), the soil samples (circa 15 g) were
weighed and transferred directly into a 66-mL stainless steel extraction cell
(Dionex, Sunnyvale, CA, USA). PTFE filters were placed at the bottom and
the top of the extraction cell. Extraction was carried out using an ASE
(Dionex, Sunnyvale, CA, USA) operating with the following parameters:
1500 psi, 50°C, static time 15 min and 120 sec purge with N2. For the first
experiment, three cycles of extraction with toluene were carried out with
the same parameters and the extracts collected separately.The extracts
were evaporated in a water bath at 60 °C under a gentle nitrogen flow until
approximately 5 ml. Finally, the extracts were filtered with 0.45 µm
regenerated cellulose filters and concentrated to a final volume of ~ 1-2 ml.
22
All experiments were performed in triplicate and non-spiked soils were
extracted with the same protocol as reference.
For the second set of experiments, a sandy soil sample spiked at the low
level (5µg/kg) was extracted using three solvents or solvent combinations.
In the first subset an extraction using toluene only was carried out based on
the results of the first experiment. ASE was used to this end with a single
extraction cycle, whereas sonication combined with shaking was carried out
using two consecutive extractions (two cycles). The next subset consisted
of ASE extraction using two consecutive cycles with acetonitrile and
toluene, respectively (the two extracts were then combined), and
sonication/shaking using three consecutive extractions: a first one with
acetonitrile, and the next two employing toluene (combining the three
extracts). Finally, the third subset consisted of an ASE extraction of the
spiked soil using two consecutive cycles: first a methanol extraction
(assuming that methanol would remove polar constitutents from the
sample; this extract was discarded), followed by a toluene extraction.
2.2.3 HPLC-MS analysis
Analyses were performed with a hybrid LTQ Orbitrap mass spectrometer
(Thermo Electron, Bremen, Germany) provided with an ESI interface and
interfaced to a Surveyor HPLC system (Thermo Electron, Bremen, Germany)
for the chromatographic separation. The separation was achieved with a
Cosmosil® Buckyprep column consisting of 3-(1-pyrenyl)propyl groups
stationary phase (4.6 mm ID x 250 mm, Nacalai-Tesque, Kyoto, Japan)
equipped with a C18 silica pre-column. The isocratic elution was obtained
with a mobile phase composition of toluene/acetonitrile (80/20 v/v) and a
flow rate of 0.8 ml/min. Post-column addition of methanol was performed
to improve ionization efficiency in ESI by means of a peek high pressure
mixing-tee at a flow rate of 200 µl/min. The injection volume was 20 µl.
With these settings, the retention times of the pristine fullerenes was 20.5
and 28.5 min for C60 and C70, respectively, whereas the functionalized
structures [60]PCBM and [70]PCBM eluted at 11.0 and 17.0 min,
respectively. The mass spectrometer operated according to the settings
described by Kolkman et al. (2013). Briefly, analysis was performed using
23
negative electrospray ionization and the capillary used was a metal needle
maintained at a temperature of 400°C. The sheath, sweep and auxiliary
gases were set to arbitrary units of respectively 30, 10, 10. A source voltage
of 3 kV and a capillary voltage of -80 V were used. The tube lens was set to
-200 V. Full scan high accuracy mass spectra were acquired in the range of
300-2000 m/z with the resolution set at 30,000 (FWHM). External
calibration curves were obtained analysing standard solutions in toluene at
concentrations ranging from 1 µg/L to 128 µg/L and quantification was
based on the sum of the peak areas of the accurate masses of the fullerene
compound and all its related adducts as described by van Wezel et al.
(2011).
2.3 Results and discussion
The experiments performed in this work were aimed to compare two
extraction techniques: accelerated solvent extraction (ASE in the further
manuscript) and a combination of ultrasonication and shaking (SSh in the
further manuscript).
The first consisted in the comparison of these techniques for the extraction
of pristine fullerenes spiked into soil at three concentrations: 80, 15 and 5
μg/kg which will be referred as high, medium and low, respectively. The
results are presented in Figure 2.1 and in the Table 2.1. In general, similar
to what was reported in previous studies (Vitek et al. 2009, Shareef et al.,
2010) the overall recoveries were acceptable (≥ 70%) for both C60 and C70 at
all the concentrations tested with the exception of one (SSh of C70 at low
concentration, 65%). Variability within the same sample was also
acceptable and generally below 10%. Furthermore the results indicate that
(I) consecutive steps of extraction had a more relevant impact on the overall
recovery in SSh than in ASE. In detail, the contribution of further extractions
after the first was 6.4%, on average, for SSh and 1.4%, on average, for ASE.
(II) For the high and medium concentrations, the overall performance of
24
SSh was better than ASE. Especially in the extraction of C60, SSh showed
recoveries of 96% on average, 25% higher than that of ASE. (III) However,
when samples at the low concentration were extracted, ASE showed slightly
better performance, 8.5% higher than SSh on average. Eventually it must be
noted that ASE showed similar performances at all the concentrations
tested whereas SSh delivered lower recoveries when tested for the
extraction of the low treatments.
Table 2.1. Recoveries of consecutive steps extraction for C60 and C70 spiked
at different concentrations into sandy soil.
ASE: Accelerated solvent extraction (toluene). SSh: Ultrasonication and shaking extraction (toluene).
25
Fig. 2.1. Performance of accelerated solvent extraction (ASE, orange) and a combination of ultrasonication and shaking (SSh, green) in the extraction of (a) C60 and (b) C70 from sandy soil samples. SSh and ASE extraction were carried out with toluene; consecutive extraction steps of ASE and SSh are indicated by different colors according to the legend.
26
From the results of the first experiment it was concluded that when using
toluene, one extraction cycle sufficed in the case of ASE, whereas for SSh
two cycles appeared to be necessary. This information was used in the
setup of the second set of experiments, where - in addition to a toluene-
only extraction with a single cycle for ASE or two consecutive cycles in the
case of SSh, the extracts of which were then combined - the influence of
additional solvents with different polarities were tested on sandy soil
samples spiked at the low level. In these second set of experiments, also
functionalized fullerenes, specifically [60]PCBM and [70]PCBM were spiked
into the soil.
In general, the recoveries obtained for the four fullerenes when using only
toluene were acceptable with both the techniques (~70%), confirming the
results observed for C60 and C70 in the first experiment (Table 2.2). For the
functionalized fullerenes recoveries were between 64 and 89%.
Table 2.2. Recovery of extraction for fullerenes and functionalized fullerenes spiked at 5 μg/kg into sandy soil.
ASE: Accelerated solvent extraction. SSh: Ultrasonication and shaking
extraction.
Although ASE, when using toluene only, provided higher recoveries for all
the compounds except for C60, the differences between the two
methodologies were within the variability of the treatment, with the
27
exception of [60]PCBM. With regard to the functionalized structures,
[60]PCBM was extracted to a higher extent in comparison with the non-
functionalized C60 and the other structures. This could be due to the higher
polarity of the functionalized C60 that resulted in a larger extractability of
the compound due to (I) a higher solubility in toluene or (II) a lower binding
to the soil particles. However, [70]PCBM was recovered to the same extent
as the pristine fullerenes, suggesting that the functional group did not play
a role in determining the overall extractability of the fullerenes in the
present study. The employment of a more polar solvent (acetonitrile), in
combination with the toluene, was expected to enhance the extractability
of the fullerenes, but resulted in a decrease of the recoveries of all the
compounds under investigation (Fig. 2.2b and Table 2.2). This can be
explained with the fact that, in addition to not recovering the fullerene
itself, the acetonitrile prevented the toluene entering the soil matrix. Pre-
washing of the samples with methanol was tested as a possible clean-up
procedure, i.e. aimed to remove the polar components of the sandy soil
that could interfere with both extraction and detection of the fullerenes.
However, in this case a large loss in the recoveries was observed (Table 2.2).
Obviously methanol also extracted a part of the fullerens from the soils
samples. The methanol pre-wash procedure was not further investigated.
In general, although SSh showed better performances for higher
concentrations and ASE was slightly better in recovering fullerenes at lower
concentrations, the two techniques provided similar results and are
therefore good options for future studies on the topic. It must be noted that
this work did not take full advantage of one of the main ASE characteristics,
which is that of operating at high temperature while organic solvents can
be kept at the liquid state, due to the high pressure generated in the ASE
cells. Since fullerene solubility is maximum around 25°C (Ruoff et al., 1993),
in this work the temperatures were relatively low in comparison with other
ASE operational procedures.
With regard to the operational procedures, which in the framework of this
study should be optimized for routine analysis, best SSh required two cycles
of extraction whereas ASE achieved good results with one cycle of
extraction only. Furthermore, ASE is an automated process and, in principle,
more reproducible. However, during this work, ASE was more time-
28
consuming and solvent requiring than SSh with regard to both sample
preparation and cleanup. Furthermore, extract volumes, which cannot be
controlled by the operator, were always larger in the case of ASE than those
obtained in SSh and resulted in further time and materials losses (e.g.
toluene, nitrogen) during the subsequent concentration step. Due to these
drawbacks, and because the performance of the two techniques are very
similar, SSh is recommended for routine analysis since it provides an overall
faster and more environmental friendly procedure. Eventually, further
tests will be required in order to assess the performance of these
procedures on soil matrices differing in physico-chemical properties such as
texture and chemical composition.
29
Fig. 2.2. Performance of accelerated solvent extraction (ASE, orange) and a
combination of ultrasonication and shaking (SSh, green) in the extraction of
fullerenes and functionalized fullerenes from sandy soil. (a) Samples
extracted with toluene and (b) samples that underwent pre-extraction with
acetonitrile.
30
2.4 Conclusions
The aim of this work was to test two extraction techniques, and specifically
accelerated solvent extraction (ASE) and a combination of ultrasonication
and shaking (SSh), for the analysis of fullerenes in soil samples. In general,
when fullerenes were spiked at the concentration of 80 and 15 μg/kg, SSh
showed higher recoveries than ASE but at the lower concentration of 5
μg/kg the two techniques were comparable. Furthermore, optimal
extraction required toluene only and could not be enhanced with the use
of more polar solvents in pre-extraction or clean-up steps. In addition of
being automated, ASE had the advantages of achieving the best results with
one cycle of extraction, whereas SSh required a double-step procedure.
Nonetheless, SSh resulted in an overall faster procedure and required less
materials and energy to be accomplished. Thus, the authors recommend
the use of SSh for the analysis of fullerenes in soils and especially for
monitoring studies where a large batch of samples is expected.
31
Chapter 3
A HPLC-UV method for the
analysis of fullerenes in soils
Published as:
Carboni A., Emke E., Parsons J. R., Kalbitz K., de Voogt P. 2013. An analytical
method for determination of fullerenes and fullerene derivatives in soil with
high performance liquid chromatography and UV detection. Analitica
Chimica Acta 807, 159-165.
32
Abstract
Fullerenes are carbon-based nanomaterials expected to play a major role in
emerging nanotechnology and produced at an increasing rate for industrial
and household applications. In the last decade a number of novel
compounds (i.e. fullerene derivatives) is being introduced into the market
and specific analytical methods are needed for analytical purposes as well
as environmental and safety issues. In the present work eight fullerenes (C60
and C70) and functionalized fullerenes (C60 and C70 exohedral-derivatives)
were selected and a novel liquid chromatographic method was developed
for their analysis with UV absorption as a method of detection. The resulting
HPLC-UV method is the first one suitable for the analysis of all eight
compounds. This method was applied for the analysis of fullerenes added to
clayish, sandy and loess top-soils at concentrations of 20, 10 and 5 µg/kg
and extracted with a combination of sonication and shaking extraction. The
analytical method limits of detection (LoD) and limits of quantification (LoQ)
were in the range of 6-10 µg/L and 15-24 µg/L respectively for the analytical
solutions. The extraction from soil was highly reproducible with recoveries
ranging from 47 ± 5 to 71 ± 4% whereas LoD and LoQ for all soils tested were
of 3 µg/kg and 10 µg/kg respectively. No significant difference in the
extraction performance was observed depending of the different soil
matrices and between the different concentrations. The developed method
can be applied for the study of the fate and toxicity of fullerenes in complex
matrices at relatively low concentrations and in principle it will be suitable
for the analysis of other types of functionalized fullerenes that were not
included in this work.
33
3.1 Introduction
Since their discovery in 1985 by Kroto et al. (Kroto et al., 1985), fullerenes
have attracted a lot of interests due to their unique structure and innovative
properties and are nowadays considered as some of the most promising
materials in nanotechnology. Fullerenes are very versatile compounds
already applied in several fields such as optics and electronics as well as
cosmetics and in medical research (Tagmatarchis et al., 2001; Guldi et al.,
2002; Burangulavet al., 2005; Kim et al., 2006) with a worldwide production
estimated in tens of tons per year, that is expected to increase in the near
future (Hendren et al., 2011; Piccinno et al., 2012). Furthermore, the
possibility to functionalize the closed cage structure by the covalent binding
of external groups to the fullerene’s surface (i.e. exohedral fullerene
derivatives) increase the solubility of these compounds in organic as well as
polar solvents and consequently widens their range of applications and
uses. Contrary to pristine compounds such as C60 and C70, that can be
naturally produced during highly energetic events such as lightening (Daly
et al., 1993) and massive wildfires (Heymann et al., 1994), functionalized
fullerenes are in all respect engineered nano-materials (ENMs). Firstly
described by Hummelen et al. in 1995 (Hummelen et al., 1995), the
fullerene derivative 1-(3-methoxycarbonyl)propyl-1-phenyl[6,6]C61, better
known as [60]PCBM, is to date one of the most studied in the field of organic
photovoltaic (OPV) materials (Dang et al., 2011) and has been proposed for
the construction of organic field-effect transistors (OFETs) (Tiwari et al.,
2007) and photo detectors (Baierl et al., 2010). In the last decade, a number
of PCBM-like chemicals differing in the substituent group (e.g. thienyl
analog of [60]PCBM, Popescu et al., 2006), number of substituents (e.g.
bisadducts, Lenes et al., 2008) or the functionalization of fullerenes other
than C60 (e.g. 70[PCBM], Wienk et al., 2003) as well as compounds with
different functionalization (e.g. C60-pyrrolidines, Marchesan et al., 2005) are
being produced and studied for their use in novel applications. Despite the
broad interest in the development of new engineered nanomaterials,
knowledge on the human safety and environmental issues of fullerenes and
their derivatives is scarce. Fullerenes entering the environment as
consequence of their production and use will presumably accumulate in soil
34
and sediments. Although functionalized fullerene derivatives have been
recently included in environmental monitoring (Sanchis et al., 2011 and
2013), most of the research so far has been focused on C60 only and no
chromatographic methods have been developed yet for the analysis of the
functionalized fullerenes structures. Among the analytical techniques that
have been applied to the analysis of fullerenes, liquid chromatography
appears to be the most feasible method for routine analysis and the main
advancements in this field have already been reviewed elsewhere (Baena
et al., 2002; Isaacson et al., 2009). In general, although octadecil silica (ODS)
stationary phases can be used to separate compounds such as C60 and C70,
better performance is achieved with other materials that offer a higher
surface for the interaction and therefore retention of fullerenes (e.g. 2-(1-
pyrenil)ethylsilica or 3-(pentabromobenzyl)oxy-propylsilylsilica) particularly
when more compounds are analyzed in a mixture.
Toluene is the most common mobile phase applied due to the high solubility
of fullerenes in this solvent at room temperature (Ruoff et al., 1993) and
can be used as only eluent when C60 is the only analyte under investigation.
When other fullerenes (e.g. C70) or functionalized fullerenes such as
[60]PCBM were included in the study, more polar solvents such as
acetonitrile (Bouchard et al., 2008), hexane or isopropanol (Deye et al.,
2008) have been used as modifiers to enhance the separation. Fullerenes
absorb light in the 300-350 nm range and UV-vis detection is a powerful tool
for their analysis in combination with HPLC because of the broad linearity
range and high sensitivity. In a recent study, Wang et al. (Wang et al., 2010)
compared UV-vis and mass spectrometry (MS) for the detection of C60 in
HPLC and concluded that, despite the higher selectivity of MS based on the
m/z ratio, the two techniques are comparable in terms of sensitivity and
UV-vis offers a larger linear range. HPLC-UV methods have been used for
the analysis of fullerenes in different matrices such as soil (Shareef et al.,
2010; Perez et al., 2013), artificial sediments (Wang et al., 2011), surface
and groundwaters (Bouchard et al., 2008) and biological matrices (Moussa
et al., 1997; Xia et al., 2006) but most of these studies were focused on C60
and occasionally higher fullerenes whereas functionalized structures were
seldom included. Furthermore, fullerenes and fullerene derivatives have
also shown to emit fluorescence at room temperature when dissolved in
35
organic solvents (Lin et al., 1995; Zhao et al., 2006) but no data are available
of fluorescence detection coupled to HPLC.
In the present study we developed a HPLC method with UV detection for
the determination of eight selected fullerenes and functionalized
fullerenes. After optimization the method was tested for the analysis of the
fullerenes in environmental matrices. Soil and sediments might act as a sink
for the accumulation of hydrophobic fullerenes after their release into the
environment but few studies have addressed yet the issue of analyzing
these compounds in these matrices (e.g. Vitek et al., 2009; Shareef et al.,
2010; Perez et al., 2013). Furthermore, in the majority of the studies that
have addressed the issue, the concentrations tested were relatively high
(hundreds µg/kg and above) with the exception of a recent study from Perez
et al. (2013). None of these studies included functionalized structures other
than [60]PCBM. Thus, in the present work three top-soils differing in their
properties as texture and organic matter content, namely sandy, clayey and
loess soils were spiked with toluene standard solutions containing all the
fullerenes under investigation to a final concentration of 20, 10 and 5 µg/kg
for each compound and analyzed using the HPLC-UV method.
3.2 Materials and Methods
3.2.1 Reagents and chemicals
Table 3.1 presents characteristics of the fullerenes in the present study.
Toluene and Acetonitrile (Biosolve, Dieuze, France) were both analytical
grade. Stock solutions of the individual fullerenes were prepared in toluene
at a concentration of 500 mg/L according to the method described by
Kolkman et al. (2013). The solutions were placed in the dark overnight on a
rotary shaker to achieve complete dissolution of the fullerenes. Diluted
solutions for the individual fullerenes and their mixture were obtained by
diluting aliquots from the individual stock solutions. The solutions were
stored at 4°C in the dark and sonicated for 2 min before use.
36
Table 3.1. Fullerene standards used in the present study.
3.2.2 Soil sampling, soil characterization and sample treatment
Sandy soil was collected in the Flevopark area, Amsterdam, the Netherlands
(52°21'55.09"N, 4°57'3.88"E), the loess soil was collected from an
agricultural field in south Limburg, the Netherlands (50°53'58"N, 5°53'16"E)
and the clayish soil was collected in Dikkebuiksweg, the Netherlands
(50°50'03"N, 5°54'27.7"E). All the soils in the present study were sampled
from top soils A horizons within the first 10-15 cm from the surface and
their texture was assessed according to WRB 2006. The samples were
placed in a freezer at -20°C overnight and lyophilized with a Scanvac
Coolsafe freeze-dryer (Labogene, Lynge, Denmark). The dried samples were
finely ground with an agate mortar and sieved.
At first we obtained an aqueous extract to measure dissolved organic
carbon (DOC). Samples of 20 g for each soil were placed in 200 ml
polyethylene bottles and 100 ml of ultrapure water were added (dilution
1:5) before to undergo shaking extraction for 2 h at 120 rpm with a
Laboshake orbital shaker (Gerhardt, Königswinter, Germany). The samples
were then transferred into 50 ml plastic tubes, centrifuged for 15 min at
2000 rpm with a Rotofix 32A (Hettich, Tuttlingen, Germany) and the
supernatants were transferred into plastic syringes and filtered with 0.2 µm
cellulose ester membrane filters (Whatman, Maidstone, United Kingdom)
37
previously rinsed with ultrapure water. The pH of the final extracts was
measured with a Consort C831 electrode (Consort NV, Turnhout, Belgium)
and DOC and IC (inorganic carbon) were determined using a TOC-VCPH
(Shimadzu, Kyoto, Japan). The carbon and nitrogen contents in the dried soil
samples were measured using a Vario EL Cube (Elementar, Hanau,
Germany). All the experiments for the soils characterization were made in
triplicate. Three samples (200 g) for each soil were placed into glass jars and
fullerenes were added by spiking a fullerene stock solution in toluene to
obtain a final concentration of 20 µg/kg, 10 µg/kg and 5 µg/kg. The soils
were then homogenized by stirring and left in the dark for 48 h to allow the
solvent to evaporate.
3.2.3 Extraction
10 g of soil from each jar were weighed and placed into a glass centrifuge
tubes, 10 ml of toluene were added and the samples were placed open into
a Bransonic 12 ultrasonic bath (Branson, Danbury CT, United states)
operating at 50 kHz for 30 min. Then, the tubes were closed with a glass
stopper and shaking extraction was performed with an orbital shaker at 160
rpm for 90 min. Subsequently, the samples were centrifuged at 2000 rpm
and the toluene supernatant was filtered through a 4-7 µm pore size
prepleated paper filter (Whatman, Maidstone, United Kingdom) into 40 ml
amber glass vials. The filter was rinsed with 3 ml of toluene and the
extraction was repeated a second time by adding 8 ml of toluene. In this
latter procedure, the samples were not centrifuged and the soil samples
were transferred directly to the paper filters. After elution of the solvent,
each sample was rinsed with 5 ml of toluene and the extracts were
evaporated in a water bath at 60 °C under a gentle nitrogen flow until
approximately 3 ml. Finally, the extracts were filtered with 0.45 µm
regenerated cellulose filters and concentrated to a final volume of ~ 0.5-1
ml. All experiments were performed in triplicate and non-spiked soils were
extracted with the same protocol as reference.
38
3.2.4 HPLC with UV and fluorescence detection
UV-vis and fluorescence spectra of the fullerenes were obtained analyzing
stock solutions of the single compounds in quartz cuvettes with an Olis DW-
2000 spectrophotometer and an Olis DM45 spectrofluorimeter (Olis, Bogart
GA, United States), both equipped with Olis SpectralWorks software. Liquid
chromatography was performed with a Shimadzu Prominence system
(Shimadzu, Kyoto, Japan) equipped with a diode-array detector and a
fluorescence detector. The wavelengths monitored for UV detection were
305 nm and 332 nm. For fluorescence detection, emission wavelengths at
400 nm, 550 nm and 700 nm were monitored with excitation wavelength
set at 286 nm, 332 nm, 400 nm or 463 nm. The data were collected with the
LCsolution software. The separation was achieved with a Cosmosil®
Buckyprep column consisting of 3-(1-pyrenil)propyl groups stationary phase
(4.6 mm ID x 250 mm, Nacalai-Tesque, Kyoto, Japan) equipped with a C18
silica pre-column at a flow rate of 1 ml/min and an injection volume of 20
µl. External calibration curves were obtained analyzing standard solutions
in toluene at concentrations ranging from 4 µg/L to 1 mg/L and
quantification was based on chromatographic peak areas whereas limits of
detection (LoD) and quantification (LoQ) were assessed observing the signal
to noise ratio (S/N) and considering LoD as the concentration with S/N=3
and LoQ as the concentration with S/N=10.
3.3 Results and discussion
3.3.1 HPLC-UV method
The separation of fullerenes in liquid chromatography necessitates the use
of an apolar mobile phase able to dissolve and elute the compounds in a
relatively short time. In this study toluene was applied as the main eluent
in the mobile phase in combination with a specific stationary-phase,
composed of pyrenyl-propyl functionalized silica (Buckyprep), that
enhances the retention of fullerenes as a result of the large ligand that can
interact with the aromatic structure of the fullerenes. This non-aqueous
39
chromatographic system can be nominally referred to as normal-phase
liquid chromatography due to the apolarity of both the mobile and
stationary phases. Since the isocratic elution with toluene as only eluent
resulted in a partial or total co-elution of some of the compounds in the
mixtures a more polar solvent, in this case acetonitrile, was added to the
mobile phase in different percentages to enhance the separation of the
analytes. The final optimized method (fig. 3.1) consisted in a gradient
elution starting with 75:25, toluene:acetonitrile (% volume) and the gradual
conversion after 6 min to 100% toluene to allow a faster elution of the more
apolar compounds. With these settings the elution of all the analytes is
obtained within 25 min while the whole method lasted 32 min to allow the
system to equilibrate prior to the next analysis.
Figure 3.1. HPLC-UV chromatogram of fullerenes and functionalized
fullerenes in toluene containing 20 ng of each analyte. [1]: bis[60]PCBM,
[2]: [60]PCBO, [3]: [60]PCBB, [4]: [60]PCBM, [5]: [60]ThCBM, [6]:
[70]PCBM, [7]: C60 and [8]: C70.
As shown in fig. 3.1, the order of elution is correlated with: 1) the number
and presence of functionalization on the cage, that increase the solubility
of the compounds in the mobile phase and decrease the surface available
for the interaction with the pyrenyl-propyl groups in the stationary phase
(functionalized elute earlier than pristine fullerenes) and 2) the size of the
40
cage (C60 structures elute earlier than C70). Thus, the double functionalized
bis[60]PCBM (Fig. 3.1, peak 1) is the first compound to elute with a relatively
broad peak, between 3.5 and 4.7 min. Its jagged peak-shape might be due
to either the presence of different isomers (60 positions are available on
the structure for the attachment of the two functional groups) or by the
formation of micelles in the solution. The four C60 derivatives (fig. 3.1, peaks
2, 3, 4 and 5), which are not baseline resolved, eluted in a cluster between
6 to 7.5 min followed by the [70]PCBM (fig. 3.1, peak 6) at time 11.8 min.
This latter peak has a shoulder that might be due to oxidized products or
the presence of different isomers. C60 and then C70 (fig. 3.1, peaks 7 and 8
respectively) are fully resolved and elute in the end of the analysis after 17.0
and 24.8 min respectively.
The separation of the mono-functionalized C60 fullerenes is challenging
because of the high similarity in the structures (Table A.1 in appendix A)
that results in the co-elution of the compounds in between 6 and 7.5 min
as shown in fig. 3.1 (peaks 2-5). Since the absorption spectra of the
compounds is very similar (discussed below) and because of the lack of
selectivity, UV detection alone cannot help in the determination of these
non-fully resolved peaks. If a more selective detection method such as mass
spectrometry is not available, the determination of the respective
compounds that are co-eluting must be achieved by improving the
chromatographic separation. In general, the elution time of the
functionalized C60 structures in this study is correlated with the aromatic
ring in the functionalizing group (phenyl-functionalized eluted before
thienyl-functionalised) and is inversely proportional with the length of the
alkyl chain in it. Therefore [60]PCBO and [60]PCBB (octyl ester and butyl
ester respectively) eluted before than [60]PCBM and [60]ThCBM (both
methyl esters but with different aromatic rings).
When a mobile phase composition of 75:25, toluene:acetonitrile was
applied (fig. 3.2B), the four compounds created two clusters, the first one
including [60]PCBO and [60]PCBB (Rs < 1) which was fully resolved from the
second one composed by [60]PCBM and [60]ThCBM (also Rs < 1). A better
resolution of the peaks in one of the clusters was obtained by modification
of the mobile phase composition, i.e the ratio between acetonitrile and
toluene, but resulted in a lower resolution in the other cluster. For instance,
41
increasing the percentage of acetonitrile (fig. 3.2A) and therefore the
polarity of the eluent resulted in a better separation of the compounds
based on the different aromatic rings (Rs ≥ 1 for [60]PCBM and [60]ThCBM)
but decreased the resolution between [60]PCBO and [60]PCBB. On the
contrary, increasing the percentage of toluene in the mobile phase (fig.
3.2C) allowed a better separation of the compounds depending on the alkyl
length, thus improving the resolution between [60]PCBO, [60]PCBB but
resulted in the co-elution of [60]PCBM and [60]ThCBM. In addition, the
variation in the polarity of the mobile phase affected the peak shape and
retention times of the analytes. A larger percentage of acetonitrile (fig.
3.2A) enhanced the separation of the jagged peaks of bis[60]PCBM but also
caused a slower elution of all the compounds whereas a more apolar eluent
(fig. 3.2C) leaded to a faster elution of all the compounds. These results
suggest that a complete separation of very similar structures such as the
ones included in this study might be achieved by the variation of the
physical parameters (e.g. length of the column, particles size) more than
chemical parameters such as the polarity of the mobile phase.
42
Figure 3.2. Chromatographic separation of fullerene derivatives at mobile phase composition of Toluene:acetonitrile; 65:15 (A), 75:25 (B) and 85:15 (C) (volume %). The fullerenes structures are numbered according to the caption of fig. 3.1.
When fullerenes are dissolved in organic solvents such as toluene,
spectrophotometric detection is a powerful tool for their analysis owing to
the strong absorption of these compounds in the UV range. The absorption
spectra of the functionalized fullerenes included in this study (fig. 3.3) are
43
comparable to those of the pristine C60 and C70 fullerenes from which they
are derived.
Figure 3.3. UV-vis absorption spectra of functionalized fullerenes at concentrations ranging from 1.5 to 2 mg/L.
As reported by Bouchard et al. (2008), the wavelength selected for the
detection during the chromatographic runs were 332 nm for C60, C70 and the
C60 mono-derivatives whereas the optimum for bis[60]PCBM and [70]PCBM
was found at 305 nm (fig. A.2 in appendix A), despite the fact that the
maximum absorbance for all the compounds was recorded at 286 nm. This
latter wavelength was not applied in the measurements owing to the
toluene absorbance in the same range that resulted in a greater baseline
noise. Thus, at these wavelengths selected the detector response was linear
(correlation coefficients > 0.99) over more than two orders of magnitude of
44
mass. The analytical method limits of detection (LoD) and limits of
quantification (LoQ) were assessed to be 120 pg (LoD) and 300 pg (LoQ) for
C60 and the C60 mono-derivatives respectively and 200 pg (LoD) and 480 pg
(LoQ) for C70 and its derivative. The presence of an interference peak at
retention time 4.0 min precluded an accurate detection and quantification
of bis[60]PCBM below 2 ng injected. Fluorescence emission spectra were
collected for all the compounds dissolved in toluene and the wavelengths
tested for the excitation were 286 nm, 332 nm and 463 nm, because of the
absorption of fullerenes at these values and 400 nm which had been
reported to excite C60 with consequent emission at 700 nm (Zhao et al.,
2006). C70 and [70]PCBM showed a weak fluorescence emission at 700 nm
when excited at 463 nm while the other compounds did not display any
clear emission signal at none of the excitation wavelengths tested.
Furthermore, the chromatograms collected recording the fluorescence
emission at 700 nm for the excitation wavelengths tested showed a very
high baseline noise and no clear chromatographic peak. Therefore,
fluorescence detection was not considered for further analysis in this study.
3.3.2 Application of the method
The analysis of fullerenes in environmental matrices can be problematic
because of the presence of matrix components in the extracts that can
absorb in the same range of wavelengths affecting the detection. The
properties of the soils used in the present study are reported in table 3.2
whereas the HPLC chromatograms corresponding to the analysis of the
three soil matrices spiked at 20 µg/kg are shown in figure 3.4.
45
Table 3.2. Physico-chemical properties of the soils used in the present study
(DOC, dissolved organic carbon; IC, inorganic carbon).
All soil extracts analyzed showed different but consistent matrix
interferences which are probably due to the extracted constituents of the
soil (e.g. hydrophobic fraction of the organic matter) that are not retained
in the column and eluted with the void peak in the beginning of the
chromatograms. The matrix effect was particularly evident in sandy soil
extracts (fig 3.4A) where the co-extractants eluted until 8 minutes and in
loess soil extracts (Fig 3.4B) with a number of small signals in the first 12
minutes of elution. In clay soil extracts (fig. 3.4C) the fullerenes peaks were
relatively clear (with higher S/N ratios) in comparison with the other two
matrices although this soil had the highest content in organic matter, DOC
and clay. The matrix constituents seemed not to affect the order of elution,
separation and retention times of the compounds. However, they
interfered with the detection of the fullerenes eluting in the beginning, i.e.
several of the functionalized C60s. Thus, while the last three fullerenes to
elute, [70]PCBM, C60 and C70, seemed not to be affected by any strong
interference in comparison with the chromatogram obtained running pure
standard solutions (fig. 3.1), bis[60]PCBM (Rt: ~4 min.) was not detected in
any of the soil extracts and the C60 mono-functionalized peaks were difficult
to quantify. Except for bis[60]PCBM, all the analytes could be detected (S/N
≥ 3) and quantified (S/N ≥ 10) in the extracts for all the three soils spiked at
46
20 and 10 µg/kg. When samples were spiked at 5 µg/kg, detection of the
compounds was still possible but no quantification could be made. In
general, C60 and C60 derivatives were more easy to detect than C70 and
[70]PCBM owing to the lower sensitivity of the detector for these latter
compounds and because of the slope in the baseline that affected the
determination in the end of the chromatograms. These results suggest that
the use of the optimized method is suitable for the analysis of fullerenes in
soils differing in clay and organic matter content especially for pristine
fullerenes such as C60 and C70. A sample clean-up before the injection may
help to remove the impurities in the extract that interfere with the analysis
of the fullerene derivatives in the beginning of the chromatograms.
47
Figure 3.4. Chromatograms of fullerenes extracts from sandy (A), loess (B) and clay (C) soils spiked at 20 µg/kg (blue, continues line). The red non-continues lines represent the non-spiked soils. The fullerenes structures are numbered according to the caption of fig. 3.1. Note that [70]PCBM was detected at 305 nm.
48
The recoveries of extraction for all the compounds spiked in the three soils
tested are reported in table 3.3. Several methods of extraction have already
been applied for the extraction of fullerenes from soil samples (e.g.
microwave-assisted extraction, sonication, soxhlet and accelerated solvent
extraction). However, it is not possible to establish which, among these
techniques, is the better because of the differences in the experimental
settings (e.g. kind of soil, concentrations) reported. Ultrasonication is a
robust method that was already investigated by Jehlicka et al. (2005), Vitek
et al. (2009) and Perez et al. (2013) and was applied in the present study in
combination with shaking extraction. As shown in table 3.3 the recoveries
for all other compounds were acceptable with good repeatability (n = 3)
except for bis[60]PCBM, that could not be recovered in any of the samples
because of the co-extracted interferences. The good repeteabilities (on
average less than 5% for the 20 µg/kg level and less than 6% for the 10 µg/kg
level) demonstrate that the method developed in the present study is
robust. Increasing the injection volume in the HPLC or extracting a larger
sample intake could further improve the recovery of extractions because of
the higher amount of fullerenes in the extracts and the limited interference
of the co-extractants for the other compounds except for bis[60]PCBM.
Fullerenes are expected to absorb to the soil matrix (Jehlicka et al., 2005)
and different soil components (e.g. clay minerals, organic carbon etc.) may
affect the extraction efficiency. In the present work, all analytes except for
one were recovered from the three soils to similar extents and this is
consistent with what already reported by Shareef et al. (2010) who did not
observe any real difference in the recovery from six soils tested in their
study. Statistical analysis performed (two way ANOVA) on the mean
recoveries (combining all concentrations tested) from the present study
revealed that there is no significant difference (P > 0.05) between the three
soils tested. Jehlicka et al., (2005) highlighted the role of fullerenes
concentration in the extraction efficiency of fullerenes from carbonaceous
matrices. They observed that the extraction efficiency decreased at
decreasing concentration of the C60 in soil and concluded that the possible
reasons for the reduction might be a decomposition or transformation of
the compounds and/or the absorption of fullerenes to the soil components.
This effect was not observed in the present study where the difference
between the recoveries for all the compounds at the two quantifiable
49
concentrations tested (table 3.3) is not significant at the 5% confident level
(P > 0.05).
Table 3.3. Comparison of the extraction recoveries of fullerenes from sandy, loess and clay soil at the concentrations of 20 µg/kg (left) and 10 µg/kg (right).
50
This could be explained by the fact that the concentrations in our study are
relatively similar (factor of 2 difference) in comparison with those tested by
Jehlicka et al. (factor of 10) and that the effect of the fullerenes
concentration on the extraction recovery is not appreciable in this small
range. Since the concentrations tested in the present work were lower than
those reported in the majority of previous studies, absorption and general
losses of the compounds during the sample treatments are a possible
explanation for the lower recoveries (from 47% to 71%) in comparison with
those already reported (e.g. 83-107% recovery with ASE, Shareef et al.,
2010). Recently Perez et al. (2013) reported a temperature dependency on
the recoveries of extraction for C60 and C70 spiked into soil at concentrations
similar to those tested in the present study. The spiking of the samples at
low concentrations in the present study allowed an accurate determination
of the LoDs and LoQs, estimated to be 3 µg/kg and 10 µg/kg respectively for
all the soil tested. Although more sensitive LC-MS methodologies have been
developed very recently, that allow the determination of fullerenes in
environmental matrices at even lower concentrations (Kolkman et al., 2013;
Sanchis et al 2013), the HPLC-UV method in the present study is a valid
alternative, cheaper and easier to interpret. Finally it must be noted that,
despite the fact that spiking of concentrated solutions in toluene is a
common procedure, it also represents a limitation because it could not
reproduce the real conditions at which fullerenes are present in the soil
environment and further efforts will be needed in the development of
alternative and more representative spiking techniques.
3.4 Conclusions
In this study a new chromatographic method was developed and optimized
for the analysis of fullerenes and functionalized fullerenes in soil using HPLC
with UV as method of detection. UV-detection showed very high linearity
for the compounds under investigation and allowed their detection at
concentrations as low as 6 µg/kg whereas fluorescence detection did not
fulfil the prerequisite for the analysis when coupled with HPLC in the
51
present study. This is the first time that such a number of functionalized and
non functionalized fullerenes are analyzed by HPLC-UV in a single run and
in principle, other functionalized structures, similar to those included in this
study, can be analyzed with the method. The analytical settings can be
optimized depending on the analyte(s) of interest e.g. by modification of
the mobile-phase composition or wavelength of detection.
The analysis of fullerenes including the functionalized derivatives extracted
from real soil samples spiked with the compounds, showed that the method
is robust and suitable for the determination of these compounds in complex
environmental matrices at concentrations in the range of µg/kg. The
extraction of the compounds with a combination of sonication and shaking
in two steps, with toluene as extracting solvent, is highly reproducible and
relatively efficient (from 47% to 71% recovery) and the method limit of
detection and limit of quantification (3 µg/kg and 10 µg/kg respectively)
are lower than those already reported by other works.
This method would allow the study of the fate and toxicity of fullerenes and
their functionalized derivatives in environmental samples at concentrations
close to those expected in the real environment. However, since the
predicted environmental concentration of fullerenes is expected to be in
the range of ng/kg (Gottshalk et al., 2009), further developments are
needed in order to apply the method to environmental monitoring,
especially for the functionalized structures whose UV detection in the
present study was affected by the matrix components. The current
limitations of the HPLC-UV method developed in the present study may be
overcome by applying preconcentration methods in combination with
clean up or using more sensitive but also more expensive detection
methods such as high resolution mass spectrometry.
52
53
Chapter 4
An UHPLC-HRMS method for the
analysis of fullerenes in soil
Published as:
Carboni A., Helmus R., Parsons J. R., Kalbitz K., de Voogt P. 2016. A method
for the determination of fullerenes in soil and sediment matrices using
ultra-high performance liquid chromatography coupled with heated
electrospray quadrupole time of flight mass spectrometry. Journal of
Chromatography A, 1433, 123–130.
54
Abstract
The increasing production of fullerenes likely means a release of these
chemicals in the environment. Since soils and sediments are expected to act
as a sink, analytical tools are needed to assess the presence of fullerenes in
these matrices. In the present work, a method was developed for the
determination of fullerenes at environmental relevant levels employing
Ultra High Performance Liquid Chromatograph coupled with High
Resolution Mass Spectrometry (UHPLC-HRMS). Chromatographic
separation was achieved with a core-shell biphenyl stationary phase that
provided fast analysis with complete baseline separation. Ion Booster
Electro Spray Ionization (IB–ESI) resulted in higher ionization efficiency and
was much less susceptible to adduct formation in comparison with standard
ESI, whereas Quadrupole Time of Flight (QTOF) MS granted high resolution
mass spectra used for accurate identification. The Instrumental method
limits of detection (ILoD) and quantification (ILoQ) were 6 and 20 fg,
respectively, for C60 and 12 and 39 fg, respectively, for C70. Matrix effects
related to co-extractants were systematically investigated in soil and
sediments extracts through standard addition method (SAM) and
monitoring the signal response during the chromatographic run of these
samples. Consequently, minor chromatographic modifications were
necessary for the analysis of matrices with high organic carbon content. The
method limit of detection (MLoD) ranged from 84 pg/kg to 335 pg/kg,
whereas limit of quantification (MLoQ) ranged from 279 pg/kg to 1.1 ng/kg.
Furthermore, the method was successfully applied for the analysis of
functionalized fullerenes (i.e. methanofullerenes). To the best of our
knowledge, this is the first analytical method for the analysis of fullerenes in
soils and sediments that employ core-shell biphenyl stationary phase as well
as IB-ESI-QTOF MS hyphenated with UHPLC.
55
4.1 Introduction
Since their discovery (Kroto et al., 1985), fullerenes and especially C60 have
been subject of a large number of studies mostly focused on their
innovative physical-chemical properties and possible applications.
Fullerenes are already used in cosmetics (as antioxidants) and in
photovoltaics (as electron acceptors) and possible fields of application
include electronics and optics as well as biomedical engineering
(Tagmatarchis et al., 2001; Guldi et al., 2002; Burangulov et al., 2006; Kim
et al., Maynard et al., 2006). Furthermore, the possibility of derivatization
of their structure e.g. with surface functionalization (Hummelen et al.,
1995) as well as the encapsulation of other chemical species (Chai et al.,
1991), is likely to increases the range of applications of these chemicals.
Precise data regarding the manufactured amounts are missing but the
worldwide production was estimated in tens of tons per year and is
expected to increase in the near future (Hendren et al., 2011; Piccinno et
al., 2012). This will inevitably mean a release into the environment and
there are several concerns about their possible accumulation and toxic
effects. Besides, fullerenes can also be naturally produced (e.g. in energetic
events such as volcanic eruptions (Heymann et al., 1994) or flame
generation (Howard et al., 1991)) but data are limited and often
contradictory (Elsila et al., 2005). Thus, the presence of fullerenes in the
environment may be due to both natural and anthropogenic origins as well
as resulting from transformation of engineered nanoparticles (e.g. fullerene
derivatives). In this context, fullerenes are nowadays considered as
emerging contaminants and analytical methods are needed for both
environmental and toxicological assessment. However, only in the last few
years have environmental monitoring and modeling studies been carried
out and the knowledge is still limited. Data modeled based on the estimated
production suggested that the concentrations (Predicted Environmental
Concentration, PEC) of C60 in the environment should range between parts
per billion and parts per trillion with higher concentrations in soil and
sediments than water and air (Gottschalk et al., 2009). Thus, soil may act as
a sink for the fullerenes whose source may be direct release and
atmospheric deposition but also water transport (in the form of nC60 and/or
56
associated with organic matter) and application of biosolids (Navarro et al.,
2013). In the last few years, a number of studies have been published
focused on the extraction of fullerenes and methods are now available that
allow the extraction of these chemicals in a large number of matrices with
acceptable if not excellent recoveries (e.g. Carboni et al., 2013; Kolkman et
al., 2013; Perez et al., 2013). High Performance Liquid Chromatography
(HPLC) in combination with UV-vis and/or mass spectrometry (MS) is the
most widely used technique for the determination of fullerenes and issues
related to the chromatographic separation of the fullerenes have been
recently reviewed elsewhere (Saito et al., 2004; Astefanei et al., 2014a).
Briefly, mobile phases usually consisted of toluene as a main eluent in
combination with a more polar solvent, such as acetonitrile or methanol,
whereas stationary phases commonly employed include standard octadecyl
silica (ODS) as well as functionalized silica. However, these methods are
often time consuming and seldom optimized for the analysis of complex
environmental samples (e.g. Carboni et al., 2013; Kolkman et al., 2013). In
this context, core-shell biphenyl is a novel stationary phase, never applied
for the analysis of fullerenes, which may provide a retention mechanism
similar to that of the pyrenylpropyl silica (Lomas et al., 2015). Regarding the
detection, although UV-vis showed promising performance (Wang et al.,
2010) for the determination of fullerenes in environmental matrices
(Carboni et al., 2013), its relatively low sensitivity and its lack of selectivity
in comparison with mass spectrometric detection make it unsuitable for the
analysis of the low concentrations expected in the environment. Thus, mass
spectrometry is considered as the detection method of choice and low as
well as high resolution methods have been described. Instruments
commonly employed make use of triple quadrupole (Sanchis et al., 2013;
Astefanei et al., 2014b), LTQ Orbitrap (van Wezel et al., 2011; Nunez et al.,
2012; Kolkman et al., 2013; Emke et al., 2015), and FTICR (Nunez et al.,
2012) coupled with HPLC systems and Quadrupole time of flight (QTOF) MS
with direct injection (Song et al., 2007). Atmospheric pressure ionization
(API) methods were usually employed in negative mode. Specifically, the
use of ESI in negative mode allowed the detection of fullerenes in soils and
water at relatively low concentrations (Kolkman et al., 2013; Sanchis et al.,
2013) but APPI was recently proposed as a better option due to the higher
sensitivity and less complex mass spectra (Nunez et al., 2012; Astefanei et
57
al., 2014b; Emke et al., 2015). Heated interfaces such as H-ESI were seldom
applied and showed efficiency of ionization generally higher than that of
standard ESI, but lower in comparison with other systems such as
atmospheric pressure photo (APPI) and chemical (APCI) ionizaton (Nunez et
al., 2012; Astefanei et al., 2014b). In this context, the Ion Booster-ESI (IB-
ESI) consists of a modified ESI source that was recently employed in the
screening of drugs in serum, showing better performance than standard ESI
(Huppertz et al., 2014; Kempf et al., 2014). The additional soft voltage and
vaporizer temperature are expected to enhance the ionization of the
fullerenes in comparison with standard ESI, i.e. to improve the sensitivity.
The analysis of fullerenes in the environment is challenging because of the
low concentrations expected and only recently monitoring studies have
highlighted the presence of these chemicals in environmental samples (e.g.
soil (Sanchis et al., 2013 and 2015), sediments (Sanchis et al., 2013;
Astefanei et al., 2014b), surface waters (Nunez et al., 2012; Astefanei et al.,
2014b), wastewater (Farre et al., 2010; Emke et al., 2015) and air (Sanchis
et al., 2012)). Furthermore, the complexity of some of the matrices that
have to be analyzed (e.g. WWTPs, soil) is an issue during both the extraction
and analysis of fullerenes (Jehlicka et al., 2005). The presence of co-
extractants, with special regard to organic matter content, could hamper a
correct quantification and it may have hindered the detection in other
recent works focused on soil (Perez et al., 2013) and water (van Wezel et
al., 2011). To date a few studies addressed the challenges of a clean-up
procedure (Sanchis et al., 2013) or accounted for the matrix effect during
the detection and quantification of fullerenes. Although fortification (i.e.
standard addition) of environmental samples under investigation was
recently included in monitoring studies, this issue was never systematically
approached and analytical methodologies such as matrix matched
calibration were recommended (Astefanei et al., 2014b; Sanchis et al.,
2015). Thus, in the present work we developed an analytical method that is
specifically aimed for the analysis of fullerenes in complex environmental
samples such as soil and sediment. The method included the use of a novel
stationary phase, i.e. biphenyl functionalized silica, and of a heated
interface (IB-ESI) in combination with high resolution mass spectrometry
(QTOF-MS).
58
4.2 Materials and Methods
4.2.1 Reagents and chemicals
Toluene, methanol, isopropanol and acetonitrile (Biosolve, Valkenswaard,
The Netherlands) were all analytical grade (LC-MS). The mobile phase
modifiers tested were acetic acid (Merck, Darmstadt, Germany), formic acid
(Biosolve, Valkenswaard, The Netherlands) and ammonium acetate (Sigma-
Aldrich, Zwijndrecht, The Netherlands) whereas sodium hydroxide was
purchased by Merck KGaA (Amsterdam, The Netherlands). Fullerenes C60
(CAS: 99685-96-8) and C70 (CAS: 115383-22-7) were purchased from Sigma-
Aldrich (Zwijndrecht, The Netherlands). Stock solutions of the individual
fullerenes were prepared in toluene, at a concentration of 5 mg/L, and
placed on an orbital shaker (Laboshake orbital shaker, Gerhardt,
Königswinter, Germany) in the dark overnight. Further solutions needed for
the experiments, including mixtures of the fullerenes, were obtained by
dilution of the stock solutions, stored at 4°C in the dark and sonicated for 2
min before use (Bransonic 12, Branson, Danbury CT, United states). The
analyses were performed using an UHPLC system (Nexera, Shimadzu, Den
Bosch, The Netherlands) equipped with a binary pump, autosampler and
column oven. Retention of the chemicals was achieved with a core-shell
Kinetex 2.6 µm biphenyl 100 Å chromatographic column (Phenomenex,
Utrecht, The Netherlands) consisting of a biphenyl stationary phase. The
UHPLC system was coupled to a high resolution Quadrupole-Time of Flight
mass spectrometer (Q-TOF; maXis 4G upgraded with HD collision cell,
Bruker Daltonics, Wormer, The Netherlands) equipped with either an
electrospray (ESI) or an Ion Booster electro spray (IB-ESI) ionization sources
(Bruker Daltonics, Wormer, The Netherlands). High purity nitrogen was
supplied by a N2 generator (Avilo, Dirksland, The Netherlands) and used for
ionization and collision gas.
59
4.2.2 Analytical method settings
Internal mass calibration was performed automatically during each analysis
in order to assure good mass accuracy for all the samples independently of
the total analysis time. This was achieved at the beginning of the analysis
(0.1-0.4 min) by infusing a 2 mM sodium acetate solution in a water
isopropanol mixture (1:1, v:v), with a loop injection of 20 µl and a loop rinse
of 20 µl. A temperature gradient was employed for the ionization source
because of the different optimal temperatures needed for the analysis of
the mass calibration solution and the analytes under investigation.
Specifically, the temperature was set at 325°C at the beginning of the run
and was gradually increased after one minute in order to reach 450°C at 2
min (Figure B.1 in appendix B). The sodium acetate cluster provided ten
points of calibration ranging from m/z 387 to m/z 1207 (Table B.2 in
appendix B) of which at least eight points (standard deviation ≤ 0.3 ppm)
were taken for the mass calibration of each sample. The final
chromatographic methods were optimized with regard to the methanol
(eluent A) and toluene (eluent B) amounts employed and consisted of the
following programs. Method A: flow rate of 400 µl, 40 sec at 100% eluent A
(focusing step), a linear gradient up to 50% eluent B in 0.3 min, an isocratic
step of 3.50 min at 50% eluent B (elution step) and then a linear gradient of
20 sec to reach 100% eluent B which was maintained for 2.5 min (cleaning
step). Method B: flow rate of 600 µl starting with 40 sec at 100% eluent A
(focusing step), a linear gradient up to 35% eluent B in 20 sec, an isocratic
step of 4 min at 35% eluent B followed by a linear gradient of 1.5 min to
reach 60% B (elution step), a linear gradient of 10 sec in order to reach 100%
eluent B which was maintained for 2.5 min (cleaning step). The
chromatograms were divided into four segments: segment 1 (from 0 min to
0.1 min) to assure the correct position of the loop, segment 2 (from 0.1 min
to 0.4 min) dedicated to the mass calibration, segment 3 (from 0.4 min to
3.4 min in method A and 4.4 min in method B) for the SRM analysis of C60
(m/z 720.0005) and segment 4 (from 3.4 min to 7.6 min for method A and
from 4.4 min to 9.6 min for method B) for the SRM analysis of C70 (m/z
840.0005). After the mass calibration was achieved, the LC flow was
diverted to the waste (0.5 min) in order to avoid the exposure of the
interface to the more polar co-extractants that eluted at the beginning of
the chromatogram. The LC flow was redirected to the MS (1.5 min in
60
method A and 2.5 min in method B) to allow the interface to equilibrate
before the elution of the chemicals and then switched again to waste after
the elution of the analytes (4.5 min in method A and 6.5 min in method B).
Fullerenes ESI and IB-ESI spectra were obtained by infusing both individual
and mixture standard solutions in toluene:methanol (1:1,v:v) at varying
concentrations. The optimized working conditions for the ESI and IB-ESI in
negative mode were: capillary voltage 1000 V, end plate offset -400 V,
charging voltage 300 V, nebulizer gas 4.1, dry gas 3.0 l/min and dry heater
200°C. Further settings optimized for the MS analysis were: funnel radio
frequency (RF) at 325.0 Vpp (voltage point to point), Multipole RF at 300.0
Vpp, collision cell RF at 1600.0 Vpp, transfer time at 50 µs and prepulse
storage time at 25 µs. The mass range analyzed in MS1 was 300 – 2000 m/z
whereas the spectra rate frequency was set at 2 Hz during all the analysis
performed. Collision energy for MS/MS was set at 100 V whereas the
isolation width was 8.00 m/z. The present settings allowed a mass resolving
power up to 80000 (0.03 m/z FWHM). The [M]-•and [M+1]-• abundances
relative to C60 and C70 were determined by analysing a standard solution at
500 ng/L repeatedly with both method A (n=7) and method B (n=7). More
details about the molecular ions and isotopic clusters during the detection
are provided in the discussion section. The Bruker Compass 1.7 software
was employed in both the data collection and data processing.
4.2.3 Samples collection and treatment
In table 4.1, the properties of the soil and sediment samples included in the
present study are reported.
61
Table 4.1. Properties of the soil and sediment samples included in the present study.
n.d. not detected
The Loess soil (matrix 1) was collected from an agricultural field in South
Limburg, The Netherlands (50°53'58"N, 5°53'16"E). Matrix 2 consisted of an
urban park soil sample collected in Vondelpark, Amsterdam, The
Netherlands (52.3580° N, 4.8680° E). River clay and sea clay (matrix 3 and
matrix 4 respectively) were available from the collection of the Earth
Surface Science research group at the University of Amsterdam. The sandy
top soil rich in organic carbon (matrix 5) was collected in Flevopark,
Amsterdam, The Netherlands (52.3611° N, 4.9492° E) whereas the dune
sand (matrix 6) was sampled in a natural park near Castricum, The
Netherlands (52.5500° N, 4.6667° E). The samples were placed in a freezer
at -20°C overnight and lyophilized with a Scanvac Coolsafe freeze-dryer
(Labogene, Lynge, Denmark) in order to remove traces of water. The dried
samples were finely ground with an agate mortar and sieved. The total
carbon, sulfur and nitrogen contents of the dried soil samples were
measured using a Vario EL Cube (Elementar, Hanau, Germany). The
inorganic carbon content was determined as reported by Wesemael (1955)
as follows: 1 g of soil was weighed into a 250 ml Erlenmeyer provided with
a silica gel lid and an excess of HCl 4 M was added. Control samples
consisted of 0.250 g of CaCO3. The samples were weighed again after 24 h
of shaking at 60 rpm (Laboshake orbital shaker, Gerhardt, Königswinter,
Germany) and the inorganic carbon content was calculated accordingly. The
soil and sediment samples were extracted with the protocol already
62
reported by our group (Carboni et al., 2013) with minor modifications, i.e.
the temperature was set at 20 ± 2 °C as recommended by Perez et al. (Perez
et al., 2013). Briefly, 10 to 15 g of each sample underwent two cycles of
extraction with toluene with each cycle consisting of a combination of
sonication for 20 min with a ultrasonic bath (Bransonic 12, Branson,
Danbury CT, United states) and shaking for 2 h (Laboshake orbital shaker,
Gerhardt, Königswinter, Germany). The extracts were concentrated
evaporating the solvent under a gentle nitrogen flow (enrichment factor
20x) and stored in a refrigerator at 4 ºC in the dark. A repeatability test for
the extraction procedure was achieved by spiking and extracting the
fullerenes into matrix 6 at the concentration of 100 ng/kg (n=8) as reported
by Carboni et al. (2013).
4.2.4 Sample analysis
Methanol (25% in volume) was added to each sample and the analyses were
performed with an injection volume of 10 µl. External calibration curves
were obtained by analyzing standard solutions at concentrations ranging
from 1 to 500 ng/L and quantification was based on chromatographic peak
areas. Instrumental method limits of quantifications (ILOQs) were assumed
as the lowest measured concentration in the linear range with a deviation
less than 30% of the theoretical concentration injected (EC commission,
2009). The instrumental method limit of detection (ILoD) was calculated as
3/10 of the ILoQ observed. The method limits of detection (MLoDs) and
quantification (MLoQs) in soil and sediment samples were extrapolated
from the signal-to-noise ratio observed in the chromatograms assuming a
70% recovery of extraction (Carboni et al., 2013). In case of absence of
matrix effect and noise, these values were assumed to be equal to the ILoD
and ILoQ. The criteria used for the identification of the analytes were: 1) the
expected chromatographic retention time (± 0.2 min), 2) a mass accuracy
threshold (≤ 5 ppm) and 3) an isotopic pattern fit threshold (≤ 50 mSigma),
where mSigma represents the goodness of fit (the smaller the better)
between the measured and theoretical isotopic pattern (Gago-Ferrero et
al., 2015). For the standard addition method (SAM) experiments, each
63
matrix was spiked with both C60 and C70 fullerenes at increasing
concentrations (25, 50, 100, 250 and 500 ng/L) in order to obtain
concentration versus response curves (fig. B.3 and B.4 in appendix B).
Standard solutions at the same concentration were also analyzed to allow
a comparison in terms of linearity, slope and intercept. In addition, the ion
suppression/enhancement due to the matrix effect was evaluated in terms
of response recovery by comparing the peak areas obtained for C60 and C70
spiked into the extracts with those obtained analyzing standard solutions at
the same concentrations. The matrix effect (ME) was further investigated
by continuously infusing a fullerenes standard (1 µg/L in toluene) into the
MS source while injecting the matrices extracts. This allowed us to monitor
the detector response during the chromatographic run, i.e. the matrix
suppression/enhancement during the analysis. For this purpose a 5 ml glass
chromatographic syringe and syringe pumps (KD Scientific, Holliston MA,
United States) operating at 0.2 ml/h were employed.
4.3 Results and discussion
4.3.1 UHPLC-HRMS
The first aim of the present work was to assess the performance of the
biphenyl stationary phase selected for the separation of the fullerenes.
When method A was applied, C60 and C70 eluted at 3.1 min and 3.8 min
respectively (fig. 4.1) with fully resolved (Rs>1.5) and highly symmetric
chromatographic peaks.
64
Fig. 4.1. Chromatographic separation of C60 (left) and C70 (right) standard at
500 ng/L. The continuous and dashed lines represent the MS1 and MS2
chromatograms respectively.
Similar to what is observed with columns that are specifically designed for
the separation of fullerenes, e.g. the pyrenylpropyl functionalized silica
(Buckyprep), with biphenyl groups the main interactions are expected to be
the pi-pi-interactions and pi-stacking between the aromatic rings in the
fullerenes and those of the stationary phase. However, as suggested by
Nunez et al. (2012) the size of the buckyballs may play a role in the retention
mechanism. The biphenyl stationary phase was an optimal compromise for
selectivity and rapidity when compared with other materials that were
usually employed providing higher retention than the standard octadecyl
silica and shorter analysis time in comparison with the pyrenylpropyl
functionalized silica. It must be noticed that the analysis time may be
shortened when using a shorter column with smaller particle size (i.e. 1.7
µm) that is currently available on the market. The “focusing step” i.e. the
application of a 100% methanol at the beginning of the chromatographic
run, resulted in a better peak shape and sensitivity and may be explained
with the fact that the analytes accumulated at the start of the column
(focused) until a stronger solvent was provided. The use of ESI-HRMS was
already investigated by some of the authors of the present work (Kolkman
65
et al., 2013). In those studies, a consistent formation of adducts during the
ionization process was reported, consisting of both oxidized products ([M +
O]-•, [M + OH] -•) and methanol and/or toluene adducts (mostly [M + OCH3]-
• and [M + C7H7O2]-•). In contrast, IB-ESI-HRMS provided mass spectra that
were dominated by the isotope cluster of the molecular ions with an only
minor abundance of [M+16]-• adducts as shown in figure 4.2. Similar to what
has already been reported in other studies, the method was more sensitive
for C60 than C70 (a factor of 2) at all the concentrations tested. Furthermore,
in the present study the IB-ESI signal response was several orders of
magnitudes higher than that of ESI stand-alone for both C60 and C70 (data
not shown). Thus, ESI was not taken into consideration for further analysis.
In the present work, sodium acetate was selected for internal mass
calibration because it can stand higher temperatures in comparison with
other commonly applied calibration solutions (e.g. sodium formate). This
allowed the determination of the accurate masses of fullerenes with errors
lower than 5 ppm. In comparison with APPI, where toluene was used as a
dopant to improve the ionization efficiency, ESI analysis required the
presence of methanol during ionization that can be added post-column as
already described (Kolkman et al., 2013). The post column addition was not
needed in the present work since methanol was one of the constituents in
the mobile phase during the elution of the chemicals. It must be noted that
fragmentation of the fullerenes could not be obtained even at extremely
high collision energies (> 100 eV). Thus, although multiple reaction
monitoring (MRM) mode was applied in the present work, the MS2 analysis
corresponded to [M]-•[M]-• transitions (pseudo-MRM) and the MS2
spectra resembled the MS1 ones (fig. 4.2b and 4.2e) in which adducts and
background masses (e.g. m/z 731.4124 in fig 4.2a) were removed.
66
Fig. 4.2. (IB)ESI-QTOF mass spectra of C60 and C70 MS (a and d respectively) and MS2 (b and e respectively). In fig. 4.2c (C60) and 4.2f (C70) a comparison between measured (top) and expected (bottom) m/z clusters.
Nunez et al. (2012) reported higher than expected relative abundances of
the isotopic cluster ions when using APPI with special regard to the m/z
[M+1]-• and an enhanced effect on larger fullerenes (Nunez et al., 2012). In
their work they demonstrated that the addition of hydrogenated products
to the peaks corresponding to the 13C natural abundance may account for
the phenomenon. Furthermore, Emke et al. (2015) showed that the
unexpected isotopic distribution was due to the presence of methanol
during the ionization, a phenomenon not observed when toluene was the
only solvent employed. If highly resolving systems, such as FTICR MS, are
not available, anomalous isotopic abundances may hinder a qualitative
detection of fullerenes. Especially when analyzing non-functionalized
structures, such as C60 and C70, the lack of MS/MS fragmentation implies
that the main identification points must be provided by the accurate mass
and cluster distribution. In the present work the C60 spectra presented
67
molecular ions at m/z [M]-•, [M+1]-•, [M+2]-• and [M+3]-• consistent with the
theoretical isotopic patterns (fig. 4.1c) and those obtained by ESI, whereas
C70 showed slightly higher than expected m/z [M+1]-• abundance in MS1 fig
(4.2a and 4.2f). In detail, C60 presented a [M+1]-•/ [M]-• ratio of 0.64 ± 0.05
(versus the expected value of 0.65) whereas C70 presented a ratio of 0.77 ±
0.03 (versus the 0.74 expected).
Identification points in the present work were provided by the
chromatographic retention times and the accurate mass detection and
isotopic clusters in both MS1 and MS2. Calibration curves, obtained
analyzing standard solutions of the fullerenes, showed high linearity
(R2>0.998) and the ILoD and ILoQ were 0.6 and 2 ng/L, respectively, for C60
and 1.2 and 4 ng/L, respectively, for C70. These results indicate that the IB-
ESI-HRMS system allowed the determination of fullerenes at concentrations
lower than those of other ESI-MS and heated ESI methods applied (van
Wezel et al., 2011) and similar to those of APPI-MS methods recently
developed (Astefanei et al., 2014b). Finally, the method was also tested for
the determination of fullerene derivatives to assess the possibility to
include functionalized structures investigated in our previous works
(Kolkman et al., 2013; Carboni et al., 2013). The method was suitable for the
analysis of the methanofullerenes that were completely resolved (Rs > 1.5)
from the related non functionalized structures at the beginning of the
chromatogram (Fig. B.5 in appendix B).
4.3.2 Matrix effects
The method we developed was tested for the determination of fullerenes
spiked into extracts of soils and sediments. Soil and sediment samples were
chosen in order to represent a range of possible environmental matrices
and textures (e.g. clay, sand) and the selection included samples with
varying inorganic and organic carbon contents whereas nitrogen and sulfur
concentrations were similar between the samples (table 4.1). After the
extraction, the sample extracts were spiked with fullerenes in order to
investigate the analysis of the chemicals in presence of the co-extractants.
The use of fullerene standards in organic solvent for the spiking of samples
has often been the subject of debate since it is not expected to represent
68
the real conditions at which the chemicals are present in the environment
(Shareef et al., 2010; Carboni et al., 2013). However, in the present work,
standard solutions in toluene were directly added to the toluene extracts,
thus the results hereby presented will help in understanding the behavior
of fullerenes that are already present in the extracts with no effect of the
extraction procedure applied to the original sample. According to a previous
study, the texture of the soil samples should be of a minor concern when
analyzing fullerenes (Carboni et al., 2013). On the contrary, the presence of
carbon and specifically organic matter in the samples is expected to play a
major role, e.g. by hindering the detection and quantification of the
chemicals (matrix effects, ME). The ME may either cause the enhancement
or the decrease of the method’s accuracy and sensitivity. It must be noted
that during the sample preparation, an addition of methanol at percentages
higher than 25% of the final volume caused precipitation in the extracts
with the highest carbon content (matrix 2). This was due to the large
content of non-polar co-extractants resulting from the extraction with
100% toluene. Therefore, although the initial optimization was achieved
with a toluene:methanol ratio of 1:1 (v:v), the final composition of each
sample injected into the UHPLC was a toluene:methanol ratio of 3:1 (v:v).
The high collision energy values (100 V) applied during the analysis led to a
“Background clean-up”, i.e. the degradation of the co-extractants, with
resulting extremely clean MS2 spectra. This allowed the unambiguous
determination of the fullerenes in all the samples, even at concentrations
as low as 25 ng/L (fig. 4.3).
69
Fig. 4.3. MS1 (top) and MS2 (bottom) spectra of C60 spiked at the concentration of 25 ng/L in presence of high organic carbon content (matrix 2).
The results of the SAM experiments are reported in Table 4.2 and more
comprehensively in Appendix B (Table B.6). The response was linear (R2 >
0.99) for both C60 and C70 in all the matrices tested (with the only exception
of C60 in matrix 5 when analyzed in MS2), and no significant difference
(P>0.05, F-test for covariances) was observed between MS1 and MS2
measurements in the same matrix. It should be noted that, although
relative small intercepts were observed, these were not significantly
different from zero (P>0.05).
70
Table 4.2. Results of the standard addition experiment for C60 and C70 with the use of the method A (left) and method B (right)a. For matrix descriptions, see section 2.3
a r2 = regression coefficient of the standard addition lines; Rec% = recovery; Matrix 2 dil 10x = matrix 2 diluted ten times; method A and method B differ in the mobile phase composition during the isocratic step (50:50,MeOH:Tol and 65:35,MeOH, respectively) and flow rate (400 and 600 ul/min, respectively).
However, a significant difference (P<0.05) was present when comparing the
response recovery of different samples. Indeed, although the majority of
the matrices showed recoveries ranging from 100% to 122%, i.e. featuring
either no ME or a slight enhancement of the response, a large signal
suppression (specifically 6% recovery for C60 and 29% recovery for C70) was
found in the matrix with the highest carbon content (matrix 2). This effect
was not observed in other matrices with a high organic carbon content
(matrix 4 and matrix 5) indicating that the percentage of organic matter in
the samples was not the only property determining the ion suppression and
that other feature such as its chemical composition may play a role.
However, when the extract from matrix 2 was diluted ten times (matrix 2
dil 10x in table 4.2), an enhancement of the response was found, which is
consistent with that observed in matrices that have a lower organic carbon
content. This suggests that although the chemical composition of the co-
71
extractants played a role in the ion suppression, their concentration was
likely the main reason for the response loss observed in the extract of
matrix 2. The MLoDs were extrapolated assuming a 70% recovery of
extraction from the matrices (see materials and methods) and ranged from
84 pg/kg for C60 to 168 pg/kg for C70 whereas MLoQs ranged from 279 pg/kg
for C60 to 559 pg/kg for C70 (method A). However, in the case of matrix 2,
which showed a strong matrix effect, these values were a factor of 30 higher
for C60 and a factor of 5 higher for C70. The smaller suppression of the C70
signal in comparison with C60 may be explained by the fact that, being more
retained during the analysis, C70 eluted in a position of the chromatogram
that was less affected by the more polar co-extractants. The matrix effect
due to the co-extractants in the samples was monitored by injecting the
matrices while infusing the fullerenes in the MS system. The signal
suppression was due to the more polar fraction of the co-extractants,
eluting at the beginning of the chromatographic run where a higher
percentage of methanol was present.
Fig. 4.4. Response of C60 post-column infused directly into the IB-ESI-MS during chromatographic runs of the soil or sediments extracts using a gradient elution of methanol:toluene (method A). The black line represents a pure solvent injection in comparison with matrix 1 (light blue), matrix 2 (red), matrix 3 (orange), matrix 4 (yellow), matrix 5 (blue) and matrix 6 (green). The start of section b corresponds to the moment the gradient reaches 100% toluene, at the start of c the gradient program returns to initial solvent composition (see 6.2.2).
72
In particular, as shown in Figure 4.4, the matrix 2 (red line) suffered a large
signal suppression at the beginning of the analysis (Fig. 4.4a) whereas
signals from other matrices with a high organic carbon content were not
suppressed (matrix 4 in yellow) or were affected to a lesser extent (matrix
5 blue line). Flushing with toluene (100%) (Fig. 4.4b), in order to remove the
more non-polar fraction that may remain in the column and affect the next
injections, ultimately restored the response to the maximum levels as
shown in Figure 4.4c. During the flushing stage of the chromatogram (b),
however, no signal was present because of the absence of any methanol
and consequent lack of ionization of the fullerenes (Fig. 4.4b) in the ESI.
Matrices 2 and 5 were selected for further experiments aimed to improve
the analytical method for the determination of fullerenes in complex
matrices with mitigation of the ion suppression. Three mobile phase
modifiers: acetic acid, ammonium acetate and formic acid, were tested at
varying concentrations with the purpose of attenuating the ion
suppression. However, since the addition of formic acid resulted in a near
or complete loss of the response whereas both acetic acid and ammonium
acetate suppressed the signal to a large extent, these modifiers were not
taken into account for further analysis. The large suppression may be due
to the susceptibility of the ESI to buffer salts in comparison with APPI as
mentioned by Nunez et al. (2012). Also the modification of the mobile-
phase composition with increasing amounts of isopropanol and acetonitrile
did not lead to any improvement in recovering the response and, in the case
of acetonitrile, caused the formation of adducts at the m/z [M+40]-• for
both C60 and C70. Subsequently, the modification of the mobile phase
composition was tested with regards to the methanol:toluene ratio. In
detail, weakening the eluotropicity, i.e. increasing the amount of methanol,
delayed the elution of the fullerenes and enhanced the separation of
interfering compounds in the biphenyl column, with a general improvement
of the signal response (Fig. B.7 in appendix B). Optimal conditions were
found at 65:35 (methanol:toluene, v:v) whereas further addition of
methanol (e.g. 70:30, v:v) increased the retention time of the fullerenes and
broadened the peak, with no further enhancement of the response. Finally,
the original method (method A) was modified accordingly by changing the
methanol:toluene ratio in the isocratic step from 50:50 (method A) to 65:35
(method B). Further optimization of the methods consisted in the addition
73
of a washing step (100% toluene) at the end of the analysis and to divert
the LC flow to the waste in order to prevent the exposure of the source to
the co-extractants at the beginning of the analysis and provide higher
sensitivity and better peak shape of the fullerenes. In the resulting method
B, the retention times of the fullerenes were delayed, with C60 and C70
eluting at 4 and 6 min respectively (Fig. B.8 in appendix B) and the ILOD and
ILOQ were 12 and 39 fg, respectively, for C60 and 23 and 78 fg, respectively,
for C70. When method B was applied, a considerable improvement in the
response recovery of C60 was observed for the matrix 2 (from 6% to 67%)
and minor but consistent improvements were obtained for C70 (from 29 to
45%), as reported in Table 4.2. This corresponded to a MLoD and MLoQ of
168 pg/kg and 559 ng/kg, respectively, for C60 and 335 pg/kg and 1.1 ng/kg,
respectively, for C70. Furthermore, no significant difference (P<0.05) was
found when comparing the performances of the two methods for the
analysis of the other matrices. Eventually, we recommend the use of
method B (10 min) for monitoring studies, since it can provide a more
reliable measurement of the fullerenes in a wider range of matrices. In
particular, method B should be employed in the analysis of soils and
sediments as well as wastewater samples that can consist of complex
matrices possessing high organic carbon contents. However, method A can
provide a faster (8 min) and more sensitive analysis for the determination
of fullerenes in standard solutions and less complex samples such as sandy
soils with low organic carbon contents.
4.4 Conclusions
In the present work we developed a sensitive and fast method for the
determination of fullerenes in soils and sediments. The biphenyl-coated
stationary LC phase provides an adequate retention and represents a valid
alternative to other stationary phase materials currently applied with
promising perspectives for the analysis of complex mixtures including
functionalized fullerenes. We also showed how heated ESI, i.e. the IB-ESI
employed in the present work, can grant an ionization efficiency higher than
74
that of standard ESI and comparable to that of other recently developed
APPI methods, with the advantage of producing isotopic patterns that
resemble the theoretical ones. In general, the high resolution and the
clarity of the mass spectra allowed an unambiguous determination of the
fullerenes in all the samples under investigation, without the need of an
additional clean up, and at low concentrations that are environmentally
relevant. The matrix effect due to the presence of co-extractants was
investigated and the method is robust and flexible with regard to the
analysis of very complex matrices such as soils having high organic carbon
contents. However, the matrix effect and specifically the ion
suppression/enhancement during the analysis remains an issue and the use
of internal standards (e.g. isotopically labeled), if available, as well as matrix
matched calibration are recommended for a more precise determination.
Due to its favorable qualitative and quantitative features, the method
developed in the present study is a valid tool for the monitoring of
fullerenes in soils and sediments and for the study of the fate of these novel
contaminants at environmentally relevant concentrations.
75
Chapter 5
Analysis of fullerenes in soils
from The Netherlands
Submitted to Environmental Pollution as:
Carboni A., Helmus R, Emke E., van den Brink N., Parsons J. R., Kalbitz K. and
de Voogt P. Analysis of fullerenes in soils samples collected in The
Netherlands. Environmental Pollution (in revision).
76
Abstract
Fullerenes are carbon based nanoparticles that may enter the environment
as a consequence of both natural processes and human activities. Although
little is known about the presence of these chemicals in the environment,
recent studies suggested that soil may act as a sink. The aim of the present
work was to investigate the presence of fullerenes in soils collected in The
Netherlands. Samples (n=91) were taken from 6 locations, and included
highly trafficked and industrialized as well as urban and natural areas and
analyzed using a LC-QTOF-MS method. In general, C60 was the most
abundant species found in the environment and detected in almost a half of
the samples, at concentrations in the range of ng/kg. Other fullerenes such
as C70 and an unknown structure containing a C60 cage were detected to a
lower extent. The highest concentrations were found in the proximity of
combustion sites such as a coal power plant and an incinerator, suggesting
that the nanoparticles were unintentionally produced during combustions
processes and reached the soil through atmospheric deposition. Consistent
with other recent studies, these results show that fullerenes are widely
present in the environment and that the main route for their entrance may
be due to human activities. These data will be helpful in the understanding
the distribution of fullerenes in the environment and for the study of their
behaviour and fate in soil.
77
5.1 Introduction
Fullerenes are carbon based nanomaterials widely researched due to their
unique properties that make them useful for a large number of applications,
including electronics and optics as well as medicine and personal care
products (Murayama et al., 2004, Mauter et al., 2008, Osawa et al., 2002).
Moreover, the possibility to derivatize the closed cage carbon structure,
with consequent production of novel nanomaterials, widens the range of
uses of these chemicals (Hummelen et al., 1995). Although the annual
production is estimated to be tens of tons per year, a large increase is
expected in the near future (Hendren et al., 2011; Piccinno et al. 2012),
which is likely to imply higher emissions into the environment. Thus,
fullerenes are nowadays referred to as emerging contaminants and there is
concern about the environmental and health implications that may arise. In
particular, fullerenes may be released directly during production or they
may leach from materials as a consequence of their use and disposal.
Furthermore, they may be produced in combustion processes and
therefore associated with both natural phenomena (e.g. forest fires,
volcanic eruptions; Howard et al., 1991; Heymann et al., 1994) and human
activities such as industrial processes (e.g. as a byproduct during
combustion of carbonaceous materials) and transport (e.g. fuel
combustion; Murr et al., 2004). Little is known about the fate of these
chemicals once they end up in the environment. Although some studies
suggested that fullerenes may be degraded due to both biotic and abiotic
processes (e.g. Avanasi et al., 2014; Tiwari et al., 2014), others highlighted
the stability of C60 that may result in their accumulation in the environment
(Jehlicka et al., 2000; Parthasarathy et al., 2008). However, fullerenes have
been shown to affect soil organisms (e.g. earthworms, van der Plog et al.,
2011) and monitoring studies are crucial in order to provide valuable
information about the occurrence of these chemicals and to determine the
potential environmental risk associated with them. In general, pristine
structures such as C60 and C70 are expected to be more abundant in the
environment because of the higher production volumes, their natural
occurrence and formation resulting from the degradation of functionalized
structures. Indeed, several studies reported the presence C60 and C70
78
fullerenes in environmental matrices (Sanchis et al., 2012, 2013 and 2015;
Nunez et al., 2012; Astefanei et al., 2014b), whereas functionalized
structures (i.e. engineered nanomaterials) were seldom detected (Astefanei
et al., 2014b). Soil has been predicted to be a sink for fullerenes (Gottschalk
et al., 2009) that may enter the terrestrial environment via atmospheric
deposition, direct release, water transport and amendment of soils (Sanchis
et al., 2012; Navarro et al., 2013). However, most of the research so far has
been focused on water and wastewater samples (van Wezel et al., 2009;
Farré et al., 2011; Nunez et al., 2012; Kolkman et al., 2013; Astefanei et al.,
2014b; Emke et al., 2015) and data regarding the occurrence of fullerenes
in soil are limited to a few studies. In particular, Sanchis et al. (2013)
reported the presence of fullerenes in soils from Saudi Arabia associated
with the petroleum refinery activities in the area. In their study, C60 was the
only compound detected, and was found in 19% of the samples at
concentrations in the low µg/kg range. Fullerenes were detected to a larger
extent (67.6% of the agricultural soils and 91% of the urban soils) during a
monitoring study in Brazil where both C60 and C70 were found at
concentrations up to 154 ng/kg (Sanchis et al., 2015). In this case, their
presence was also correlated to combustion activities, particularly the
deposition of atmospheric particulate resulting from combustion processes.
Fullerenes were also found in aerosol particulates (Sanchis et al., 2011) and
urban atmosphere (Laitinen et al., 2014) as well as resulting from coal
combustion (Utsunomiya et al., 2002), suggesting that atmospheric
deposition could represent a main route for their entrance in the terrestrial
environment. In the present work, an environmental survey was carried out
with the aim of investigating the presence of fullerenes in soil samples
collected in The Netherlands. For this purpose, a method employing Ultra
High Performance Liquid Chromatography (UHPLC) coupled with High
Resolution Mass Spectrometric (HRMS) detection was used (Carboni et al.,
2016). The main goal was to investigate the presence of C60 and C70 in highly
trafficked and industrialized areas, i.e. those areas where the generation of
fullerenes may be related to human activities, in order to assess the
influence that these possible sources may have in the occurrence of these
chemicals in the environment. The selection of the samples was done in
order to represent diverse situations. Thus, soil samples were collected in
79
proximity of expected sources such as a coal power plant (Utsunomiya et
al., 2002) as well as in uncontaminated areas (i.e. natural park).
5.2 Materials and Methods
5.2.1 Reagents and chemicals
Toluene, methanol, isopropanol and acetonitrile (Biosolve, Valkenswaard,
The Netherlands) were all analytical grade (LC-MS). Fullerenes C60 (CAS:
99685-96-8) and C70 (CAS: 115383-22-7) standards were purchased by
Sigma-Aldrich (Steinheim, Germany). Stock solutions of the individual
fullerenes, at the concentration of 5 mg/L, were prepared in toluene and
placed on an orbital shaker (Laboshake orbital shaker, Gerhardt,
Königswinter, Germany) in the dark overnight. Further solutions needed for
the experiments, were obtained by dilution of the stock solutions, and
stored at 4°C in the dark and sonicated for 2 min before use (Bransonic 12,
Branson, Danbury CT, United States).
5.2.2. Sample collection and treatment
The surface soil samples (top 5 cm, at least 125 g each) were collected in
250 ml amber glass bottles. Each sample consisted of the composite of five
sub-samples (25-30 g each) collected at the corners and centre of a 1 m2
squared area. A total of 91 surface soils were sampled in 6 locations in The
Netherlands:
Location A was an urban area in proximity of a coal power plant in
Amsterdam (15 samples, labelled A_01 - A_15); Location B was a rural and
urban area nearby the AVR incinerator in Duiven (16 samples, labelled B_01
– B_16); Location C was an area surrounding the Amsterdam’s “Ring”
highway (26 samples, labelled C_01 – C_25); Location D was in green areas
(e.g. parks, flower beds) in the city of Amsterdam (16 samples, labelled
D_01 – D_16); Location E was a natural park situated near Castricum (6
80
samples, labelled E_01 – E_06) whereas Location F samples were collected
in proximity of a runway at the Eindhoven airport (12 samples, labelled F_01
– F_12). Additional information regarding the distance of the sampling
locations from the expected sources and their coordinates are reported in
the appendix C (Table C.1). The wind direction was assumed as the main
wind direction recorded during the three days before the sampling was
performed. The samples were placed in a freezer at -20 °C overnight and
lyophilized with a Scanvac Coolsafe freeze-dryer (Labogene, Lynge,
Denmark) in order to remove traces of water. The dried samples were
ground and homogenized with an agate mortar and sieved before
extraction with the protocol already reported by our group (Carboni et al.
2013) with minor modifications, i.e. the temperature was set at 20 ± 2 °C as
recommended by Perez et al. (Perez et al., 2013) and the extraction was
performed in 40 ml custom made glass vial in order to allow the extraction
of larger volumes. Briefly, circa 25 g of each sample underwent two cycles
of extraction with toluene with each cycle consisting of a combination of
sonication for 20 min with a Bransonic 12 ultrasonic bath (Branson, Danbury
CT, United states) and shaking for 2 h (Laboshake orbital shaker, Gerhardt,
Königswinter, Germany). Negative controls consisted of quartz sand
samples (25 g each) extracted and analyzed with the same procedure. The
extracts were stored in a refrigerator at 4 °C in the dark and sonicated for
30 sec before to be analyzed.
5.2.3 Sample analysis
The analyses were performed with the method recently developed in our
research group for the identification of fullerenes in soil and sediment
samples as described previously (Carboni et al., 2016), using an UHPLC
system (Nexera, Shimadzu, Den Bosch, The Netherlands) equipped with a
binary pump, autosampler and column oven. Retention of the chemicals
was achieved with a core-shell Kinetex 2.6 µm biphenyl 100 Å
chromatographic column (Phenomenex, Utrecht, the Netherlands)
consisting of a biphenyl stationary phase. The UHPLC system was coupled
to a high resolution Quadrupole-Time of Flight mass spectrometer (Q-TOF;
maXis 4G, Bruker Daltonics, Wormer, The Netherlands) equipped with an
Ion Booster electro spray (IB-ESI) ionization source (Bruker Daltonics,
81
Wormer, The Netherlands) as described elsewhere (Carboni et al. 2016).
High purity nitrogen was used as collision gas (Avilo, Dirksland, The
Netherlands). Automatic mass calibration was performed for each sample
by injecting a 2 mM sodium acetate solution at the beginning of the
chromatographic run. Additional information regarding the analytical set up
and operational parameters are reported in the appendix C.2. In addition to
the pristine fullerenes structures C60 and C70, the samples were screened for
the presence of six methanofullerenes, such as [60]PCBM and [60]bisPCBM,
as reported in Carboni et al. (2016). The criteria used for the identification
of the analytes were: 1) the expected chromatographic retention time (±
0.2 min), 2) a mass accuracy threshold (≤ 5 ppm) and 3) an isotopic pattern
fit threshold (≤ 50 mSigma), where mSigma represents the goodness of fit
(the smaller the better) between the measured and theoretical isotopic
pattern in both MS and MS/MS modes (Carboni et al., 2016). The analyses
were performed with an injection volume of 10 µl after adding methanol
(25% in volume) to each sample prior to injection. External calibration
curves were obtained after analyzing standard solutions at concentrations
ranging from 500 ng/L to 1 ng/L and quantification was based on
chromatographic peak areas. The Bruker Compass 1.7 software was
employed in both the data collection and data processing. The method
allowed the determination of C60 and C70 fullerenes in the samples with
method limit of detection (MLOD) of 67 and 134 pg/kg for C60 and C70,
respectively, and limits of quantification (MLOQ) of 223 and 446 pg/kg for
C60 and C70, respectively. The matrix effect, i.e. the suppression or
enhancement of the signal due to the presence of co-extractants in the
samples, was investigated by the addition of a C60 and C70 standard to the
extracts at the concentration of 25 ng/l (appendix C.3). In order to study the
unknown C60-like fullerene, a larger amount (100 g) of the samples from the
incinerator area (location B) were extracted to increase its concentration.
These samples were additionally analyzed with a modified UHPLC-IB-ESI-
QTOF method (appendix C.2). For statistical analysis, a linear model was
used that considered the relationship between the concentration of the
samples and the distance from the possible source. Further analyses were
conducted where the sample location with regard to the possible source
(i.e. upwind or downwind) were taken into account.
82
5.3 Results
The high accuracy (< 5 ppm), resolution (80000 FWHM) and clarity of the
MS/MS spectra allowed the unambiguous determination of the fullerenes
in the soil samples. The UHPLC chromatograms of the soil extracts (fig. 5.1b)
resembled those obtained with standard solutions (Fig. 5.1a) with slightly
higher background noise at the beginning of the chromatograms. Fullerenes
were found to be present in almost half of the soil samples analyzed and C60
was the most detected specie (48% of the samples). C70 fullerene was
detected less frequently (7% of the samples) and mostly associated with the
presence of C60 whereas methanofullerenes structures (e.g. [60]PCBM])
were not detected in any of the samples under investigation.
An additional C60 peak was found in some of the samples (fig. 5.1c) that
showed mass, isotopic pattern and MS/MS spectra similar to those of the
C60 standard. This suggested the presence of other fullerenes species in the
extracts. Since the chemical structure has not been unequivocally
elucidated, we will refer to this compound as “unknown”. Specifically, the
unknown was found only in samples collected in the incinerator area and
only if C60 was also present. The unknown had an accurate mass
corresponding to C60 but eluted earlier in UHPLC (at a retention time of 3.1
min) suggesting the presence of a C60-like species (e.g. a functionalized
fullerene) that, although separated chromatographically, underwent in-
source fragmentation during the ionization process (i.e. the loss of the
external functionalization) with formation of pristine C60. As shown in figure
5.1c, the addition of C60 standard to the original sample resulted in the
enhancement of the C60 peak area (at retention time 4 min) but had no
effect to the additional peak (at retention time 3.1 min), confirming that the
peak at 3.1 min is due to the presence of another C60-like species in the
samples. Modification of the analytical method with regard to the
chromatography as well as the mass spectrometric method (i.e. by
decreasing the temperature and/or the capillary voltage) were not able to
prevent the fragmentation of the unknown and had the only effect of
reducing the ionization efficiency. Eventually, the extraction of a larger
amount of samples, i.e. the analysis of more concentrated extracts, did not
lead to the identification of the unknown and did not show any presence of
possible precursors.
83
Fig. 5.1. Chromatographic peak and mass spectra of A) a C60 standard at concentration 8 ng/L, B) a soil sample extract from the highway (location C) and C) a sample from the incinerator area (location B) presenting an additional C60 peak at retention time 3.1 min. The black line represents the signal of the sample spiked with standard.
84
Samples collected close to the coal power plant (location A) revealed the
presence of fullerenes in 53% of the extracts. As shown in figure 5.2, C60 was
the compound detected most often at concentrations ranging from 0.20 to
1.44 ng/kg whereas C70 was found at similar concentrations but only in two
of the samples. A larger occurrence was found downwind of the powerplant
(samples A_7-A_15) at distance up to 1.8 km from the chimney whereas no
fullerenes were detected upwind in proximity of the powerplant (samples
A_1-A_3). However, the highest concentrations of both C60 and C70 was
found close to a coal storage site (sample A_6).
In the samples collected in the incinerator area (Location B), C60 was
detected in the majority of the samples (69%) at concentrations ranging
from 0.15 to 3.14 ng/kg (Fig. 5.3). Although C70 was not found in any of the
samples under investigation, an additional peak representing a C60-like
structure (the unknown product discussed above) was detected in 44% of
the samples and was always accompanied by the presence of C60. In the
case of location B, the highest concentrations were found in proximity of
the incinerator (sample A_1-A_3), whereas lower concentrations were
found mostly downwind in the surrounding area. Similar to what was
observed in the power plant location (Fig. 5.2), samples collected
immediately upwind of the chimney did not show any presence of
fullerenes (samples B_7-B_8).
Fullerenes were found in 62% of the samples collected in proximity of the
highway in Amsterdam (Location C). C60 was the most abundant species also
in this case (in 62% of the samples at concentrations between MLOQ and
0.77 ng/kg), whereas C70 was found in 8% of the samples. In the soils
collected in the Amsterdam urban area (location D), C60 was the only
fullerene found in 18% of the samples at concentrations ranging from 0.43
to 0.87 ng/kg. In contrast, no fullerenes were detected in the samples
collected in the natural area in Castricum (Location E) whereas samples
collected close to the runway of Eindhoven Airport (Location F) showed the
presence of fullerenes in 33% of the soils at concentrations usually below
the MLOQ. However, the only quantifiable samples had the relatively high
concentrations of 2.14 and 2.17 ng/kg for C60 and C70, respectively.
85
Fig. 5.2. Concentrations of fullerenes in the coal power plant area (location A) in Amsterdam. Top: concentration of C60 (orange) and C70 (blue) in the samples. Bottom: sampling locations and range of concentrations of C60. The star represents the powerplant’s main chimney.
86
Fig. 5.3. Concentrations of fullerenes in the incinerator area (location B) in Duiven. Top: concentration of C60 (orange) in the samples, C70 was not detected in any sample. Bottom: sampling locations and range of concentrations of C60. The star represents the powerplant’s main chimney.
87
Fig. 5.4. Concentrations of fullerenes in the highway area (location C) in Amsterdam. Top: concentration of C60 (orange) and C70 (blue) in the samples. Bottom: sampling locations and range of concentrations of C60.
88
5.4 Discussion
Fullerenes were present in almost a half of the surface soils sampled in the
present study and C60 was the species detected most often (48% of the
samples) in comparison with C70 (7%). However, it must be noted that
method detection limits of C70 were higher compared to C60, which may
have hindered the detection of lower concentrations of C70 in the samples.
The average concentrations, excluding the samples at concentrations below
the MLOQ, were 0.7 and 0.5 ng/kg for C60 and C70, respectively. In general,
a higher occurrence of fullerenes was observed in intensively trafficked and
industrialized areas (e.g. power plant, incinerator and in proximity of the
highway) where fullerenes were found in 61% of the samples in average.
The incinerator was the most contaminated area with regard to both the
occurrence (69% of the samples) and concentration (1.2 ng/kg of C60 in
average) whereas lower concentrations (< 1 ng/kg in average) were found
in the other locations sampled. Along the airport’s runway, the highest
occurrence of C60 was found in coincidence with the touch-down spot, thus
suggesting that the fullerenes may result from the contact of tires with the
runway during landing. In particular, the fullerenes may be directly released
from the tires or produced during the landing due to the high temperatures.
In general, our results are consistent with previous observations and
modeled data (Gottshalk et al., 2009) and suggest that C60 is the most
abundant fullerene species found in the environment at concentrations in
the range of ng/kg in soil. In particular, Sanchis et al. recently reported
similar results, with regard to both concentration and occurrence (Sanchis
et al., 2015), when monitoring trafficked and industrialized areas and
suggested that the more frequent occurrence of C60, at relatively higher
concentrations, may be due to its higher stability in the environment in
comparison with other pristine and functionalized structures (Sanchis et al.,
2013). It must be noted that, to the best of our knowledge, fullerenes are
not intentionally produced close to the sampled areas and that no products
containing fullerenes are intensively used or disposed in those areas.
Furthermore, no natural sources (e.g. forest fires) are known to the authors
that occurred during the last years in the sampling locations and that may
account for the presence of fullerenes in the surrounding soils. Therefore,
89
the presence of the fullerenes is likely due to human activities and
particularly the result of combustion processes involving waste, coal and
other fossil fuels or, in the case of the power plant, associated with the
presence of coal stocks on the soil. Thus, although it is not possible to
exclude an engineered origin, the fullerenes found in the present work were
likely unintentionally produced i.e. not manufactured nanomaterials.
Although higher concentrations and occurrence were usually found
downwind and close to the expected sources (e.g. incinerator or power
plant chimney), statistical analysis did not highlight either a significant
correlation between the concentration and the distance or a role of the
wind in the distribution of the fullerenes (P>0.05). However, it must be
noticed that, in the locations sampled, an assessment of the fullerenes
origin is complicated by the presence of multiple sources. To the best of our
knowledge no analytical method currently available is able to discriminate
between the several sources of the fullerenes detected in the environment.
In this context the ratio between fullerene species could be helpful in
understanding the origins, as different sources (e.g. coal or fossil fuel
combustion) may produce different ratios. In the present work, the
[C70]/[C60] ratio in the samples was relatively constant in both the location
A and C, with ratios of 68.9 ± 3.6 and 59.4 ± 8.8 percent, respectively.
However, the limited number of samples containing C70 did not allow a
more comprehensive comparison. The C60-like unknown specie was only
found in samples collected in the incinerator area which may be due to
either specific combustion processes (e.g. waste combustion) or to changes
in the chemical composition of pristine C60 after release in the environment.
Although characterization of the chemical was not possible in the present
study, the presence of a specific C60 chromatographic peak, at a retention
time different than that of the pristine C60, suggests the presence of a
functionalized-C60 structure that underwent an in-source fragmentation
during the ionization process. Furthermore, the unknown product is
apparently more polar than C60 as it eluted earlier than C60 in reverse-phase
chromatography. Since the functionalized fullerenes (i.e. the
methanofullerene [60]PCBM) do not undergo in-source fragmentation
when analyzed with the same method (Carboni et al., 2016), the energy
associated with the bonding of the external group in the unknown
compound is probably lower than that generally observed in the
90
commercially available functionalized fullerene derivatives. These
observations suggest that the unknown product may be a functionalized
fullerene such as the oxides recently characterized by Tiwari et al. (2014)
during the ozonation of C60 aerosol.
Finally, although the recovery of pristine C60 during the analysis may be
useful for the identification of unknown species in environmental samples,
an alternative method should be tested for their characterization. In
particular, atmospheric pressure ionization methods such as APPI may
provide similar ionization efficiency for the fullerenes (Emke et al., 2015),
but preventing or minimizing the in-source fragmentation of the
compounds.
5.5 Conclusions
The present work demonstrated the presence of fullerenes in the terrestrial
environment and especially in locations close to highly industrialized and
trafficked areas in Netherlands. Similarly to what has been reported by
previous studies in different locations, C60 was the most abundant fullerene
and was present at higher concentrations in comparison with other species.
However, the presence of an unknown C60-like structure(s), which may be
linked to either the unintentional release of different fullerene species or
the modification of pristine fullerenes in the environment, highlights both
the complexity in the environmental study of these chemicals and the need
for novel analytical strategies. Although an assessment of the origin of the
fullerenes is not possible with the current methodologies, we found no
indications that the level of soil contamination with fullerenes is related to
the intentional production and / or use of engineered nanomaterials at the
sites. Our results rather indicate that fullerenes are unintentionally
released, enter the environment particularly by combustion processes and
were likely deposited on the soil from the atmosphere. These results will be
helpful in the understanding of fullerenes occurrence and behaviour in the
environment and provide useful data for both modelling studies and for the
planning of forthcoming monitoring campaigns.
91
Chapter 6
Incubation of solid state C60
fullerene under environmentally
relevant conditions
Submitted to Chemosphere as:
Carboni A., Helmus R., Parsons J. R., Kalbitz K. and de Voogt P. Incubation of
Solid State C60 fullerenes under environmentally relevant conditions.
92
Abstract
Carbon-based nanomaterials, such as C60 fullerenes are expected to
accumulate in soil due to direct release and deposition from the
atmosphere. Although little is known about their environmental fate, these
nanoparticles may be susceptible to photochemical and microbial
degradation. In the present work, C60 was incubated for a period of 28 days
and irradiated with UVA light. Three experiments were carried out where
the fullerenes were either spiked onto a glass surface or added to quartz
sand or sandy soil samples. At specific time intervals samples were extracted
and analysed by liquid chromatography coupled to UV or high resolution
mass spectrometric (HRMS) detection. The fullerenes were degraded in all
the treatments and the decay followed a pseudo-first-order rate law. In
absence of a solid matrix, the half-life (t1⁄2) of the C60 was 13.1 days, with an
overall degradation of 45.1% that was accompanied by the formation of
functionalized C60-like structures. Furthermore, mass spectrometric analysis
highlighted the presence of a large number of fulleroid products that were
not directly related to the irradiation and presented opened cage and
oxidised structures. When C60 was spiked into solid matrices the
degradation occurred at a faster rate (t1⁄2 of 4.5 and 0.8 days for quartz sand
and sandy soil, respectively). Minor but consisted losses were found in the
non-irradiated samples, presumably due to biotic or chemical processes in
these samples. The results of this study suggest that light-mediated
transformation of the fullerenes will occur in the environment but that an
accurate assessment of their fate is complicated by the large number of
products that may derive.
93
6.1 Introduction
Fullerenes, including C60, are a class of carbonaceous nanomaterials
displaying a closed-cage structure composed of five- and six-membered
rings of carbon (Kroto et al., 1985). Due to their innovative properties,
production and application of these chemicals is developing rapidly and
fullerene-based consumer products are already commercially available
(Hendren et al., 2011; Maynard et al., 2012). Increased production and
application will inevitably mean an increased release into the environment
and there is concern for the possible negative effects on humans and the
ecosystem. Although fullerenes can occur naturally (e.g. lightning strikes,
wildfires, e.g. Howard et al., 1991) or be unintentionally produced, (e.g. as
by-products of combustion or during oil refinery, e.g. Utsunomiya et al.,
2002; Tiwari et al., 2016), due to the large scale production, the potential
release of purposefully manufactured nanomaterials is expected to play a
major role in determining the occurrence of these novel contaminants in
the environment. In this context, C60 released in the environment is
expected to accumulate in soil more than in water and the atmosphere
(Gottschalk et al., 2009). In particular, fullerenes may enter the terrestrial
environment directly as a consequence of their use and disposal as well as
due to amendment of soil with bio-solids (Navarro et al., 2013) and
indirectly by deposition from the atmosphere (Laitinen et al., 2014; Tiwari
et al., 2016). Although little is known about their environmental fate,
fullerenes may undergo functionalization, polymerization, degradation and
mineralization reactions due to the interaction with ozone, biota and solar
radiation (Panina et al., 1997; Lee et al., 2009; Avanasi et al., 2013; Tiwari
et al., 2014). With regard to their photochemistry, fullerenes are well
known to be photosensitive and to absorb light in the UV range (Carboni et
al., 2013). In particular, interaction of the fullerenes with UV-A light, which
represent the main UV irradiation reaching the Earth surface, will likely play
a major role in determining their environmental fate (Hwang et al., 2010).
Previous studies have shown that C60 dissolved in organic solvent (e.g.
benzene) undergo photo-oxidation with consequent formation of epoxides,
oxides and more polar unidentified products (Taylor et al., 1991; Wood et
al., 1991; Cregan et al., 1992), whereas photo-polymerization was observed
94
under oxygen-limiting conditions (Sun et al., 1995). Oxidative pathways
were also observed when C60 was dissolved in water (i.e. in the form of
aqueous nano-aggregates, nC60) and irradiated by UV-A (Hwang et al.,
2010), UV-C (Lee et al., 2009) or sunlight (Hou et al., 2009). In particular,
studies carried out at environmentally relevant conditions reported half-
lives from 19 to 41 h and surface functionalization of C60 (oxygenation and
hydroxylation) as well as the formation of unidentified water soluble
intermediates. However, most of the research was focused on fullerenes
dissolved in solvents. To the best of our knowledge, no research was
conducted yet that irradiates C60 nanoparticles dispersed onto a surface or
into a solid matrix (e.g. soil), which may represent the main form in which
these nanoparticles are present in the environment. The characterization of
transformation pathways for fullerenes is challenging because of the large
number of possible products that they may create (e.g. polymerization,
functionalization and cage break-down) (Taylor et al., 1991; Hwang et al.,
2010) and possible strategies have been recently reviewed by Pycke et al.
(2012). In particular, spectroscopic detection is an effective tool for the
analysis of fullerenes, due to the strong absorptivity of these chemicals in
the UV range and the lack of selectivity that may help in the identification
of the totality of the species in a sample (Carboni et al., 2013). Mass
spectrometric analysis instead can provide structural information needed
for the identification of transformation products resulting from
fragmentation (Lee et al., 2009) and oxidation (Tiwari et al., 2014). In this
context, high resolution mass spectrometry (HRMS) was successfully
employed for the detection of fullerenes and functionalized fullerenes, also
in environmental matrices (Astefanei et al., 2014b). In the present work, the
fate of fullerenes at environmentally relevant conditions was studied by
incubating C60 for a period of 28 days. The fullerenes were spiked in soild
matrices and the effect of UVA light irradiation was investigated.
95
6.2 Materials and Methods
6.2.1 Reagents and chemicals
Toluene (analytical grade), methanol (ULC/MS grade) and acetonitrile (LC-
MS grade) were obtained from Biosolve B.V. (Valkenswaard, The
Netherlands). Fullerenes C60 (CAS: 99685-96-8) and C70 (CAS: 115383-22-7)
were purchased by Sigma-Aldrich (Steinheim, Germany). Stock solutions of
the individual fullerenes were prepared in toluene and placed on an orbital
shaker (Laboshake orbital shaker, Gerhardt, Königswinter, Germany) in the
dark overnight. Further solutions needed for the experiments were
obtained by dilution of the stock solutions, stored at 4°C in the dark and
sonicated for 2 min before use (Bransonic 12, Branson, Danbury CT, United
States). Quartz sand (silicon dioxide, SiO2) was purchased from Sigma-
Aldrich (Zwijndrecht, the Netherlands). The sandy soil consisted of a top-
soil (top 10 cm) collected in Oude Schulpweg, Castricum, the Netherlands
(52° 32’ 39.689”N, 4° 39’ 5.623”E). The soil was placed in an oven at 65 °C
for one week in order to remove traces of water and then sieved with a 1.68
mm mesh.
6.2.2 Sample preparation and incubation
The sample preparation took place into a fume hood, and the laboratory
windows were equipped with UV-filters. For the preparation of the samples,
100 µl of a C60 solution in toluene (10 mg/L) was spiked at the centre of the
glass Petri dishes. Then the dishes were covered with the quartz domes
(custom made) and the solvent let dry for 30 min. Quartz sand and sandy
soil samples were prepared as following: 2 ml of a C60 solution in toluene
(20 mg/L) were spiked into 100 g of sample that was then homogenized by
stirring and let to dry. Then 300 g were added while stirring in order to
obtain a final mass of 400 g of sample at the concentration of 100 µg/kg of
C60. Eventually, 10 g were placed in the glass dishes, resulting in a sample
thickness of circa 1.5 mm. The incubation took place in a 1 m2 area provided
with three UV lamps (UVP, Keswick, Australia) set at 365 nm and placed at
a height of 80 cm. This resulted in a uniform irradiation of UVA light (350-
96
390 nm range) whose maximum intensity was 9 µW/cm2/nm at circa 360
nm (fig. D.1 in appendix D). The light measurements in the incubator were
achieved with the aid of a USB2000+ spectrometer and the Spectrasuite
software (both Ocean Optics, Duiven, The Netherlands). Non-irradiated
samples were placed into the incubator but covered with aluminium foil in
order to prevent exposure to light. The incubator was covered with a 100%
obscurant curtain and the temperature was held constant at 25 ± 0.5 °C.
6.2.3 Extraction and analysis
The samples (n=3) from each treatment were extracted at the beginning of
the experiment and at specific time intervals of 1, 3, 7, 14 and 28 d as
following: the samples were taken out of the incubator and 50 µl of a C70
solution in toluene (internal standard, 10 mg/L) were added. After
equilibration time of 30 min, 5 ml of toluene was used for rinsing the petri
dishes and then transferred into a 10 ml vial. The procedure was repeated
twice. In the case of quartz sand and sandy soil, the samples were
homogenized by stirring after spiking of the internal standard and
underwent extraction with the protocol previously described by our group
(Carboni et al., 2013). The extracts were analysed with the HPLC-UV method
described in Carboni et al. (2013) with minor modifications. Briefly, the
method employed a pyrenylpropyl silica stationary phase (Buckyprep),
whereas the mobile phase consisted of toluene and acetonitrile. Elution
was achieved with an isocratic method employing a 80:20
toluene:acetonitrile mixture. Quantification was based on the
chromatogram peak areas as described in the appendix D.2. The UHPLC-
HRMS analyses were performed using an UHPLC system (Nexera, Shimadzu,
Den Bosch, The Netherlands) equipped with a binary pump, autosampler
and column oven. Retention of the chemicals was achieved with a core-shell
Kinetex 2.6 µm biphenyl 100 Å chromatographic column (Phenomenex,
Utrecht, the Netherlands) consisting of a biphenyl stationary phase whereas
methanol (A) and toluene (B) were used as mobile phase. A gentle elution
program was used to enhance separation of compounds with varying
polarity range: from 0 – 5 min B was kept at 0%, following a linear increase
to 75% at 21 min and a final holding step until 25 min. The flow rate was set
to 0.4 ml/min, whereas the column temperature was kept at 30°C. The MS
97
methodology was adopted from (Carboni et al., 2016). Mass detection was
carried out with a high resolution Quadrupole-Time of Flight mass
spectrometer (Q-TOF; maXis 4G equipped with HD collision cell, Bruker
Daltonics, Wormer, The Netherlands) coupled to the UHPLC system
described earlier. Compounds were ionized using an Ion Booster
electrospray ionization source (IB-ESI) operating in negative mode using the
following settings: capillary voltage 1000V, end plate offset -400V, charging
voltage 300V, dry heater 200°C, nebulizer gas 4.1 bar and dry gas 3 l/min.
Nitrogen was used for ionization and collision gas, and obtained from a N2
generator (Avilo, Dirksland, The Netherlands). Ion transfer settings were as
follows: funnel radio frequency (RF) 325 Vpp (voltage point to point) and
multipole RF 300 Vpp. Mass calibration was achieved as reported by
Carboni et al. (2016) with minor modifications and is described in the
appendix D.3. The collision cell RF, transfer time and prepulse storage time
were varied within four time segments throughout the analysis. The first
two segments were dedicate to low and high masses calibration,
respectively. Consequently, compounds eluting within the first ten minutes,
i.e. relative polar, were measured with settings optimized for low masses
(segment 3) whereas fulleroid and other very non-polar compounds eluting
after ten minutes were analysed with settings optimized for high masses
(segment 4). Mass spectra were recorded at 2 Hz with a range of 50 – 2000
m/z. Fragmentation data of the most intense peaks were automatically
acquired with Auto MS/MS mode using a maximum cycle time of 3 seconds.
Smart exclusion and active exclusion were enabled to limit acquisition of
continuous background and increase uniqueness of precursor selection,
respectively. More information regarding the MS settings for the segments
and Auto MS/MS are reported in the appendix D.4. An extensive list of
masses was compiled from background compounds found in solvent and
sample blanks , and set as exclusion masses (+/- 0.05 Da window) to
improve quality of mass spectra. Unknown transformation products (TPs)
in a sample were screened and identified according a five step procedure.
First, a list of chromatographic peaks of interest was compiled from manual
inspection of base peak chromatograms and by use of the software based
Auto MS/MS peak finder. Secondly, for each peak of interest, a list of
masses of interest was manually generated from averaged mass spectra.
For these masses, an extracted ion chromatogram (EIC) was generated to
98
verify its origin from the chromatographic peak. Thirdly, a candidate list of
chemical formulas was created based on accurate mass (≤ 5 ppm deviation)
and isotopic fit (≤ 50 mSigma) with SmartFormula. Fourthly, candidates
were removed if present in solvent or sample blanks. Finally, tentative
identification of candidates was performed from MS/MS fragmentation
data, if present. Mass spectral data were processed with DataAnalysis 4.3
(Bruker Daltonic, Wormerveer, The Netherlands).
6.3 Results and discussion
6.3.1 Incubation
Losses of C60 fullerenes occurred during irradiation with UV-A light in all the
experiments performed, as shown in fig. 6.1. Furthermore, similar to what
was observed by Hwang et al. (2010) when irradiating aqueous C60 with UV-
A light, the decay of the fullerenes followed a pseudo first order reaction
rate in all the irradiated treatments (r2 ≥ 0.85, Table 6.1).
Table 6.1. Degradation characteristics derived from photolysis experiments with C60 fullerene. The results of the UVA irradiated and non-irradiated incubations are reported on the left and right side of the table, respectively.
t½ : half-lives, k: pseudo-first-order reaction constant, r2: regression coefficient.
When the C60 was added onto glass dishes (“glass” in the further
manuscript), degradation of the fullerenes started after 3 d and a total loss
of 45.1% was observed after 28 d of incubation (fig. 6.1a). The negligible
99
losses in the non-irradiated glass samples indicate that, similar to what was
previously reported in water (Hwang et al., 2010; Hou et al., 2009) and
organic solvents (Taylor et al., 1991), the degradation of the C60 was due to
a photochemical process. These results suggest that fullerenes released in
the environment will interact with the ultraviolet portion of the solar
radiation and will be degraded as a result. However, it must be noted that
the half-life of 13.1 d in the glass incubation was much longer than those
previously reported for the dissolved C60, estimated in the range of 10-40 h
(Hou et al., 2009; Taylor et al., 1991), and could be explained with a lower
reactivity of the fullerenes in the solid state in comparison with the
dissolved structures or a role of the solvent in the process.
When C60 was spiked into solid matrices, the observed losses in the
irradiated samples were higher and reached 88.1% and 100% for quartz
sand and sandy soil, respectively (fig. 6.1b and 1c), at the end of the
incubation. These correspond to half-lives of 4.5 and 0.8 days, respectively
(Table 6.1), and suggest that other processes took place, in addition to the
irradiation, that enhanced the degradation in these samples. Especially in
the sandy soil samples, the matrix-enhanced loss may be explained with a
biotic degradation due to microorganisms present in the soil or by other
processes such as a matrix-related photosensitisation (e.g. due to organic
matter). Thus, these results indicate that once deposited onto soil, the fate
of fullerenes will be mostly determined by factors other than the
irradiation. This is supported by the fact that, in contrast to the glass
incubation, the degradation of the fullerenes started at the beginning of the
incubation and that consistent losses over time were found also in absence
of light (Table 6.1). In particular, also the decay of C60 in the non-irradiated
samples followed a pseudo first order reaction rate (r2 ≥ 0.80) with half-lives
of 14.3 and 6.7 days for quartz sand and sandy soil, respectively.
100
Fig. 6.1. Concentration of C60 fullerenes over time in (a) fullerenes dried on top of a glass surface (“glass”), (b) spiked into quartz sand and (c) spiked into sandy soil at the concentration of 100 µg/kg. The red line represents the sample irradiated with UV-A light whereas the blue line represents the samples incubated in the dark.
101
6.3.2 Transformation products
6.3.2.1 C60-like products
In contrast to the HPLC-UV chromatograms, that were dominated by the C60
and C70 peaks (fig. D.5 in appendix D), UHPLC-HRMS chromatograms
revealed the presence of additional peaks (fig. 6.2a). In particular, three
structures, namely “C60-I”, “C60-II” and “C60-III”, showed accurate masses
and isotopic cluster distributions equal to that of C60, but eluted later at
retention times 19.2, 20.0 and 22.5 min, respectively (fig 6.2a and 6.2c).
Fig. 6.2. UHPLC-HRMS analysis of glass incubation samples irradiated with UV-A light. (a) Chromatogram presenting the C60 peak at retention time 15 min and smaller C60-containing peaks (C60-1, C60-II and C60-III) eluting later. (b) Several unknowns peaks eluting before C60 (blue frame). (c) A zoom of the 18-24 min range (red frame), highlighting the presence of C60-containing structures.
102
These products could not be characterized in the present work and their
composition has not been elucidated yet. However, the detection of a C60
cage structure suggests the presence of surface functionalized fullerenes
undergoing in-source fragmentation during the ionization process.
Consequently, modification of the ionization method with regard to the
source temperature and capillary voltage (i.e. to promote ‘softer
ionization’) did not lead to the identification of the precursor masses and
resulted in a near or complete loss of the signal. The detection of C60On
traces together with the C60-like peaks (Fig. D.6 in appendix D), suggests that
these products may consist of oxidized products similar to those observed
by Tiwari et al., (2014) who recently reported the elution of C60 oxides (C60O,
C60O2 and C60O3) to occur after that of C60 with a methodology similar to that
of the present work (i.e. reverse phase chromatography with functionalized
silica stationary phase). Quantitation of these structures was hindered by
the lack of analytical standards. However, analysis of the peak areas showed
that, in the glass incubation samples, the decrease in concentration of C60
corresponded to the increase of the C60-like transformation products over
time (Fig. 6.3). In particular, the abundance of all the structures increased
from the baseline signal after 3 d of incubation, in correspondence with the
decrease of C60, and reached a maximum after 14 d. The decrease to lower
levels at the end of the incubation suggests the formation of intermediates
in the transformation of fullerenes irradiated with UV-A light. It must be
noted that, in quartz sand and sandy soil extracts, these products were
either not detected or did not show a different trend between irradiated
and non-irradiated treatments. This could be due to several reasons,
including 1) different processes (e.g. biotic transformation) that took place
in the solid matrices, 2) the extraction of these chemicals was hindered by
the sample matrix and / or 3) the products were degraded at a faster rate
and were already removed at the first sampling step.
103
Fig. 6.3. Abundance (peak area) over time of (a) C60-I, (b) C60-II and (c) C60-III products in the glass incubation. The blue and red lines represent the irradiated and non-irradiated samples, respectively.
104
6.3.2.2 Unknown products
Auto MSMS analysis (see section 6.2.3) highlighted the presence of several
structures that eluted immediately before C60, between 12.2 and 14.4 min
(fig. 6.2b), and will be referred as “unknowns” in the further manuscript. An
example of the Auto MS/MS analysis is provided in the appendix D.7. In
general, base peaks were identified as the most abundant traces at a certain
retention time and, within each base peak, several m/z traces were
automatically selected for MS/MS. A tentative identification of the
unknowns was based on the MS1 and MS2 accurate masses and isotopic
cluster distribution, but complicated by the low signals intensity and the co-
elution of more structures. Furthermore, in-source fragmentations may
have occurred similar to what was reported above (section 6.3.2.1). A
summary of the m/z values relative to the unknowns found in the glass
incubation is reported in table 6.2 with a tentative identification of their
chemical formula.
Analysis of the mass spectra highlighted an ionization mechanism (i.e. the
formation of a radical molecular ion [M]-•) and isotopic cluster distribution
similar to that of the fullerenes. Furthermore, the mass (as measured m/z)
was always larger than that of the C60, suggesting functionalization of the
carbon cage. These fulleroid structures displayed both carbon addition and
loss from the pristine C60 cage. The addition of carbon was always in the
form of (CH3O)n groups and has already been reported to occur as a
consequence of methanol adduction to the fullerenes (Kolkman et al.,
2013). However, since the methodology applied in the present work is less
prone to the formation of adducts in comparison with other ESI-MS
techniques (Carboni et al., 2016), the (CH3O) clusters suggests a
methoxylation of the fullerenes. This hypothesis is supported by the fact
that the number of (CH3O) additions were not associated with all the
transformation products detected. On the other hand, carbon loss occurred
with the formation of both C59 and C58 species. The loss of carbon, and
specifically C2 units, has already been observed in the mass spectrometric
analysis of functionalized C60 (e.g. fullerols; Chao et al., 2011) and indicates
the opening of the cage.
105
Table 6.2. Summary of the unknown products found in the glass incubation. The base peak indicates the most abundant structure detected in MS1 at a certain retention time (Rt). Within each base peak, some m/z traces were automatically selected (Auto MSMS) in MS1 and sent to the MS2.
Rt: retention time
106
Additional functionalization mostly consisted of hydrogen and oxygen that
could be located in hydroxylic, ketons and epoxides groups. We found no
evidence of clusterization of the fullerenes, such as the formation of C120O
observed by Taylor et al. (1998). These results indicate that the unknowns
found in the present work could be similar to those observed during UV and
sunlight irradiation of C60 dissolved in water and organic solvents (Taylor et
al., 1991; Hou et al., 2009; Lee et al., 2009). Similar to the C60-like structures
(see section 3.2.1), the unknowns were more abundant in absence of a
matrix. However, the analysis of the peak areas did not show a direct
correlation between irradiation of the samples and their concentrations.
Indeed, although not present in the spiking solutions and non-spiked
samples (fig. D8 in appendix D), some of the products were found to be
already present at the beginning of the incubation and may have been
formed during the sample preparation. Furthermore, some unknowns
increased in abundance in the non-irradiated treatment only (Fig. D.9 in
appendix D) indicating that some processes either did not occur in presence
of light or that the products were too short-lived to be appreciated at the
time intervals in the present study. Finally, it must be noted that no
evidence of transformation products was found at the beginning of the
chromatograms (between 0 and 12 min) where the more polar species are
expected to elute. This could be due to a limitation of the present method
that did not allow their detection or, similar to what reported by Taylor et
al. (1991), that too polar products may not have been extracted with
toluene. In general, alternative methodologies should be tested that may
overcome the limitations of this work. In particular, we recommend 1) the
use of higher concentrations that, although not mimicking environmental
conditions, would simplify the determination of the fulleroid species in the
samples, 2) a second step of extraction with a more polar solvent in order
to recover polar products that were likely excluded in this work and 3)
improve identification by the use of alternative MS ionization conditions
and/or interfaces to avoid in-source fragmentation of precursor ions.
In general, the results of the present work show that fullerenes will be
degraded in the environment and that the removal from the soil
compartment could be relatively fast. In this context, the data hereby
presented will be helpful for modelling and environmental studies in
general. In particular, we have shown that the assessment of fullerenes fate
107
and occurrence is complicated by the formation of a large number of
transformation products that will likely display different properties in
comparison with the original structures (e.g. enhanced mobility). Thus,
future research addressing these topics will need screening analysis such as
those hereby presented in order to identify the totality of the species
present in environmental samples as well as to further elucidate the
pathways of degradation in the environment.
6.4 Conclusions
The effect of UVA light irradiation on fullerenes incubated at the solid state
was investigated. When C60 was directly irradiated, i.e. in absence of a
matrix, the irradiation had a clear effect on the degradation. However,
when the C60 was spiked into a quartz sand or sandy soil samples, much
faster degradation occurred suggesting that, once deposited onto the soil,
their fate will more likely be determined by other factors such as the
interactions with soil microbiota, including bioturbation and
biotransformation. The results indicate that fullerenes could undergo an
oxidative pathway similar to that observed for C60 dissolved in water, and
can result in the break-down of the carbon cage. The present study
highlighted the complexity of these processes and the need for analytical
strategies for their understanding. For instance, less selective methodology,
such as UV detection, can be suitable for the quantitation of the totality of
fullerenes in the samples, whereas HRMS-based techniques are required for
the characterization of the many species that likely result from their
transformation. Eventually, the results hereby presented will be helpful in
the assessment of the environmental fate of fullerenes and in defining
future strategies for their study.
108
109
Chapter 7
Synthesis
110
Fullerenes are considered to be novel environmental contaminants and the
characterization of their occurrence and fate in the environment is
fundamental for the general assessment of their possible effects on humans
and the environment. At the start of the present work, several studies
claimed the lack of analytical methodologies able to fulfil these goals and in
one of the first critical reviews on the topic, Isaacson et al. (2009) suggested
that “Sensitive and mass-selective detection, such as that offered by mass
spectrometry when combined with optimized extraction procedures, offers
the greatest potential”. In this work, such methodologies were developed
to address two specific scientific issues, the analysis of fullerenes in
environmental samples and the study of the presence and fate of these
chemicals in soil.
7.1 Development of analytical methods for the analysis of fullerenes in the
terrestrial environment
The work achieved in this thesis contributed to overcome some limitations
in the soil analysis of fullerenes and particularly (I) the development of
routine extraction procedures (Chapter 2), (II) the analysis of mixtures of
pristine and functionalized structures (Chapter 3), (III) the determination of
these nanoparticles at environmentally relevant concentrations (Chapters
4 and 5) and (IV) the analysis of transformation products (Chapters 5 and
6). These are discussed in the following synthesis.
With regard to the extraction methodology, Chapters 2 and 3 show that the
combination of ultrasonication and shaking extraction is a robust and
reproducible technique to recover fullerenes from soil independent of the
particle size distribution (texture) of the soil under investigation.
Furthermore it has the advantages of being applicable to large batches of
samples with relatively low costs in terms of materials and energy. It was
observed that functionalized fullerenes (Chapters 2 and 3) and unknown
fulleroid structures (Chapters 4 and 5) can be recovered to a similar extent
of the pristine C60, making this procedure suitable for the study of both
engineered and naturally occurring nanoparticles as well as transformation
111
products that may result in the environment. A current drawback of this
technique is that it likely alters the conditions of the fullerenes in the soil,
i.e. may not be representative of their natural state (homo- or hetero-
aggregated).
Non-aqueous reverse phase (ultra) high performance liquid
chromatography can provide sub-optimal separation of several fullerenes
and functionalized fullerenes species (Chapters 3 and 4) and, in
combination with selective detection methods such as high resolution mass
spectrometry (Chapter 4), can provide the unambiguous determination of
the fullerenes, even in complex mixtures where co-extractants and similar
structures are present. In particular, the experiments reported in this thesis
highlighted the performance of novel stationary phases such as core-shell
biphenyl (Chapter 4) and pyrenylpropyl silica (Chapter 3). Although the
retention mechanism has not been completely elucidated yet, the main
retention is likely provided by pi-pi interactions between the aromatic rings
of both the fullerenes and the functional groups of the stationary phases.
This indicates that such materials can be applied for the analysis of most of
the fullerenes and related structures, but also that some functionalized
species such as the poly-hydroxylated fullerols may not be retained. A direct
comparison of the column’s performances is not possible due to the
differences in the overall analytical setup they were employed in. The
chromatographic study reported in Chapter 3 was optimized for the
separation of more structures, for which pyrenylpropyl silica is more
suitable than byphenyl by virtue of the larger surface available for the
interaction with the fullerenes. However, the higher retention is achieved
at the expense of longer analysis time and this is not always desirable,
especially in routine analysis. Thus, further methods development, aimed
to monitoring studies, employed the core-shell biphenyl stationary phase
that can provide much shorter analysis with complete resolution of the
pristine structures only (Chapter 4), which are the most abundant in the
environment (Chapter 5). Chapter 4 and 6 also show how methodologies
employing this stationary phase can be optimized with regard to the
separation of co-extractants and functionalized structures, respectively.
Eventually, the work in this thesis showed that, when coupled to high
performance liquid chromatography, both UV and high resolution mass
112
spectrometry detection can be successfully applied in the analysis of
fullerenes. However, for environmental studies these techniques must
necessarily address different goals. In particular, UV detection lacks the
specificity and sensitivity required for the analysis of the low concentrations
present in real soil samples. Nevertheless, since both fullerenes and
functionalized structures generally display similar absorptivity behaviour
(Chapter 3), UV detection can still be a valuable tool for the identification
of unknown species, i.e. products of transformation during incubations and
ecotoxicological studies, and in general in experiments where higher
concentrations are applied (Chapter 6). Mass spectrometry can fulfil the
requirements of specificity and sensitivity necessary for an accurate
determination in soil matrices and is therefore the detection method of
choice. In particular, most experiments carried out in this thesis rely on a
high resolution instrument, the quadruple time-of-flight (Q-TOF) mass
spectrometer. Chapters 4 and 5 show how Q-TOF-based methodologies can
be applied for the investigation of fullerenes in soil and sediment matrices
and in environmental surveys addressing these chemicals. Furthermore,
software-based data collection and analysis represent a valuable tool for
the search and identification of non-target species in the samples (Chapter
6).
Ionization of the fullerenes (singly-charged molecular ions) was obtained
with a heated electrospray ionization (H-ESI) interface, operating in
negative mode. This is a hard ionization technique in comparison with
standard ESI and particularly fits the analysis of fullerenes due to the
resistance of these chemicals to high temperatures. As shown in Chapter 4,
the Ion-Booster ESI (IB-ESI) interface applied in this study presents several
advantages in comparison with existing methodologies and specifically: (I)
a higher ionization efficiency and (II) a lower tendency to create adducts
than standard ESI, and (III) the production of an isotopic pattern distribution
better matching the theoretical pattern in comparison with other
techniques. This latter point is particularly important in the analysis of
fullerenes. Indeed, one of the peculiarities in their mass spectrometric
analysis is the difficulty (impossibility in this work) to obtain fragmentation
of the closed-cage structure and, although exohedralic species (e.g.
[60]PCBM) can undergo fragmentation of the functional group from the
cage, this limitation is especially relevant in the analysis of pristine species.
113
Thus, correct and highly resolved isotopic clusters in combination with the
accurate mass provided by the Q-TOF can compensate these drawbacks and
provide identification criteria needed for the unambiguous determination
(Chapters 4 and 5). Nonetheless, the lack of fragmentation allows analysis
at high collision energies with the advantage of a “background clean-up” in
MS2, i.e. the removal of co-extractants from the samples. As shown in the
illustration of MS1 and MS2 spectra in Chapter 4, this result in extremely
clean mass spectra and is especially useful during the analysis of complex
matrices. A further advantage of this approach is that in-source
fragmentation of functionalized fullerenes allows identification of
fullerenes that would be otherwise not detected by target analysis e.g. the
C60-like products found in environmental samples (Chapter 5) and
incubation studies (Chapter 6). Such “defunctionalization” strategy is
similar to that suggested by Pycke et al., (2012) and can represent a valid
tool for future analysis of heterogeneous mixtures as well as colloidal nC60
structures, were fullerenes congeners may represent the majority of the
species in the samples. In this context the main drawback of the IB-ESI Q-
TOF methodology hereby proposed is the impossibility to prevent the
defunctionalization, since any attempt to prevent the fragmentation (i.e.
with “softer” analysis) was inconclusive. Thus, further studies should
address such limitations with the optimization of the current methods or its
combination with alternative techniques. Eventually, although optimized
for the study of soil samples, the methodologies developed in this study
should in principle be applicable to the analysis of fullerenes extracted from
other media (e.g. biological matrices).
Eventually, it must be noted that the current methodologies/strategies
present several limitations that future studies should address. One is
represented by the lack of proper internal standards for quantitative
purposes. Although standard addition methods (Chapters 4 and 5) and the
use of other fullerenes as internal standard (Chapter 6) can allow a proper
quantitation in certain experimental conditions, there is a need for high
purity, isotopically labelled, materials that are currently unavailable on the
market at reasonable prices. A second limitation, especially in the analysis
of complex matrices, is the lack of optimized clean-up procedures that can
improve the quality of the extracts and allow a better identification of the
fullerenes. In conclusion, further enhancements of the current strategies
114
for the characterization of fullerenes in soil, and generally in environmental
matrices, will necessarily involve the combination, and possibly
hyphenation, of more analytical techniques. Indeed, characterization of
fullerenes’ behaviour in the environment necessarily relies on the study of
the interaction with natural components that will determine transport,
accumulation and the general fate of these nanoparticles. Methods such as
those hereby proposed provide qualitative and quantitative data about the
presence of fullerenes but cannot completely describe these interaction.
Thus, imaging techniques (e.g. transmission electron microscophy, Goel et
al., 2004) and novel methodologies such as the recent coupling of field flow
fractionation (FFF) with HRMS (Herrero et al., 2014) could be employed in
order to characterize these processes with regard to properties such as the
composition, size, size distribution and morphology.
7.2 Occurrence and fate in the environment
Some of the main research questions that formed the basis of this project
were: “To which extent are fullerenes present in the soil compartment?”
and “what is the contribution of engineered nanomaterials to their overall
concentration?”. The answers were mostly hindered by the lack of
monitoring studies that allow estimation and modelling of the
environmental concentrations on the basis of empirical data.
The environmental survey reported in Chapter 5 shows that fullerenes are
widely present in the environment but at relatively low concentrations. In
particular, pristine structures occur in the soil in the part per billion range
and, although a precise assessment of their presence will need further
work, similar studies carried out in the last two years support these
observations. These findings are also in line with the amounts predicted in
soils due to use and production of engineered nanoparticles (Gotthschalk
et al., 2009). However, although the presence of manufactured materials
cannot be excluded a priori, the occurrence of fullerenes in the Dutch soils
investigated in this thesis was likely due to incidental sources, i.e. due to
combustion processes of carbonaceous materials such as coal and fuel.
Thus, the overall occurrence in the terrestrial environment may nowadays
be of anthropogenic origin but related to incidental sources more than the
115
production and application of ENMs. This hypothesis is also supported by a
recent modelling study (Tiwari et al., 2016), which suggests that the global
emission of incidental C60 may be several orders of magnitude higher than
that of manufactured C60. However, no methodologies are available yet that
can clarify the origin of the nanoparticles detected in the environment and,
although some species could be linked to a source (e.g. [60]PCBM in
photovoltaics application), this is not valid for pristine structures (e.g. C60,
C70) that can result from both natural and anthropogenic activities as well
as resulting from transformations of precursors and other species in the
environment. In Chapter 4 it is proposed that an assessment of the ratio at
which different species occur in the environment may be helpful in
clarifying their origin, but this hypothesis is hindered by the lack of similar
environmental surveys and the fact that most studies are focused on the
determination of C60 only. Thus, the best strategy for the unequivocal
identification of fullerenes’ origins may be the characterization of source-
related (or process-related) products, such as the unknown C60-like
structure presented in Chapter 5 that, although not characterized yet, may
represent a marker for nanoparticles production during incinerators
activity.
The incubation studies in Chapter 6 show that, upon release in the
environment, C60 will interact with the ultraviolet portion of the sunlight
irradiation and that it will be degraded as a consequence. However, this
process will take place at a relatively slow rate and will likely affect only the
fullerenes that are released in the atmosphere and those that deposit on
the top-soil (i.e. those that are directly exposed to the light). Once mixed
into soil, other biotic and abiotic phenomena will likely determine their fate
to a larger extent. In particular, biotic factors could determine faster decay
rates similar to those observed for C60 incubated into sandy soil in this study.
The study in Chapter 6 also suggests that, upon release in the environment,
fullerenes in the “solid” state will be degraded by abiotic factors and that
the transformation pathways will be similar to those already observed
during ozonation and irradiation of the colloidal structures. However, it is
not possible to clarify yet whether biotic factors will determine similar
pathways and if they will proceed until mineralization of the fullerenes. The
figure below shows a putative pathway of degradation of C60 in the
environment.
116
Fig. 7.1. Possible degradation pathway of C60 upon release in the environment. Light and oxygen related reactions on the closed cage structure (1) can lead to the functionalization of the fullerenes (2) with formation of oxidized species (3). Destabilization of the fullerenes can determine carbon removal and opening of the cage (4) that could in turn trigger the fragmentation of the cage (5) with the production of transformation products or mineralization (6).
In general, reactions on the cage, enhanced by interactions with light and
oxygen species, are expected to involve the carbon(s) in the junctions
between two hexagonal rings (Diederich, 1997). This can lead to the
oxidation of the fullerenes with consequent production of oxidized
functionalized structures that can in turn determine a destabilization of the
fullerenes and their progressive fragmentation. Such hypothesis is
supported by the detection reported in Chapter 6 of fulleroid traces that
display carbon loss and several degrees of oxidation, hydroxylation and
methoxylation. These species are more polar than the pristine fullerenes
and will likely exhibit enhanced transport in soil. In order to characterize the
life-cycle of fullerenes in the environment, further studies will need to focus
on such oxidative pathway and the interaction of the transformation
products with environmental components such as atmosphere particulates
and organic matter.
117
7.3 Concluding remark
The studies reported in this thesis provide tools and pioneering data about
the occurrence and fate of fullerenes in soil. Hopefully, these will be helpful
for future studies and will enhance the assessment, regulation and the
general understanding of these nanoparticles in the environment. A further
ambition of this work is that of being helpful for environmental studies
focussed on other carbon-based nanomaterials, such as graphene and
carbon nanotubes, whose assessment is at least as difficult and whose
production and application is emerging as well.
Nonetheless, future perspectives about fullerenes occurrence and fate are
very uncertain. To date, the majority of these nanoparticles in the
environment is likely due to incidental sources (both natural and
anthropogenic), but the production of engineered nanomaterials will play a
major role in determining both the amount and the number of species that
will enter the environment. This is particularly relevant for the colloidal
nC60, whose behaviour and fate are expected to differ from the “solid”
structures and that could easily find industrial application in the near future
due to the impressive properties that they display and the relative ease and
low costs of production. In addition, while C60 by-product release may
decrease in the future, due to current and future policies aimed to limit the
global emissions and the use of coal and fossil fuels (e.g. Kyoto protocol),
engineered nanomaterials will likely find increasing applications and uses.
In this context is at least interesting to note that sustainable energy
technologies, such as photovoltaics, could strongly rely on the application
of engineered fullerenes. Eventually, an increasing contribution of the
manufactured fullerenes to the global load could present new scenarios
where their presence is enhanced in a wider variety of ecosystems. For
instance, while incidental species are necessarily dispersed onto soil by the
atmosphere, resulting in a more homogeneous distribution in the
environment, manufactured nanomaterials will likely present hotspots of
accumulation close to production, use and dumping sites. In general, future
studies focussed on the characterization of fullerenes in the environment
will necessarily need to characterize (I) such hotspots of accumulation, (II)
the origin of fullerenes found in the environment, with particular regard to
118
the ENMs and (III) to identify the totality of the fulleroid species that may
result from nanotechnology as well as from natural processes.
119
Appendices
120
Appendix A:
Supportive information chapter 3 Table A.1. The fullerenes included in the study.
121
Fig. A.2. HPLC-UV chromatogram of the fullerenes. In blue detection at 332 nm, in red detection at 305 nm.
122
Appendix B:
Supportive Information Chapter 4
Fig. B.1. Temperature gradient of the IB-ESI interface during the analysis. Notice that, although the Y axis indicates APCI heater, the interface employed was a heated ESI.
Mass calibration
Table B.2. An example of the mass calibration obtained from sodium acetate cluster.
Reference m/z Resulting m/z Intensity Error [ppm]
387.0261 387.0261 245506 -0.033
469.0292 469.0293 598888 0.147
551.0323 551.0322 1142349 -0.241
633.0354 633.0354 1651113 0.104
715.0384 715.0386 1805570 0.182
797.0415 797.0413 920056 -0.28
879.0446 879.0447 494061 0.136
961.0477 961.0477 331092 0
1043.0507 1043.0507 155736 -0.022
1125.0538 1125.0538 70091 0.005
Standard deviation of
the calibration curve:
0.279
1 2 3 4 5 6 7 Time [min]
325
350
375
400
425
450
[°C]
AP
CI H
eate
r
123
An example of the SAM results in the present work.
Figure B.3. Chromatograms of the standard addition method (SAM) in matrix 5. Left: C60 at the concentrations of 25 (yellow), 50 (purple), 100 (orange), 250 (brown) and 500 ng/L (red). Right: C70 at the concentrations of 25 (grey), 50 (black), 100 (violet), 250 (green) and 500 ng/L (blue).
Fig. B.4. C60 response versus concentration calibrations obtained during the standard addition method experiments using method B.
124
Analysis of several fullerene derivatives with the method developed in the
present study.
Fig. B.5. Chromatograms of C60, C70 and six methanofullerene structures at the concentration of 500 ng/l analyzed with the method B.
125
Table B.6. Standard addition method results in the present study.
126
Fig. B.7. The response of C60 spiked at 500 ng/L into matrix 2 with toluene:methanol ratio of A) 50:50 (v:v), B) 40:60 (v:v) and C) 35:65 (v:v).
Chromatographic separation obtained applying the method B.
Fig. B.8. Chromatographic separation of C60 (Rt 4 min) and C70 (Rt 6 min) obtained with the method B. The orange and red lines indicate the MS1 and MS2 respectively for C60 whereas the green line and blue line are relative to MS1 and MS2 signal respectively for C70.
127
Appendix C:
Supportive Information Chapter 5
Table C.1. List of the samples included in the environmental survey.
128
129
130
131
132
C.2 UHPLC-IB-ESI QTOF analysis
The chromatographic method employed a core-shell biphenyl stationary
phase and a mobile phase consisting of methanol (eluent A) and toluene
(eluent B). The eluent was set to flow at 600 μl/min and programmed to
start with 40 sec at 100% eluent A (focusing step), a linear gradient up to
35% eluent B in 20 sec, an isocratic step of 4 min at 35% eluent B followed
by a linear gradient of 1.5 min to reach 60% B, a linear gradient of 10 sec
was in order to reach 100% eluent B which was maintained for 2.5 min. The
chromatograms were divided into four segments: segment 1 (from 0 min to
0.1 min) to assure the correct position of the loop and loading the mass
calibration solution during the LC equilibration, segment 2 (from 0.1 min to
0.4 min) dedicated to the mass calibration, segment 3 (from 0.4 min to 4.4
min) for the SRM analysis of C60 (m/z 720.0005) and segment 4 (from 4.4
min to 9.6 min) for the SRM analysis of C70 (m/z 840.0005). The optimized
working conditions for the IB-ESI operating in negative mode were: capillary
voltage 1000V, end plate offset -400 V, charging voltage 300 V, nebulizer
gas 4.1 bar, dry gas 3.0 l/min and dry heater 200 °C. The present settings
allowed a mass resolving power up to 80000 (0.03 m/z FWHM). An
additional UHPLC-IB-ESI-QTOF method was employed for the analysis of the
samples collected in the incinerator area (location B) that was specifically
aimed to the characterization of the Unknown C60-like fullerene found in
the samples. The method consisted of a longer analysis (25 min) with the
following program: the first minute was a focusing step with 100% eluent
A, then a gradient up to 21 min in order to reach 75% eluent B which was
maintained for 4 min. The analysis were performed with a column
temperature of 30 °C and with injection volume of 10 µl. The
chromatograms were split in three segments, segment 1 (from 0 min to 0.1
min) to assure the correct position of the loop, segment 2 (0.1-0.4 min) for
mass calibration with 2 mM sodium acetate solution and segment 3 (0.4-25
min) for the MS detection of the fullerenes. The IB-ESI parameters were the
same as reported above with the exception of 1) the method was run at
different temperatures and specifically 275, 300, 350, and 475 °C and 2)
both Auto MSMS mode and broad band collision induced dissociation mode
(bbCID) were tested. Furthermore, several capillary voltages (i.e. 800 V vs
1000 V) were tested.
133
C.3. Matrix effect in the samples
The matrix effect (ME) in the soil extracts was calculated as following:
𝑀𝐸 =𝐶𝑠𝑝𝑖𝑘𝑒
∗ − 𝐶𝑠𝑎𝑚𝑝𝑙𝑒∗
𝐶𝑠𝑝𝑖𝑘𝑒 (1)
where C*spike is the concentration observed after the addition of the
fullerenes standard. C*sample is the concentration observed in the extracts
and Cspike is the concentration added to the samples. The concentration in
the extracts (Csample) was calculated as:
𝐶𝑠𝑎𝑚𝑝𝑙𝑒 = 𝐶𝑠𝑎𝑚𝑝𝑙𝑒
∗
𝑀𝐸 (2)
134
Appendix D:
Supportive Information Chapter 6
Fig. D.1. The spectrum of the lamps used for the irradiation in the present study.
135
D.2 Quantitation of the C60 in the samples
A calibration line was obtained by running levels with fixed C70
concentration (50 µg/L) and increasing C60 concentration (from 4 to 128
µg/L). The resulting line can be described as:
(1) 𝑌 = 𝑎𝑋 + 𝑏
where a and b are the slope and intercept of the line, respectively; Y is the
ratio of C60 and C70 chromatographic peak areas and X is the ratio between
C60 and C70 concentration (conc) in the calibration levels. Accordingly, the
equation (1) can be rearranged as:
(2) 𝐶60 𝐴𝑟𝑒𝑎
𝐶70 𝐴𝑟𝑒𝑎= 𝑎
𝐶60 𝑐𝑜𝑛𝑐
𝐶70 𝑐𝑜𝑛𝑐+ 𝑏
The concentration of C60 in the samples was obtained as following
(3) 𝐶60 𝑐𝑜𝑛𝑐 =(
𝐶60 𝐴𝑟𝑒𝑎
𝐶70 𝐴𝑟𝑒𝑎−𝑏)
𝑎
136
D.3. Mass calibration
In general, mass calibration was achieved as reported in Carboni et al., 2016
with minor modifications. In order to allow the analysis of both the mass
calibration solution and the analytes, at the start of each analysis the source
heater temperature was 325°C for mass calibration and quickly ramped to
450°C afterwards during analysis of chromatographed compounds. Internal
mass calibration was automatically performed by loop injection of a 2 mM
sodium acetate solution dissolved in 1:1 ultrapure water-isopropanol. The
total amount (20 μl) was discharged into the MS source in two subsequent
steps with roughly the same volume. This resulted in two separate ‘mass
calibrant peaks’ (figure S3), where the former was measured with MS
methodology optimized for a low mass range (circa 100 - 715 m/z, fig. S4),
and the latter with settings optimized for a high mass range (circa 450 - 1050
m/z, fig. S5). Depending on the m/z range of interest, mass data were
recalibrated either with the first or second mass calibrant peak, allowing a
wide mass range to be monitored during one injection.
Fig. D.3.1. Chromatogram relative to the mass calibration solution. The peak at Rt 0.12 min was used for low masses range whereas the one at 0.4 min was used for the high masses range.
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Time [min]
0
1
2
3
5x10
Intens.
20150306-silica28 3-1_1-37_01_3158.d: EIC 537.0206±0.05 -All MS
137
Fig. D.3.2. MS spectra relative to the low-masses (m/z) calibration solution.
Fig. D.3.3. MS spectra relative to the high-masses (m/z) calibration solution.
141.0169
223.0200
305.0230
387.0262
469.0291
551.0323
633.0353
715.0384
20150306-solvent-8_1-1_01_3237.d: -MS, 0.1-0.1min #10-12
0
1
2
3
4
5
5x10
Intens.
100 200 300 400 500 600 700 800 900 m/z
551.0382
633.0394
715.0401
797.0424
879.0447
961.0480
1043.0528
20150306-solvent-8_1-1_01_3237.d: -MS, 0.3-0.3min #35-36
0
1
2
3
5x10
Intens.
300 400 500 600 700 800 900 1000 1100 m/z
138
D.4. MS and Auto MS/MS settings
Table D.8.1. Q-TOF spectrometer parameters during the analysis. The chromatograms were split into four segments, the first two dedicated to mass calibration and the latter two to the analysis of the fullerenes and their transformation products.
Table D.8.2. Q-TOF spectrometer parameters in the Auto MS/MS analysis
139
Fig. D.5. An example of the chromatograms obtained during HPLC-UV analysis. C60 and C70 are eluting at 7.5 and 12.5 min respectively. No additional peaks were present in the sample.
Fig. D.6. Mass spectrum showing the presence of C60O and C60O3 traces in correspondence of the C60-II peak at retention time 20.0 min. (14 days sample of the glass incubation).
140
Fig. D.7. An example of the AutoMSMS analysis in the present work. (A) Auto MSMS detection of m/z 831.0251 (purple line) and 784.0115 (blue line) in the chromatograms, the grey line represents the base peak chromatogram obtained from the m/z 831.0261 trace. Below the MS2 spectra relative to the m/z 831.0251 (in purple, B) and m/z 784.0115 (in blue, C).
141
Fig. D.8. Chromatograms of the spiking solution (top) and a non-spiked glass sample extract (bottom) were the extracted ion chromatograms (EIC) of C60 and all the unknowns are highlighted.
Fig. D.9. An example of an unknown structure that showed increasing concentration in the non-irradiated samples (red line) but not in the irradiated ones (blue line). (m/z 860.0772, retention time 14.4 min, sandy soil incubation).
142
143
Fullerenes nanoparticles in soil:
Analysis, occurrence and fate
Summary
Fullerenes are carbon-based nanomaterials that can occur in the
environment due to both natural events and human production. Once
released, little is known about the fate of these chemicals and the
assessment is complicated by the large number of species that may occur
and the low concentrations at which they are present. Thus, the aim of this
work was to study the occurrence and fate of these chemicals in the soil
compartment, which is expected to act as a sink. The research consisted of
two parts. First, analytical methods were developed for the analysis of
fullerenes in soil samples, then these methodologies were applied in
environmental survey and fate studies.
The developed methodologies made use of a combination of ultra-
sonication and shaking extraction techniques. This resulted in good
recoveries for both the fullerenes and the functionalized structures and
allowed the processing of large batches of samples in relatively short time
and low energy costs. High performance liquid chromatography (HPLC) was
extensively investigated for the separation of mixtures of fullerenes and
was optimized for the analysis of complex soil matrices. Two stationary
phases, namely pyrenyl-propyl silica and core-shell biphenyl were tested
that can grant good retention of the fullerenes and sub-optimal separation
of similar structures. Eventually the methodologies were optimized with
regard to the detection techniques. Although the fullerenes displayed
strong absorbance in the UV range, mass spectrometric detection provided
144
the selectivity and sensitivity characteristics needed for the analysis of more
species and at environmentally relevant concentrations. Thus, most of the
analyses in this study relied on high resolution mass spectrometry (HRMS)
coupled to a heated-electrospray ionization (H-ESI) interface that allowed
unambiguous determination of the fullerenes at concentrations below the
parts per billion range.
The analysis of soil samples collected in The Netherlands showed that
fullerenes are extensively occurring in the environment and that C60 is the
most present and abundant specie. Furthermore, their origin is likely
anthropogenic and originates from emissions of traffic and combustion
processes (by-product), whereas the contribution of engineered
nanomaterials (ENMs) is expected to be comparatively low. Subsequently,
incubation studies showed that, once released in the environment,
fullerenes will interact with the solar radiation and will be degraded as a
result. However, once deposited onto soil, their fate will likely be
determined by other factors such as the action of microorganisms. The
degradation of the fullerenes will follow an oxidative pathway with the
possible formation of a large number of transformation products that will
presumably lead to the fragmentation of the closed-cage structure.
The research work in this thesis provided valuable tools for the analysis of
fullerenes in environmental samples and pioneering observations about
their occurrence and fate in the environment.
145
Samenvatting
Fullerenen zijn uit koolstof opgebouwde nanomaterialen die door zowel
natuurlijke processen als kunstmatige productie in het milieu voor kunnen
komen. Over het lot van deze stoffen in het milieu is nog weinig bekend en
de studie hiervan is gecompliceerd gezien de vele chemische vormen en
veelal lage concentraties waarin deze stoffen aanwezig kunnen zijn.
Verwacht wordt dat fullerenen accumuleren in de bodem en het doel van
deze studie was daarom ook om de aanwezigheid en het lot van deze
stoffen in de bodem te onderzoeken. Het onderzoek was tweeledig:
allereerst werd een analytische methode ontwikkeld om fullerenen in
bodem te meten, die daarna werd toegepast om de aanwezigheid en het
lot in het milieu te monitoren.
De ontwikkelde methode voor monsteropwerking bestaat uit een
combinatie van extractie met ultrasoon en uitschudden. Dit resulteerde in
bijna volledige extractie voor zowel fullerenen met en zonder functionele
groep en de methode is geschikt voor de verwerking van hoge aantallen
monsters binnen een relatief korte tijd en met lage (energie)kosten. High
Performance Liquid Chromatography (HPLC) werd uitgebreid getest en
toegepast voor de chromatografische scheiding van fullerenen en de
methode werd geoptimaliseerd voor de analyse van deze stoffen in
complexe bodem matrices. De stationaire fases pyrenyl-propyl silica en
core-shell biphenyl werden getest en dit resulteerde in beide gevallen in
volledige scheiding van fullerenen en gedeeltelijke scheiding van
vergelijkbare componenten. Uiteindelijk werden de detectiemethoden
geoptimaliseerd. Hoewel fullerenen sterke absorptie in het UV bereik
vertonen, werd alleen met massaspectrometrische detectie de selectiviteit
en gevoeligheid behaald welke nodig zijn voor de analyse van deze en
vergelijkbare componenten bij in het milieu voorkomende concentraties.
Om die reden zijn de meeste analyseresultaten in deze studie verkregen
met hoge resolutie massa spectrometrie (HRMS) gekoppeld met een
heated-electrospray ionizatie (H-ESI) interface welke eenduidige bepaling
146
van fullerenen met concentraties beneden het delen per miljard (ppb)
niveau toelaten.
Uit de analyse van in Nederland verzamelde bodemmonsters bleek dat
fullerenen op grote schaal voorkomen in het milieu, waarbij C60 het meeste
voorkomt en in de hoogste concentraties. Het is zeer waarschijnlijk dat deze
stoffen van antropogene herkomst zijn. Aangenomen wordt dat de
oorsprong voornamelijk ligt bij uitlaatgassen (bijvoorbeeld van auto’s en
vrachtwagens) of als bijproduct van andere verbrandingsprocessen
(bijvoorbeeld verbrandingsovens) en de in de bodem aangetroffen
fullerenen in mindere mate afkomstig zijn van geproduceerde
nanomaterialen. Door de uitgevoerde incubatie-experimenten werd
aangetoond dat fullerenen in het milieu worden afgebroken onder invloed
van zonlicht. Wanneer fullerenen in de bodem terecht komen zal de afbraak
door zonlicht echter in mindere mate optreden en zullen andere
afbraakprocessen, bijvoorbeeld door micro-organismen een grotere rol
spelen. De afbraak van fullerenen vindt waarschijnlijk plaats door een
oxidatieproces, waarbij mogelijk een groot aantal omzettingsproducten
gevormd worden als gevolg van het openbreken van de C60 structuur.
De onderzoeksresultaten die in dit proefschrift worden gepresenteerd
vormen een waardevol hulpmiddel voor de analyse van fullerenen in
bodem en bevatten unieke en innovatieve waarnemingen over het
voorkomen en het lot van deze stoffen in het milieu.
147
References
Astefanei A., Núñez O., Galceran M. T. 2014a. In: S.B. Ellis (Ed.), Fullerenes,
Chemistry, Natural Sources and Technological Applications, Nova Science
Publishers, New York, pp. 35-63.
Astefanei A., Núñez O., Galceran M. T. 2014b. Analysis of C60-fullerene
derivatives and pristine fullerenes in environmental samples by ultrahigh
performance liquid chromatography–atmospheric pressure
photoionization-mass spectrometry, Journal Chromatography A 1365, 61-
71. -63.
Avanasi R., Jackson W. A., Sherwin B., Mudge J. F., Anderson T. A. 2014. C60
Fullerene Soil Sorption, Biodegradation, and Plant Uptake. Environmental
Science and Technology, 48, 2792−2797. dx.doi.org/10.1021/es405306w.
Baena J. R., Gallego M., Valca´rcel M. 2002. Fullerenes in the analytical
sciences. Trends in Analytical Chemistry 21, 187-198.
Baierl B., Fabel B., Gabos P., Pancheri L., Lugli P., ScarpaG. 2010. Efficient
indium-tin-oxide (ITO) free top-absorbing organic photodetector with
highly transparent polymer top electrode. Organic Electronics 11, 1199–
1206.
Bakry R, Vallant R. M., Najam-ul-Haq M., Rainer M., Szabo Z., Huck C. W.,
Bonn G. K. 2007. Medicinal applications of fullerenes. International Journal
of Nanomedicine 2:4, 639-649.
Becker L., Bada J. L., Winans R. E., Hunt J. E., Bunch T. E. and French B.M.
1994. Fullerenes in the 1,85-billion-year-old Sudbury impact structure.
Science, 265: 642–645.
148
Benn T. M., Westerhoff P., Herckes P. 2011. Detection of fullerenes (C60 and
C70) in commercial cosmetics. Environmental Pollution. 159 (5), 1334-1342.
Bhatt I. and Triphathi B. N. 2011. Interaction of engineered nanoparticles
with various components of the environment and possible strategies for
their risk assessment. Chemosphere 82 (2011) 308–317
Bouchard D., Ma X. 2008. Extraction and high-performance liquid
chromatographic analysis of C60, C70, and [6,6]-phenyl C61-butyric acid
methyl ester in synthetic and natural waters. Journal Chromatography A,
1203, 153-159.
Boxall, A. B., Tiede, K., Chaudhry, Q. 2007. Engineered nanomaterials in
soils and water: How do they behave and could they pose a risk to human
health? Nanomedicine 2, (6), 919‐927.
Buhl M. and Hirsch A. 2001. Spherical aromaticity of fullerenes. Chemical
Reviews. 101, 1153-1183.
Burangulov N., Moravsky A.P., Kulikova Y.V., Loutfy R.O., Dyachuk G.I. 2005.
Cosmetic composition containing fullerene clusters. U.S. Patent Application
20050136079.
Buseck P.R., Tsipursky S. J., Hettich R. 1992. Fullerenes from the geological
environment. Science 257, 215-217.
Cantrill S. 2011. Endohedral fullerenes: Water behind walls. Nature
Chemistry 3, 753. doi:10.1038/nchem.1168
149
Carboni A., Emke E., Parsons J. R., Kalbitz K., de Voogt P. 2013. An analytical
method for determination of fullerenes and fullerene derivatives in soil with
high performance liquid chromatography and UV detection. Analitica
Chimica Acta 807, 159-165.
Carboni A., Helmus R., Parsons J. R., Kalbitz K., de Voogt P. 2016. A method
for the determination of fullerenes in soil and sediment matrices using
ultra-high performance liquid chromatography coupled with heated
electrospray quadrupole time of flight mass spectrometry. Journal of
Chromatography A. 1433, 123–130.
Chai, Y.,Guo T., Jin C., Haufler R. E., Chibante L. P., Felipe; Fure J., Wang L.,
Alford J.M., Smalley R.E. 1991. Fullerenes with metals inside. Journal of
Physical Chemistry 95 (20), 7564–7568.
Chao T.-C., Song G., Hansmeier N., Westerhoff P., Herckes P., Halden R. U.
2011. Characterization and Liquid Chromatography-MS/MS Based
Quantification of Hydroxylated Fullerenes. Analytical Chemistry 83, 1777–
1783. dx.doi.org/10.1021/ac1031379
Christian P., Von der Kammer F., Baalousha M., Hofmann Th. 2008.
Nanoparticles: structure, properties, preparation and behaviour in
environmental media. Ecotoxicology 17:326–343
Creegan, K. M., Robbins, J. L., Robbins, W. K., Millar, J. M., Sherwood, R. D.,
Tindall, P. J., Cox, D. M. 1992. Synthesis and characterization ofC60O, the
first fullerene epoxide. Journal American Chemical Society 114, 1103–1105.
Daly T. K., Buseck P. R., Williams P., Lewis C. F. 1993. Fullerenes from a
fulgurite. Science 259, 1599–1601.
150
Dang M. T., Hirsch L., Wantz G. 2011. P3HT:PCBM, Best Seller in Polymer
Photovoltaic Research. Advanced Materials 23, 3597-3602.
Deguchi S., Alargova G. R., and Tsujii K. 2001. Stable Dispersions of
Fullerenes, C60 and C70, in Water. Preparation and Characterization.
Langmuir 17, 6013-6017.
Deye J., Shiveley A. N., Oehrle S. A., Walters K. A.. 2008. Separation of
Substituted Fullerenes using Normal-Phase HPLC-MS. Journal
Chromatography A 1181, 159–161.
Diederich F. 1997. Covalent fullerene chemistry. Pure and applied chemistry
69:3, 395-400.
Ebbesen T. W., Lezec H. J., Hiura H., Bennett J. W., Ghaemi H. F., Thio T.
1996. Electrical conductivity of individual carbon nanotubes. Nature, 382,
54‐56.
Elsila J.E., de Leon N.P., Plows L., Buseck P.R., and Zare R.N. 2005 Extracts of
impact breccia samples from Sudbury, Gardnos, and Ries impact craters and
the effects of aggregation on C60 detection. Geochimica Cosmochimica Acta
69, 2891–2899.
Emke E., Sanchís J., Farré M., Bäuerlein P. S., de Voogt P. 2015.
Determination of several fullerenes in sewage water by LC HR-MS using
atmospheric pressure photoionisation. Environmntal Science Nano 2, 167-
176. doi: 10.1039/C4EN00133H.
151
European Commission, Commission Directive 200/90/EC. 2009. laying
down technical specifications for chemical analysis and monitoring of water
status, Official Journal of the European Union L201, 36-38.
European Commission, Commission recommendation of 18 October 2011
on the definition of nanomaterial (2011/696/EU). Official Journal of the
European Union 2011, 54, 38‐40.
Farre M., Perez S., Gajda-Schrantz K., Osorio V., Kantiani L., Ginebreda A.,
Barcelo D. 2010. First determination of C60 and C70 fullerenes and N-
methylfulleropyrrolidine C60 on the suspended material of wastewater
effluents by liquid chromatography hybrid quadrupole linear ion trap
tandem mass spectrometry, Journal of Hydrology 383, 44-51.
Gago-Ferrero P., Schymansky E. L., Bletsou A. A., Aalizadeh R., Hollender J.,
Thomaidis N. S. 2015. Extended Suspects and Non-Target Strategies to
Characterize Emerging Polar Organic Contaminants in Raw Watewater with
LC-HRMS/MS, Environmental Science and Technology 49, 12333-12341.
Goel A., Howard J. B., J. B. Vander Sande. 2004. Size analysis of single
fullerene molecules by electron microscopy. Carbon 42, 1907-1915.
Gottschalk F., Sonderer T., Scholz R. W. 2009. Modeled Environmental
concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT,
Fullerenes) for different regions, Environmental Science and Technology 43,
9216–9222.
Gottschalk F., Soonderer T., Scholz R. W., Nowack B. 2010. Possibilities and
limitations of modelling environmental exposure to engineered
152
nanomaterials by probabilistic material flow analysis. Environmental
Toxicology and Chemistry, Vol. 29, No. 5, pp. 1036–1048.
Guldi D.M., Martin N. 2002. Fullerenes: From Synthesis to Optoelectronic
Properties, Kluwer Academic Publishers, Norwell, MA.
Haftka J. J.-H., Bäuerlein P. S., Emke E., Lammertse N., Belokhovstova D.,
Hilvering B., de Voogt P., ter Laak T. L. 2015. Colloidal stability of
(functionalised) fullerenes in the presence of dissolved organic carbon and
electrolytes. Environmental Science Nano 2, 280-287. DOI:
10.1039/C4EN00206G.
Hassellov M., Readman J. W., Ranville J .F., Tiede K. 2008. Nanoparticle
analysis and characterization methodologies in environmental risk
assessment of engineered nanoparticles. 2008. Ecotoxicology 17, 344–361.
DOI 10.1007/s10646-008-0225-x
Hendren, C. O., Mesnard, X., Dröge, J., Wiesner, M. R. 2011. Estimating
Production Data for Five Engineered Nanomaterials as a Basis for Exposure
Assessment. Environmental Science and Technology 45, (7), 2562–2569.
Henglein A. 1993. Physicochemical Properties of Small Metal Particles in
Solution – Microelectrode Reactions, Chemisorption, Composite Metal
Particles, and the Atom-To-Metal Transition. Journal of Physical Chemistry
97, 5457–5471.
Herrero P., Bauerlein P. S., Emke E., Pocurull E., de Voogt P. 2014.
Asymmetrical flow field-flow fractionation hyphenated to Orbitrap high
resolution mass spectrometry for the determination of (functionalised)
aqueous fullerene aggregates. Journal of Chromatography A 1356, 277-282.
153
Heymann D., Dressler B.O., Knell I., Thiemens M.H., Buseck P.R., Dunbar
R.B., Mucciarone D. 1999. Origin of carbonaceous matter, fullerenes, and
elemental sulfur in rocks of the Whitewater Group, Sudbury impact
structure, Ontario, Canada. In Large Meteorite Impacts and Planetary
Evolution II, Special Paper, vol. 339, Dressler, B. O. and Sharption, V. L.
(eds.), 345–360. Boulder, CO: Geological Society of America.
Heymann D., Chibante L.P.F., Brooks R.R., Wolbach W.S., Smalley R.E. 1994.
Fullerenes in the cretaceous-tertiary boundary-layer. Science 265, 645–647.
Hirsch A. 2005. The Chemistry of Fullerenes, Wiley-VCH, Weinheim,
Germany.
Hou W.-C., Jafvert C. 2009. Photochemical transformation of aqueous C60
clusters in sunlight. Environmental Science and Technology 43, 362-367.
Howard J.B., Mc Kinnon J.T., Makarovsky Y., Lafleur A.L., Johnson M.E. 1991.
Fullerenes C60 and C70 in flames, Nature 352, 139 – 141.
Hummelen J.C., Knight B.W., Lepeq F., Wudl F., Yao J., Wilkins C.L. 1995.
Preparation and Characterization of Fulleroid and Methanofullerene
Derivatives, Journal of Organic Chemistry 60, 532-538.
Huppertz L. M., Kneisel S., Auwarter V., Kempf J. 2014. A comprehensive
library-based, automated screening procedure for 46 synthetic
cannabinoids in serum employing liquid chromatography-quadrupole ion
trap mass spectrometry with high-temperature electrospray ionizaton,
Journal of Mass Spectrometry 49, 117-127.
154
Hwang Y. S., Li Q. 2010. Characterizing photochemical transformation of
aqueous nC60 under environmentally relevant conditions. Environmental
Science and Technology 44, 3008-3013.
Isaacson C. W., Kleber M., Field J.A. 2009. Quantitative Analysis of Fullerene
Nanomaterials in Environmental Systems: A Critical Review. Environmental
Science and Technology 43 (17), 6463-6474.
Jafvert C. T., Kulkarni P. P. 2008. Buckminsterfullerene’s (C60) octanol-water
partition coefiicient (Kow) and aqueous solubility. Environmental Science
and Technology 42, 5945-5950.
Jehlicka J., Frank O. , Hamplova´ V., Pokorna´ Z., Juha L., Bohacek Z.,
Weishauptova Z. 2005. Low extraction recovery of fullerene from
carbonaceous geological materials spiked with C-60. Carbon 43, 1909–
1917.
Kempf J., Traber J., Auwarter V., Huppertz L. M. 2014. ‘Psychotropics caught
in a trap’ – Adopting a screening approach to specific needs, Forensic
Science International 243, 84-89.
Kim Y., Cook S., Tuladhar S. M., Choulis S. A., Nelson J., Durrant J. R., Bradley
D. D. C., Giles M., McCulloch I., Ha C., Ree M. 2006. A strong regioregularity
effect in self-organizing conjugated polymer films and high-efficiency
polythiophene: fullerene solar cells. Nature Materials 5, 197–203.
Klaine S. J., Alvarez P. J. J., Batley, G. E., Fernande T. F., Handy R. D., Lyon D.
Y., Mahendra, S., McLaughlin M. J., Lead J. R. 2008. Nanomaterials in the
environment: Behavior, Fate, Bioavailability, and Effects. Environmental
Toxicology and Chemistry 27, (9), 1825‐1851.
155
Kolkman A., Emke E., Bauerlein P. S., Carboni A., Tran D. T., ter Laak T. L.,
van Wezel A. P., de Voogt P. 2013. Analysis of (functionalized) fullerenes in
water samples by liquid chromatography coupled to high-resolution mass
spectrometry, Analytical Chemistry 85, 5867–5874.
Kroto, H. W., Heath, J. R., O’Brien, S. C., Curl, R. F. and Smalley, R. E. 1985.
C60: Buckminsterfullerene. Nature, 318, pp.162-163.
Kroto H. W., Taylor R., Walton D.R.M. 1994.The structure and reactivity of
C60. Pure & Applied Chemistry 66, 2091-2094.
Laitinen T., Petaja T., Backman J., Hartonen K., Junninen H., Ruiz-Jimenez J.,
Worsnop D., Kulmala M., Riekkola M.-L. 2014. Carbon clusters in 50 nm
urban air aerosol particles quantified by laser desorption-ionization aerosol
mass spectrometer. International journal of mass spectrometry 358, 17-24.
Lee J., Cho M., Fortner J. D.,Hughes J. B., Kim J.-H. 2009. Transformation of
Aggregated C60 in the Aqueous Phase by UV Irradiation. Environmental
Science and Technology 43, 4878-4883.
Lenes M., Wetzelaer G. A. H., Kooistra F. B. , Veenstra S. C. , Hummelen J.
C., Blom P. W. M. 2008. Fullerenes bisadducts for enhanced open-circuit
voltages and efficiencies in polymer solar cells. Advanced Materials 20,
2116-2119.
Li C., Yipab H., Jen A. K. Y. Jen. 2012. Functional fullerenes for organic
photovoltaics. Journal of Materials Chemistry 22, 416.
156
Lin S.-K., Lin S., Shiu L., Chien K., Luh T., Lin T. 1995. Fluorescence of fullerene
derivatives at room temperature. Journal of Physical Chemistry 99, 105 –
111.
Lomas S. 2015. Identifying the Increased Scope of Core-Shell Technology for
HPLC and UHPLC Chromatographers, Chromatography Today (Feb/Mar
2015) 43-44.
Lux Research. 2004. The Nanotech Report 2004. New York: Lux Research.
Marchesan S., Da Ros T., Prato M. 2005. Isolation and characterization of
nine tris-adducts of N-methylfulleropyrrolidine derivatives. Journal of
Organic Chemistry 70, 4706–4713.
Mauter M. S. and Elimelech M., 2008. Environmental Applications of
Carbon-Based Nanomaterials. Environmental Science and Technology 42
(16), 5843–5859. DOI: 10.1021/es8006904
Maynard A. D. 2006. Nanotechnology: A Research Strategy for Addressing
Risk. WoodrowWilson International Center for Scholars,Washington, DC.
Maynard A. and Michelson E. 2012. Woodrow Wilson International Centre
for Scholars, The project on Emerging Nanotechnologies. Consumer
Products Inventory of Nanotechnology Products.
http://www.nanotechproject.org/inventories/consumer (5‐12‐2012)
Moussa F., Trivin F., Ce´olin R., Hadchouel M., Sizaret P.Y., Greugny V., Fabre
C., Rassat A., Szwarc H. Early effects of C60 admininstration in swiss mice: a
157
preliminary account for in vivo C60 toxicity. 1997. Fullerenes Nanotubes and
Carbon Nanostructures 21–29.
Mueller N. C., Nowack B. 2008. Exposure modeling of engineered
nanoparticles in the environment, Environmental Science and Technology
42, 4447–4453.
Murayama H., Tomonoh S., Alford J.M., Karpuk E., 2004. Fullerene
production in tons and more: from science to industry. Fullerenes
Nanotubes and Carbon Nanostructures 12, 1-9.
Murr L. E., Bang J. J., Esquivel E. V., Guerrero P. A., Lopez D. A., 2004. Carbon
nanotubes, nanocrystal forms, and complex nanoparticle aggregates in
common fuel-gas combustion sources and the ambient air. Journal of
Nanoparticle Research 6: 241–251.
Navarro D.A., Kookana R.S., Kirby J.K., Martin S.M., Shareef A., Du J., Mc
Laughlin M.J. 2013. Behaviour of fullerenes (C60) in the terrestrial
environment: potential release from biosolids-amended soils, Journal of
Hazardous Materials 262, 496-503.
Nowack B., Bucheli T. D., 2007. Occurrence, behaviour and effects of
nanoparticles in the environment. Environmental Pollution 150, (1), 5‐22.
Núñez O., Gallart-Ayala H., Martins C. P., Moyano E., Galceran M. T. 2012.
Atmospheric Pressure Photoionization Mass Spectrometry of Fullerenes,
Analytical Chemistry 84, 5316-5326.
158
Osawa, E., 2002. Perspectives of Fullerene Nanotechnology. Kluwer
Academic Publisher, Dordrecht.
Owen, R., Handy R.. 2007. Viewpoint: Formulating the Problems for
Environmental Risk Assessment of Nanomaterials. Environmental Science
and Technology 41, (16), 5582‐5588.
Pakaninen K. 2013. Carbon nanoparticles in aquatic environments: Fate of
fullerenes (C60) in freshwaters and their effects on organisms. Publication of
the University of Eastern Finland.
Pal T., Sau T. K., Jana N. R. 2010. Reversible Formation and Dissolution of
Silver
Nanoparticles in Aqueous Surfactant Media. Langmuir, 13, (6), 1481‐1485.
Pan B., Xing B. 2012. Application and Implication of manufactured
nanoparticles in soils: a review. European journal of soil science 63, 437-
456.
Panina L. K., Kurochkin V. E., Bogomolova E. V., Evstrapov A. A., Spitsyna N.
G. 1997. Biotransfromation of fullerenes. Bocklady Biological Sciences
357,530-532.
Parthasarathy G., Bhandari N., Vairamani M., Kunwar A.C., 2008. High-
pressure phase of natural fullerene C60 in iridium-rich Cretaceous-Tertiary
boundary layers of Deccan intertrappean deposits, Anjar, Kutch, India.
Geochimica Cosmochimica Acta 72, 978-987.
159
Peralta-Videa J. R., Zhao L., Lopez-Moreno M. L., de la Rosa G., Hong J,
Gardea-Torresdey J. L. 2011. Nanomaterials and the environment: A review
for the biennium 2008–2010 Journal of Hazardous Materials 186 1–15
Perez R. A., Albero B., Miguel E., Tadeo J. L., Sanchez-Brunete C. 2013. A
Rapid Procedure for the Determination of C60 and C70 Fullerenes in Soil and
Sediments by Ultrasound-assisted Extraction and HPLC-UV. Analytical
Science 29, 533-538.
Piccinno F., Gottschalk F., Seeger S., Nowack B. 2012. Industrial production
quantities and uses of ten engineered nanomaterials in Europe and the
world. Journal Nanoparticles Research 14, 1109
Popescu L. M., van’t Hof P., Sieval A. B., Jonkman H. T., Hummelen C.. 2006.
Thienyl analog of 1-(3-methoxycarbonyl)propyl-1-phenyl-[6,6]-
methanofullerene for bulk heterojunction photovoltaic devices in
combination with polythiophenes. Applied Physics Letters 89, 213507.
Powell B. R., Bloink R. L., Eickel C. C. 1988. Preparation of Cerium Dioxide
Powders for
Catalyst Supports. Journal American Ceramic Society, 71 (2), C‐104‐C‐106.
Pycke B. F. G., Chao T.-Z., Herckes P., Westerhoff P., Halden R. U. 2012.
Beyond nC60: strategies for identification of transformation products of
fullerene oxidation in aquatic and biological samples. Analytical
Bioanalytical Chemistry, DOI 10.1007/s00216-012-6090-8.
Reed M. A. 1993. Quantum Dots Scientific American. 268, (1), 118‐123.
160
Roco, M. C., Bainbridge, W. S. 2005. Societal implications of nanoscience
and nanotechnology: Maximizing human benefit. Journal of nanoparticles
Research 7(1):1-13.
RS & RAE, Royal Society and Royal Academy of Engineering. 2004.
“Nanoscience and nanotechnologies: opportunities and uncertainties”.
Ruoff R. S., Tse D. S., Malhotra R., Lorents D. C. 1993. Solubility of C60 in a
variety of solvents. Journal Physical Chemistry 97, 3379-3383.
Saito Y., Ohta H., Jinno K. 2004. Chromatographic separation of fullerenes,
Analytical Chemistry 76, 266A-272A.
Sanchis J., Berrojalbiz N., Caballero G., Dachs J., Farre M., Barcelo D. 2012.
Occurrence of Aerosol-Bound Fullerenes in the Mediterranean Sea
Atmosphere, Environmental Science and Technology 46, 1335-1343.
Sanchis J., Bozovic D., Al-Harbi N. A., Silva L. F., Farre M., Barcelo D. 2013.
Quantitative trace analysis of fullerenes in river sediment from Spain and
soils from Saudi Arabia, Analytical Bioanalytical Chemistry 405, 5915-5923.
Sanchis J., Oliviera L.F., Leão F.B., Farre M., Barcelo D. 2015. Liquid
chromatography–atmospheric pressure photoionization–Orbitrap analysis
of fullerene aggregates on surface soils and river sediments from Santa
Catarina (Brazil), Science Total Environment 505, 172-179.
Shareef A., Li G., Kookana R. S. 2010. Quantitative determination of
fullerene (C-60) in soils by high performance liquid chromatography and
161
accelerated solvent extraction technique. Environmental Chemistry 7, 292-
297.
Song L., Wellman A.D., Huifang Y., Adcock J. 2007. Electron capture
atmospheric pressure photoionization mass spectrometry: analysis of
fullerenes, perfluorinated compounds, and pentafluorobenzyl derivatives,
Rapid communications mass spectrometry 21, 1343-1351.
Sun, Y. P., Ma, B., Bunker, C. E., Liu, B. 1995. All-cabon polymers
(polyfullerenes) from photochemical reactions of fullerene clusters in
room-temperature solvent mixtures. Journal American Chemical Society
117, 12705–12711.
Tagmatarchis N. and Shinohara H. 2001. Fullerenes in medicinal chemistry
and their biological applications. Mini-Reviews in Medicinal Chemistry 1 (4),
339–348(MA).
Taylor, R., Parsons, J. P., Avent, A. G., Rannard, S. P., Dennis, T. J., Hare, J. P.,
Kroto, H. W., Walton, D. R. 1991. Degradation of C60 by light. Nature 351,
277.
Taylor R., Barrow M. P., Drewello T. 1998. C60 degrades to C120O. Chemical
Communications, 2497-2498. DOI: 10.1039/A806726K
Tiwari J.A., Morris J.R., Vejerano E.P., Hochella M.F., Marr L.C., 2014.
Oxidation of C60 aerosol by atmospherically relevant levels of O3.
Environmental Science and Technology 48, 2706-2714.
162
Tiwari J.A., Ashraf-Khorassani M., Marr L. C. 2016. C60 fullerenes from
combustion of common fuels. Science of the Total Environment 547, 254–
260
Tiwari S. P., Namdas E. B., Rao V. R., Fichou D., Mhaisalkar S. G. 2007.
Solution-Processed n-Type Organic Field-Effect Transistors With High on
/off Current Ratios Based on Fullerene Derivatives. IEEE Electron Device
Letters 28, 880-883.
Utsunomiya S., Jensen K. A., Keeler G. J., Ewing R. C., 2002. Uraninite and
fullerenes in atmospheric particulates. Environmental Science and
Technology 36 (23), 4943-4947.
Van der Ploeg M. J. C., Baveco J. M., van der Hout A., Bakker R., Rietjens I.
M. C. M., van den brink N. W. 2011. Effects of C60 nanoparticle exposure on
earthworms (Lumbricus rubellus) and implications for population dynamics.
Environmental Pollution 159, 198-203.
van Wesemael J.C. 1955. De bepaling van het calciumcarbonaatgehalte van
gronden, Chem Weekblaad 51, 35-36.
van Wezel A. P., Morinière V., Emke E., ter Laak T., Hogenboom A. C. 2011.
Quantifying summed fullerene nC60 and related transformation products in
water using LC LTQ Orbitrap MS and application to environmental samples,
Environment International 37, 1063–1067.
Vítek P., Jehlička J., Frank O., Hamplová V., Pokorná Z., Juha L., Boháček L.
2009. Optimizing Conditions for Ultrasound Extraction of Fullerenes from
Coal Matrices. fullerenes nanotubes carbon nanomaterials 17, 109-122.
163
Wang C., Shang C., Westerhoff P. 2010. Quantification of fullerene
aggregate nC60 in wastewater by high-performance liquid chromatography
with UV-vis spectroscopic and mass spectrometric detection. Chemosphere
80, 334–339.
Wang J., Cai Q., Fang Y., Anderson T.A., Cobb G.P. 2011. Determination of
fullerenes (C60) in artificial sediments by liquid chromatography. Talanta 87,
35–39.
Wienk M. M., Kroon J. M., Verhees W. J. H., Knol J., Hummelen J. C., van Hal
P. A., Janssen R. A. 2003. Efficient methano[70]fullerene/MDMO-PPV bulk
heterojunction photovoltaic cells. Angewandte Chemie International
Edition, 42, 3371-3375.
Wijnhoven S. W. P., Dekkers S., Kooi M., Jongeneel W. P., Jong W. H. 2010.
Nanomaterials in consumer products. Update of products on the European
market in: National Institute for Public Health and the Environment: 2010.
Wood, J. M., Kahr, B., Hoke, S. H., Dejarme, L., Cooks, R. G., Ben-Amotz, D.
1991. Oxygen and methylene adducts of C60 and C70. Journal American
Chemical Society 113, 5907–5908.
WRB, World Reference Base for Soil Resources, FAO, 2006.
Xia X. R., Monteiro-Riviere N. A., Je R. 2006. Trace analysis of fullerenes in
biological samples by simplified liquid–liquid extraction and high-
performance liquid chromatography. Journal Chromatography A, 1129,
216–222.
164
Yadav B.C. and Kumar R. 2008. Structure, properties and applications of
fullerenes International Journal of Nanotechnology and Applications ISSN
0973-631X Volume 2, Number 1, pp. 15–24
Zhang K., Xu C. H., Wang C. Z., Chan C. T., Ho K. M. 1992.Systematic study
of structures and stabilities of fullerenes. Physical Review B 46, 11, 7333-
7336.
Zhao Y., Fang Y., Jian Y. 2006. Fluorescence study of fullerene in organic
solvents at room temperature. Spectrochimica Acta A 64, 564–567.
165
List of papers used in this thesis
I. Carboni A., Emke E., Parsons J. R., Kalbitz K., de Voogt P. 2013. An analytical method for determination of fullerenes and fullerene derivatives in soil with high performance liquid chromatography and UV detection. Analitica Chimica Acta 807, 159-165. Sample collection: A. Carboni Standard preparation: A. Carboni, E. Emke LC-UV/FLU analysis: A. Carboni Writing: A. Carboni Supervision and reviewing: J.R. Parsons, K. Kalbitz, P. de Voogt
II. Carboni A., Helmus R., Parsons J. R., Kalbitz K., de Voogt P. 2016. A
method for the determination of fullerenes in soil and sediment matrices using ultra-high performance liquid chromatography coupled with heated electrospray quadrupole time of flight mass spectrometry. Journal of Chromatography A, 1433, 123–130. Sample collection: A. Carboni Standard preparation: A. Carboni LC-MS analysis: A. Carboni, R. Helmus Writing: A. Carboni Supervision and reviewing: J.R. Parsons, K. Kalbitz, P. de Voogt
III. Carboni A., Helmus R., Emke E., van den Brink N., Parsons J.R., Kalbitz K., de Voogt P. Analysis of fullerenes in soils samples collected in The Netherlands (Environmental Pollution, in revision) Sample collection: A. Carboni, N. van den Brink, E. Emke Standard preparation: A. Carboni, E. Emke LC-MS analysis: A. Carboni, R. Helmus Writing: A. Carboni Supervision and reviewing: J.R. Parsons, N. van den Brink, K. Kalbitz, P. de Voogt
IV. Carboni A., Helmus R., Parsons J. R., Kalbitz K., de Voogt P. Incubation of solid state C60 fullerene under environmentally relevant conditions (submitted to Chemosphere) Experiments: A. Carboni LC-MS analysis: A. Carboni, R. Helmus Writing: A. Carboni Supervision and reviewing: J.R. Parsons, K. Kalbitz, P. de Voogt
166
Curiosities
Fullerenes were named in homage to Sir Richard Buckminster Fuller (1895
– 1983), the inventor of the geodesic domes which resemble these
molecules. For this C60 is commonly named Buckminsterfullerene or even
Buckyball. Sir Fuller was likely among the most interesting personalities of
the last century and one of the first concerned about sustainability and
human survival under the existing socio-economic system (yet he was
optimistic about the future of humans). In addition to being an architect,
systems theorist, designer and inventor (28 patents) Sir Fuller was also an
excellent writer (he published more than 30 books) and author of some
amazing quotes like the one at the beginning of this book. My absolute
favourite is:
“When I am working on a problem, I never think about beauty but when I
have finished, if the solution is not beautiful, I know it is wrong.”
R. Buckminster Fuller
The size ratio between fullerenes (C60) and a soccer ball is similar to that
between a soccer ball and the planet Earth.
Fullerenes are the most symmetric molecules in the world. There are 120
symmetry operations, like rotations around an axis or reflections in a plane,
which map the molecule onto itself. This makes C60 the molecule with the
largest number of symmetry operations.
167
Like many other interesting things, the discovery of fullerenes happened by
chance. It seems that the first 3d structure was obtained by the authors
while playing with paper hexagons and pentagons, basically an origami.
The discovery of fullerenes (in 1985), a new allotrope of carbon out of the
blue, was quite an event the scientific community and the discoverers will
receive a Nobel Prize for it (in 1996). However, at that time the possible
applications and consequences for humans were still unknown and
argument of debate. Shortly after the discovery a member of the British
House of Lords remarkably commented “It (C60) does nothing in particular
and does it very well”.
Considering that it was published on Nature, was cited some thousands
times, it won a Nobel prize and basically started a new entire field of
research, the first publication about fullerenes was pretty amazing. The title
is short and sweet (‘C60:Buckminsterfullerene’), the paper runs to less than
two pages, there is a photograph of a soccer ball but no supplementary
information, and in places the text is written in an informal style that is hard
to imagine appearing in a journal today.
Even though C60 is relatively soft under normal conditions it can be
compressed at 320 000 atmospheric pressure to create a substance so hard
it can dent a diamond (it could be the hardest material on Earth).
When a molecule of C60 is attached to twelve molecules of nitrous oxide,
the resulting structure can explode in a controlled reaction. Surprisingly, the
main application of this “Buckybomb” could be medical.
According to the rules for making icosahedron, an infinite number of
(always larger) fullerenes can exist. The smallest is a C20 (actually the
smallest is a C28…C20 is unstable). In addition, you also get “Buckyonions”,
168
i.e. multi-layered round carbon particles where fullerenes are one into the
other like an onion peel.
The solubility of fullerenes in certain solvents is weird and maxed at room
temperature. It’s something like putting sugar in your hot tea and see it
dissolving while the tea cools down.
There’s a rock (actually a mineraloid) relatively rich in fullerenes. It’s called
Shungite, has a 98 weight percent of carbon, it’s very black, relatively rare
and mostly found in Russia.
Fullerenes are also weird to work with in the lab. When dissolved into
solvents (which is something I did on a daily basis while working at the
experiments in this thesis) different molecules gives different colours in
different solvents. For instance, C60 is violet in toluene and yellow in water.
C70 instead is dark red in toluene and pink into water.
About nanoparticles in general, nanotechnology is the (recent) ability to
manipulate matter at the nano size. However, ancient populations have
used nanoparticles since thousands years ago, e.g. introducing them into
utensils in order to produce a glittering effect.
Nanoparticles sounds small but they are not that small. For instance, they
are much bigger that most of the other stuff you breath in the traffic
(oxygen, methane, NOXs, benzene, PAHs). However, for being particles,
they are absolutely small.
Nanoparticle can actually be pretty big. This is due to the current
terminology that defines nanoparticles to be so if they are very small (less
than 100 nm) in at least one dimension.
This means that a nano-wire could be very thin but also very very long. For
the same reason, a nano-sheet could be 1 nm thick but have a surface large
enough to wrap the planet. However, this is not likely to happen.
169
Acknowledgements
First, my deepest gratitude for my supervisors and promotors, Pim de
Voogt, Karsten Kalbitz and John R. Parsons for supporting my research and
trusting me and my ideas.
I gratefully aknowledge the Dutch government and the NanoNextNL project
which supported and funded my doctoral work.
I am thankful and grateful to all my colleagues that participated, inspired
and helped this project. Most especially Chiara Cerli, Joke Westerveld, Rick
Helmus Leo Hoitinga and Peter Serne at the UvA and Erik Emke, Annemarie
Kolkman and Patrick Bauerlein at the KWR.
Many thanks to my colleagues and friends Vittorio, Manuel, Christian and
Sebastian whom I can always rely on and who have been travel companions.
Many thanks also to the NanoNextNL and NanoSENSE colleagues, it was
wonderful to work with you.
The deepest thanks are to my family and loved ones, my partner Cinzia and
my son Leonardo.
170
Notes