Development of a method of extraction and quantification
of Polycyclic Aromatic Hydrocarbons
from a bituminous matrix
Spinosi Reno
Dissertação para obtenção do Grau de Mestre em
Química
Orientadores: Profª. Maria Matilde Soares Duarte Marques
Profª Silvia Zamponi
Júri
Presidente:
Prof. Pedro Paulo de Lacerda e Oliveira Santos
Vogais:
Prof. Corrado Bacchiocchi
Profª. Maria Matilde Soares Duarte Marques
Novembro de 2014
1
Acknowledgements
I would like to thank all those who helped me in the realization of this thesis work. Thanks to all the
laboratory of “API refinery of Ancona”, where I have conducted all my studies, the Doctor Claudio Sorana
who helped me in all the consideration and all the discussion of the work. I would like to thanks the
professors Silvia Zamponi, and Matilde Marques who gave me the opportunity to perform this work outside
the university.
I would like to thank my family, who helped me not only in the economical part of the work but also
helped me in all the stressed moments.
I would like to thank Diletta, she is the first person who really believed in my personal skills, she has
always spurred me to have more by myself. In all the stressed, worst, discouragements moments, she has
always helped me, despite off she was so far from me.
And the last thank is for me, thanks Reno.
2
ABSTRACT
This thesis is focused on the development of a method for the extraction and quantification of polycyclic
aromatic hydrocarbons (PAHs) from a bituminous matrix using the tools and materials available in the
laboratory. The experimental work was carried out in the laboratory of the “API Refinery of Ancona”, and
the samples used were refined by Vis-breaking processes of the refinery itself. The extraction was carried out
using an ultrasonic solubilization, the solubilized product was filtered and purified by column
chromatography, using as stationary phase a combination of silica gel and alumina. The eluted fraction
collected was concentrated, to 1 mL, in a sample concentrator with a constant temperature of 60 °C under a
nitrogen flow and subsequently injected in a GC-MS system to be quantified. A five-point calibration curve
was created using a linear regression of known concentration of internal standard, surrogate compounds and
target compounds. The concentrations used were 5, 10, 20, 50 and 100 ppb. Deuterated internal standards
were used as quantifiers and surrogate compounds were used as reference of the efficiency of the analysis
carried out. The extraction method developed showed a good ability for the extraction and quantification of
the analytes. Further studies should be conduced to improve the extraction phase of PAHs using lower
volumes of solvents and also to improve the separation step.
KEYWORDS: PAH, Bitumen, GC-MS, Extraction.
RESUMO
Esta tese aborda o desenvolvimento de um método de extracção e quantificação de hidrocarbonetos
policíclicos aromáticos (PAHs) de uma matriz betuminosa usando as metodologias e equipamentos
disponíveis no laboratório.O trabalho experimental decorreu no laboratório da Refinaria API de Ancona e as
amostras utilizadas tinham sido refinadas por processos de vis-breakingna própria refinaria.A extracção foi
efectuada mediante solubilização com ultra-sons, o produto solubilizado foi filtrado e depois purificado por
cromatografia em coluna, usando uma combinação de silica gel e alumina como fase estacionária. A fracção
do eluato recolhida foi concentrada a 1 mL a uma temperatura constante de 60 °C em atomosfera de azoto e
subsequentemente injectada num sistema de GC-MS para quantificação. Foi construída uma curva de
calibração com cinco pontos usando uma regressão linear de concentrações conhecidas de padrões internos,
compostos teste e compostosalvo. As concentraçõesutilizadasforamde 5, 10, 20, 50 e 100 ppb.Foram
utilizados padrões internos deuterados para quantificação e compostos teste para referência da eficiência da
análise. O método de extracção desenvolvido demonstrou possuir uma boa capacidade na extracção e
quantificação dos analitos. Deverá proceder-se a estudos adicionais com o objectivo de melhorar a fase de
extracção dos PAHs usando volumes menores de solventes e também de melhorar o passo de separação.
PALAVRAS-CHAVE: PAH, Betume, GC-MS, Extracção.
3
Index
1
Introduction ..................................................................................................................... 10
2
Bitumen ............................................................................................................................ 12
3
2.1
Definition.................................................................................................................... 12
2.2
The different types of bitumen ................................................................................... 12
2.3
Manufacturing processes ............................................................................................ 13
2.4
Chemical composition and colloidal structure ........................................................... 16
2.5
Rheological Properties ............................................................................................... 19
2.6
Grade System ............................................................................................................. 21
2.7
Test Methods .............................................................................................................. 23
Polycyclic Aromatic Hydrocarbons (PAHs) ................................................................. 26
3.1
Definition.................................................................................................................... 26
3.2
Sources of PAHs ........................................................................................................ 29
3.3
Physico-chemical properties....................................................................................... 31
3.4
Toxicity ...................................................................................................................... 33
3.5
Toxicokinetics ............................................................................................................ 35
4
Aim of the work ............................................................................................................... 37
5
Experimental Techniques ............................................................................................... 38
6
7
5.1
Extraction ................................................................................................................... 38
5.2
Concentration ............................................................................................................. 40
5.3
Quantification ............................................................................................................. 40
Development of the method ............................................................................................ 43
6.1
Preliminary tests ......................................................................................................... 43
6.2
Calibration Curves ...................................................................................................... 63
6.3
Study of the best operating conditions ....................................................................... 76
Conclusions and future developments ........................................................................... 97
References ............................................................................................................................ 99
4
Index of Scheme
Table 2.1 Average elemental composition of bitumen manufactured from a variety of crude
oils ............................................................................................................................................ 17
Table 2.2 Paving grade bitumen specifications for grades from 20 x 0,1 mm to 220 x 0,1 mm
penetration - Properties applying to all paving grade bitumen listed in this table ................. 22
Table 3.1 Structure of the PAHs studied .................................................................................. 26
Table 3.2 Physico-chemical properties of selected PAHs a 298 K from Chemspider. ............ 32
Table 6.1 Inlets Parameters of IPA_SIMSCAN_0404 ............................................................. 43
Table 6.2 Oven programmed temperature of the method IPA_SIMSCAN_0404 ..................... 43
Table 6.3 Concentration of target compound from the different test method performed ........ 54
Table 6.4 Oven programmed temperature of the method IPA_SIMSCAN_RENO .................. 63
Table 6.5 Oven programmed temperature of the method IPA_SIMSCAN_RENO1 ................ 63
Table 6.6 Oven programmed temperature of the method IPA_SIMSCAN_RENO2 ................ 64
Table 6.7 Inlets Parameters of IPA_SIMSCAN_RENO used for the calibration curve .......... 68
Table 6.8 Oven programmed temperature of the method IPA_SIMSCAN_RENO used for the
calibration curve ...................................................................................................................... 69
Table 6.9 Internal standard characteristic ions used for the calibration curve ...................... 70
Table 6.10 Surrogate compounds characteristic ions used for the calibration curve ............. 70
Table 6.11 Target compounds characteristic ions used for the calibration curve .................. 70
Table 6.12 Analytical performance of the calibration curve created. ..................................... 75
Table 6.13 Concentration of the surrogate and target compound in SARA 5-6-2014 and SARA
9-6-2014 ................................................................................................................................... 80
Table 6.14 Concentration of the surrogate and target compound in SARA 11-6-2014 and
SARA 12-6-2014 ....................................................................................................................... 84
Table 6.15 Concentration of the surrogate and target compound in SARA 16-6-2014 ........... 87
5
Table 6.16 Concentration of the surrogate and target compound in SARA 19-6-2014 a and
SARA 19-6-2014 b .................................................................................................................... 91
Table 6.17 Concentration of the surrogate and target compound in SARA 23-6-2014 ........... 93
Table 6.18 Concentration of the surrogate and target compound in SARA 25-6-2014 ........... 96
6
Index of Figure and Spectra
Figure 2.1 Atmospheric Distillation......................................................................................... 14
Figure 2.2 Vacuum Distillation ................................................................................................ 15
Figure 2.3 Asphaltene example of Unit Sheet .......................................................................... 18
Figure 2.4 Resin example ......................................................................................................... 18
Figure 2.5 Saturates example................................................................................................... 19
Figure 2.6 Laboratory equipment for the Ring & Ball test ...................................................... 21
Figure 5.1 Example of the different affinity of two molecules. ................................................ 39
Spectrum 6.1 Bitumen extraction Zek method without chromatography purification ............. 45
Spectrum 6.2 Bitumen extraction with toluene Zek method ..................................................... 46
Spectrum 6.3 Bitumen extraction using cyclohexane ............................................................... 49
Spectrum 6.4 Bitumen extraction using toluene and alumina .................................................. 51
Spectrum 6.5 Bitumen extraction with dichloromethane ......................................................... 53
Spectrum 6.6 Bitumen extraction using the SARA method....................................................... 57
Spectrum 6.7 SARA method with a different programmed temperature .................................. 59
Spectrum 6.8 First eluted part of the SARA method ................................................................ 60
Spectrum 6.9 Test using the IPA_SIMSCAN_RENO method ................................................... 65
Spectrum 6.10 Test using the IPA_SIMSCAN_RENO1 method ............................................... 66
Spectrum 6.11 Test using the IPA_SIMSCAN_RENO2 method ............................................... 67
Figure 6.1 Plot of the internal standard Naphthalene-d8 ........................................................ 71
Figure 6.2 Retention time list of the analyte taken from the computer interface of the
instruments ............................................................................................................................... 72
Figure 6.3 Plot of the surrogate compounds p-terphenyl-d14 .................................................. 72
Figure 6.4 Calibration curve of the fluorene compound ......................................................... 73
Figure 6.5 Calibration curve of the dibenzo[a,h]anthracene compound ................................ 74
7
Spectrum 6.12 Bitumen extraction SARA 5-6-2014 ................................................................. 78
Spectrum 6.13 Bitumen extraction SARA 9-6-2014 ................................................................. 79
Spectrum 6.14 Bitumen extraction SARA 11-6-2014 ............................................................... 82
Spectrum 6.15 Bitumen extraction SARA 12-6-2014 ............................................................... 83
Spectrum 6.16 Bitumen extraction SARA 16-6-2014 ............................................................... 86
Spectrum 6.17 Bitumen extraction SARA 19-6-2014 a ............................................................ 89
Spectrum 6.18 Bitumen extraction SARA 19-6-2014 b ............................................................ 90
Spectrum 6.19 Bitumen extraction SARA 25-6-2014 ............................................................... 95
8
Abbreviations
PAHs – Polycyclic Aromatic Hydrocarbons
BaP – Benzo[a]Pyrene
CYP- Cytochrome P450
GC-MS – Gas Chromatography-Mass spectrometry
TLC – Thin Layer Chromatography
9
1 Introduction
Bitumen is popularly called the “scrap” of petroleum refining, but this epithet is misleading because bitumen
is a product of the distillation of crude oil. Bitumens are dark, viscous liquids or semi-solids that are non-volatile
at ambient temperature and are composed principally of high molecular weight hydrocarbons.
Petroleum bitumen is known by different names throughout the world. In Europe terms like “bitumen” or
“asphaltic bitumen” are used, whereas in North America the same product is called “asphalt binder” or “asphalt
cement”. The final mixture of binder and aggregate is then called “asphalt” if we are in Europe, whereas the term
“asphalt concrete” is used in North America1.
In the 2011, it was estimated that the world use of bitumen has been of was approximately 102 million tons
per year 2. Bitumen could can be used for several purposes; among which these the primary use is for roadpaving applications (85%). When it undergoes oxidation or is mixed with polymers, it forms oxidized bitumen
which is used for roofing applications (10%). The rest of the bitumen is used for a variety of purposes, which,
however, represent a small percentage of the total usage, approximately 5%. This sector is referred to as
“Secondary Uses”2.
Figure 1.1 Global bitumen use and application areas1
Bitumen should not be confused with coal-derived products such as coal tar products which are distinctly
different substances. Coal tar products are among the by-products of the process of coal carbonization at a
temperature above 1000°C, usually normally used to make coke or to make coal gas. Coal tar products on the
one hand have a higher content of polycyclic aromatic hydrocarbons (PAHs) than bitumen, particularly in the
three- to seven-ring size range, and, on the other hand, they have a lower concentrations of paraffinic and
naphthenic hydrocarbons.
Polycyclic aromatic hydrocarbons (PAHs) PAHs are a large group of organic compounds with two or more
fused aromatic rings. These substances have been detected in air, soil, reservoir water, marine sediments, and in
some types of food products. They have a relatively low solubility in water, but are highly lipophilic.
10
PAHs are formed mainly as a result of the incomplete combustion of organic materials during industrial
activities, such as processing of crude oil and coal, and combustion of natural gas. PAHs are also formed by
other human activities including heating, combustion of refuse, vehicle traffic, cooking and tobacco smoking.
Polycyclic aromatic hydrocarbons represent a danger due to their potential carcinogenic and mutagenic
capabilities potential. In the past, chimney sweepers and tar workers were dermally exposed to substantial
amounts of PAHs and there is sufficient evidence that skin cancers developed by many of these workers were
caused by PAHs . The first discovered carcinogenic PAH was the benzo[a]pyrene from tobacco smoking.
In the last years, the amount presence of the PAHs in the final products raised the interest of petrochemical
companies. Because there is no well-established reference analytical procedure, there aren’t are no imposed
limits of PAHs to PAH concentrations in bitumens. Moreover, the analysis carried out by the few state
administrations which have actually started worrying about the problem are not easily comparable because they
result from the results of different matrix treatment methods, adjusted to fit the bituminous compound materials.
The real problem is not the quantification itself but the extraction used for the complex bituminous matrix.
11
2 Bitumen3
2.1 Definition
The definition of bitumen has varied in time, and the terminology of bitumen is the result of many years of
research. The Bitumen is one of the final products derived from the manufacturing of crude oil, and it is a
dark/black, visco-elastic material. Its adhesive, sealant and waterproofing properties have long been recognised
and are the reasons why bitumen has been used in constructions and for preservation for more than 3000 years.
Due to the waterproofing property of bitumen, discovered in the middle of the 19th century , the use of
bitumen has increased mainly in road construction. In the latter decade of 20th century the first quality
specification was therefore developed (penetration).
2.2 The different types of bitumen
The differences in raw materials allow the categorization of bitumen into different types:
1.
Lake asphalt: When subterranean bitumen (from which asphalt is mostly derived) leaks to the
surface, it creates a large puddle or lake, improperly called tar pits. It is the most extensively used
and best-known form of “natural” asphalt. It is found in well-defined surface deposits, the most
important of which is located in Trinidad.
2.
Rock asphalt: It is formed by the impregnation of calcareous rocks such as limestone or sandstone
with seepages of natural bitumen. Rock asphalt is mainly composed by stone and only 5-15% of
the total composition is asphalt. Asphalt is more easily extracted from this kind of rocks compared
to the extraction from other types of rock which can be an expensive and time consuming process.
The principal sources of these deposits are in France, Switzerland and Ragusa (Italy).
3.
Tar: It is derived from the manufacture of coke from coal and seems to be comparable with
bitumen derived from crude oil. Tar is very similar to bitumen and is used also for similar
applications. It can be described as a liquid obtained when natural organic materials such as coke
and wood are carbonised or destructively distilled in the absence of air. The tar is refined by
fractional distillation which separates it into a number of distillates and a residue, called pitch. Due
to different origins the two materials differ both in physical and chemical properties. Chemically,
(coal) tars are composed mainly of highly condensed-ring aromatic hydrocarbons and pose a
health hazard in their application due to their carcinogenicity.
4. Bitumen: Bitumen is manufactured from the heavier fractions of crude oils. There are nearly 1500
different crudes produced world-wide. Based on the yield and the quality of the product produced,
only 10-20% of them are considered suitable for the manufacture of bitumen They all differ in
their physical and chemical properties. From a physical point of view they vary from viscous black
liquids to free-flowing straw coloured liquids. Their main sources can be found in the Middle East
and in South America.
12
2.3 Manufacturing processes
Bitumen is manufactured from crude oil. The first process in the refining of crude oil is fractional distillation,
which is carried out in a crude oil distillation unit (CDU). The heaviest part of crude leaves the CDU at the
bottom, and is called Long Residue (LR).
The long residue is distilled at reduced pressure in a high vacuum distillation unit (HVU). The heaviest part
of the short residue will be used as feedstock for different grades of bitumen.
The manufacturing process is composed of various unit operations mainly divided in distillation, extraction,
conversion and blending.

Distillation/Flashing
Atmospheric distillation: Crude oil is a complex mixture of hydrocarbons differing in molecular weights and
boiling ranges. The refining of crude oils consists of several steps of physical separation and/or chemical
treatment. The first step in the process is the fractional distillation carried out in a crude oil distillation unit.
Before entering the CDU the crude oil is heated to temperatures between 350 and 380°C at a pressure slightly
above the atmospheric one. The material entering the atmospheric column is a mixture of liquid (the higher
boiling fractions of the crude) and vapour (the lower boiling fractions). In the crude oil distillation the raw
material is fractionated by differences in boiling point.
In figure 2.1 it is possible to observe the different fractions obtained from the atmospheric distillation. At the
top of the column the Gas fraction represents the volatile components, such as methane and propane, which are
the first to leave the CDU. Going down the column are depicted the remaining fractions, by increasing boiling
temperature; the Long Residue (LR) stream is the last fraction to be removed from the column. The LR
represents the heavier fraction and requires further processing before it can be used as a feedstock for the
manufacture of bitumen.
13
Gas
Naphta
Crude oil
C
D
U
Kerosene
Gasoil
Long
Residue
Figure 2.1 Atmospheric Distillation3
Vacuum Flashing: The Long residue is a mixture of vapour and liquids characterized by high boiling
temperatures and in order to increase the separation of all these components, a high vacuum distillation in a high
vacuum unit (HVU) is needed. Typical process conditions are 10 to 100 mm Hg pressure and furnace outlet
temperatures between 350 and 425°C.
In order to maintain the vacuum in the column, the non-condensable gas that enters the HVU with the feed is
removed using steam ejectors, sometimes in combination with a water ring pump.
Depending on the mode of operation, the fractions obtained by vacuum distillation of the long residue may be
applied to the manufacturing of lubrication oil or used as conversion feedstocks.
In the former case besides vacuum gas oil (VGO - the lightest product of an HVU) two or three lubricating
oil fractions (light, medium, heavy) are also produced. In the latter case, all the side streams are combined into
one flashed distillate (FD) which can be used as conversion feedstock.
From the lowest part of the HVU a stream called short residue (SR) is produced. Depending on the quality
requirement and most of all on the priority of the process, the short residue can take different ways. The SR can
be used in several ways: as bitumen feedstock, as a component for residual fuel oil, as residue conversion
feedstock (e.g. thermal cracking or residue hydroconversion), for the production of an even heavier luboil
component, and as conversion feedstock in a deasphalting unit.
From figure 2.2 is possible to observe the different types of products derived from the vacuum flashing
process
14
VGO
LFD
MFD
HFD
LR
DWO
SR
Figure 2.2 Vacuum Distillation3
As figure 2.2 shows, in some units, in order to foster the separation between the short residue and the lighter
products, a recycling of dirty washoil (DWO) is used.

Air-blowing or Oxyconversion
The blowing process is an oxidation process which involves the blowing of air through the bitumen. This
process produces a harder product with lower penetration and higher softening point. The aim of this method is
to improve the quality of potential bitumen feedstocks with greater attention to rheological characteristics such
as the Penetration Index. In this way it is possible to increase the number of crudes available and suitable to
bitumen manufacturing.
The process of bitumen blowing can be described as a conversion process in which oxidation,
dehydrogenation and polymerisation take place. It has been discovered that all the oxygen taken up by bitumen
can be accounted for by the formation of hydroxyl, carbonyl, acid and ester groups; no ether oxygen has been
detected3.
The main effect of this process results in the conversion of the short residue in their maltenes (resins,
aromatic and saturates) phase into (blown) asphaltenes. The ester family is particularly important because esters
serve as a link of two different molecules and thus contribute to the formation of materials of higher molecular
weight. This mechanism, together with the carbon-carbon bond formation, results in increased asphaltene
content.
However, the transfer of oxygen from the gaseous phase to the liquid phase is often not very satisfactory
since, due to the relatively high viscosity of the liquid bitumen, the distribution of the air bubbles may be poor.
15
The blowing process can be carried out batchwise or in continuous mode. The former has a cost advantage over
continuous blowing when bitumen demand is low.
With increasing blowing temperatures the batch-blown products may be of inferior quality than the
continuously blown products mainly due to the fact that, on average, longer residence time in batch mode may
induce some cracking.
Separately, a special variant of the continuous process also exists. It is called “catalytic blowing” and it is a
method in which phosphoric acid is used as a chemical modifier during the blowing process. The main
advantage of using a (selected) catalyst in the blowing process is the shift obtained towards the so-called higher
selectivity levels, not achievable in conventional blowing. This higher selectivity offers the opportunity to
manufacture a specific final product from harder feedstocks.

Thermal cracking/Visbreaking
As mentioned before, short residue (SR) can be used as a conversion feedstock. This means that it will be
partly converted into lighter products with higher hydrocarbon values. The difference in value between that of
the feedstock and that of the total product package is called upgrading.

Blending
The blending process, the mixture of two or more products/components to form a product with intermediate
properties, is achieved by filling a tank with such products and then by recirculating or agitating the tank
contents until a homogeneous blend has been obtained.
A modern alternative for this operation is the “in-line blender”, where the components are pumped together
in a blending line, with or without a mixing nozzle, in a ratio which is controlled by a ratio controller, which
governs control valves in the product lines.
Typical blending applications in manufacturing bitumen are the blending of:
1.
Overblown bitumen with blowing feed to form intermediate grades;
2.
A harder and a softer (finished) bitumen grade to form bitumen of an intermediate quality;
3.
Propane bitumen with short residue and/or semi-blown bitumen to form a finished penetration
grade;
4.
A semi-blown bitumen with a heavy distillate (Wash Oil) to form blowing feedstock for
second-stage blowing (to form industrial grades);
5.
Overblown product from second-stage blowing with heavy distillate form softer industrial
grades.
The advantages of using an “in-line blender” are represented by a higher flexibility, a better economy of
storage capacity, and a more constant blowing operation which increases the homogeneity of blown bitumen
tanks.
2.4 Chemical composition and colloidal structure
16
Bitumen consists of a complex chemical mixture of molecules of predominantly hydrocarbon nature with a
minor amount of heterocyclic structures and functional groups containing sulphur, nitrogen and oxygen atoms. It
also contains traces of metals such as vanadium, nickel, iron, magnesium, and calcium which are present in the
form of inorganic salts and oxides or in large organic (porphyrine) structures.
Element
Carbon
Hydrogen
Sulphur
Oxygen
Nitrogen
Composition (%)
82-88
8-11
0-6
0-0.5
0-1
Table 2.1 Average elemental composition of bitumen manufactured from a variety of crude oils3
The chemical composition/constitution is rather complex, it is possible to describe bitumen as a colloidal
system of asphaltenes dispersed or dissolved in an oily medium, maltenes. The maltenes are, by definition,
soluble in n-heptane. They are a mixture of aromatic and non-aromatic compounds, with a certain aromaticity
(peptising test). Maltenes can be further subdivided into resins, aromatics, saturates (very often called wax),
and sometimes olefins.
Asphaltenes: They are large, highly aromatic molecules with molecular weights in the range from 1000 to
100000. By definition, they are the n-heptane insoluble fraction, but soluble in toluene. Figure 2.3 shows an
example of the Unit Sheets (US’s). They are the building blocks of asphaltenes. Structurally, they are polycyclic
aromatic compounds (PAC’s) with saturated chains, heteroatoms and often one or more polar groups. A higher
concentration of bitumen’s metals is incorporated in the asphaltenic fraction than the maltenic fraction. The US’s
blocks are stacked, due to dispersive interactions between their flat PAC parts moieties, followed by further
agglomeration of the stacks as a result of Van der Waals forces and hydrogen bonding. All these effects depend
on the temperature and on the aromaticity of the surrounding medium.
17
S
N
Figure 2.3 Asphaltene example of Unit Sheet
Resins: Resins are aromatic molecules which contain hetero-atoms, are soluble in n-heptane and have
molecular weights in the range between 500 and 50000. They are very polar and are dispersing agents or
peptisers for the asphaltenes.
OH
N
O
OH
Figure 2.4 Resin example
Aromatics: They comprise the lowest molecular weight aromatic compounds in the bitumen and represent the
major proportion in the maltenes. The average molecular weights range between 300 and 5000. They consist of
non-polar carbon chains in which the unsaturated ring systems dominate and they include the class of PAHs.
Saturates: They comprise straight and branch-chain aliphatic hydrocarbons together with alkyl-naphthenes.
The average molecular weight is similar to that of the aromatics.
18
n
Figure 2.5 Saturates example
Olefins: They are hydrocarbons containing two or more double bonds. Olefins are formed during cracking
(decomposition) of large saturated hydrocarbons. They are more reactive than saturates, and typically undergo
polymerization. Olefins can also react forming cyclic hydrocarbons.
The characteristic colloidal structure of bitumen is given by asphaltenic micelles, which are formed by
asphaltenes and resins. In this particular agglomeration the resins act as a stabilising solvating layer, whereas the
asphaltenes are the heavier part.
In the presence of sufficient quantities of aromatic molecules of adequate peptising power, the asphaltenes
are fully peptised with a decrease of association, whereas a decrease in peptising power will increase the
asphaltenes association.
2.5 Rheological Properties
The fundamental rheological characterization of bitumen involves the determination of the stiffness modulus
and viscosity under fixed conditions of stress, strain temperature and loading time.
Due to the high time-consuming and the expensive laboratory equipment required for the characterization of
bitumen, a simple test of an empirical nature has been developed for a more practical characterization.
The primary rheological properties are penetration, softening point and viscosity. The results obtained from
this test can be interpreted using mathematical models, in terms of linear stress-strain relationship.
The studies on the rheological properties are very important because they have demonstrated the dependence
of constitution on rheology. It has been also found that:
1.
By maintaining a constant ratio of resins to aromatics and by increasing the saturates
bitumen is softened;
2.
On the contrary, the addition of resins hardens the bitumen, reduces the penetration index,
shears temperature susceptibility but increases viscosity
3.
Increasing the concentration of asphaltenes hardens the bitumen and increases the
viscosity significantly.

Penetration and Penetration Index
The penetration test method was first designed by Bowen in 1889, and in a modified way, is still in regular
use. In the modified method a needle of specified shape is allowed for five seconds to penetrate into a bitumen
sample under a load of 100 grams. The penetration value at 25°C is often used to indicate the grade of bitumen.
19
The temperature at which the analysis is carried out influences the properties of bitumen, for example,
increasing the temperature will increase the softness and consequently the penetration value will be higher.
Pfeiffer in 1936 found a linear relationship between the logarithm of the penetration and temperature 4.
𝑳𝒐𝒈 𝑷𝒆𝒏 = 𝑨𝑻 + 𝑪
2.1
Where A is the temperature susceptibility and varies from 0.015 to 0.06, and C stands for the “softness” of
the bitumen. Pfeiffer and Van Doormaal preferred an expression for the temperature susceptibility which would
assume a value of about zero for road bitumens. For this reason they defined the so-called penetration index
(PI) as:
PI 
20  500 A
1  50 A
2.2
The penetration index is a function of the temperature susceptibility, with a range from -3 (highly
temperature susceptible bitumens) to +7 (least susceptible bitumens). The values of the two factors can also be
derived from penetration measurements at two temperatures (T 1 and T2).
A

log Pen (T1 )  log Pen (T2 )
T1  T2
2.3
Softening point (Ring & Ball)
A characteristic property of bitumen is that it does not possess a sharp melting point, but it gradually softens
by heating. To obtain a characteristic similar to melting point, different methods were studied but the best known
method is the Ring & Ball (R&B) Softening point test. Under specified condition, a ring filled with bitumen is
heated in a water bath until the bitumen sags over a certain distance under the load of a steel ball.
20
Figure 2.6 Laboratory equipment for the Ring & Ball test

Viscosity
Viscosity is a measure of the internal friction of bitumen; from a rheological point of view it describes the
behaviour of a liquid at a given temperature and/or over a temperature range.
If the liquid is present between two parallel plates with a surface area “a” at a distance “d”, and the plates
slide with respect to each other over a path “x” when a force “f” is applied in the direction of the plates during
time “t” the viscosity, η , is:

f a
x d  t
=
𝒔𝒉𝒆𝒂𝒓 𝒔𝒕𝒓𝒆𝒔𝒔
𝒓𝒂𝒕𝒆 𝒐𝒇 𝒔𝒉𝒆𝒂𝒓
2.4
In other terms, it is the shear stress divided by the rate. The basic unit of the absolute or dynamic viscosity is
Pascal second (Pa∙s); 1 Pa∙s = 10 P (Poise)
From equation 2.5 it is possible to describe bitumen as a Newtonian liquid, due to an independence of the
dynamic viscosity from the shear applied.
Kinematic viscosity, expressed in m2/s, is used for many purposes and it is related to the absolute viscosity
by,
𝑲𝒊𝒏𝒆𝒎𝒂𝒕𝒊𝒄 𝒗𝒊𝒔𝒄𝒐𝒔𝒊𝒕𝒚 (𝒎𝟐 ⁄𝒔) =
𝒅𝒚𝒏𝒂𝒎𝒊𝒄 𝒗𝒊𝒔𝒄𝒐𝒔𝒊𝒕𝒚 ( 𝑷𝒂𝒔)
𝒎𝒂𝒔𝒔 𝒅𝒆𝒏𝒔𝒊𝒕𝒚 (𝑲𝒈⁄𝒎𝟑 )
2.5
The unit obtained from equation 2.5 is m2/s but nowadays, for easiness of calculation in conversions, mm2/s
is used (1 mm2/s=1 cSt, centistoke).
2.6 Grade System
The currently used bitumen grade system is based on penetration and has a softening point as the principal
property.
Pen-grades, Penetration or Paving grades: These grades are usually produced by vacuum flashing of crudes,
followed, in some cases, by a partial oxyconversion. They are principally used for road surfacing but they can
also be used for industrial applications and roofing. Grades are normally designed by their specified penetration
limits.
21
Table 2.2 Paving grade bitumen specifications for grades from 20 x 0,1 mm to 220 x 0,1 mm penetration - Properties applying to all paving grade bitumen listed in this table 5
Property
Test
method
Unit
20/30
30/45
30/45
40/60
50/70
70/100
100/150
160/220
Penetration at 25 °C
EN 1426
0,1
mm
20–30
30–45
35–50
40–60
50–70
70–100
100–150
160–220
Softening
EN 1427
°C
55–63
52–60
50–58
48–56
46–54
43–51
39–47
35–43
%
≥ 55
≥ 53
≥ 53
≥ 50
≥ 50
≥ 46
≥ 43
≥ 37
°C
≤8
≤8
≤8
≤9
≤9
≤9
≤ 10
≤ 11
≤ 10
≤ 11
≤ 11
≤ 11
≤ 11
≤ 11
≤ 12
≤ 12
%
≤ 0.5
≤ 0.5
≤ 0.5
≤ 0.5
≤ 0.5
≤ 0.8
≤ 0.8
≤ 1.0
Resistance to hardening at 163 °C
Retained penetration
Increase in soft. Point-Severity 1
or
Increase in soft. point-Severity 2a
EN 12607-1
°C
Change of massb (absolute value)
a
Flash point
EN ISO
2592
°C
≥ 240
≥ 240
≥ 240
≥ 230
≥ 230
≥ 230
≥ 230
≥ 220
Solubility in toluene
EN 12592
%
≥ 99,0
≥ 99,0 ≥ 99,0
≥ 99,0
≥ 99,0
≥ 99,0
≥ 99,0
≥ 99,0
When Severity 2 is selected it shall be associated with the requirement for Fraass breaking point or penetration index or both measured
on the unaged binder
b
Change in mass can be either positive or negative.
22
R-grades, Blown, Roofing or Industrial grades: Blown or oxidised grades are produced by passing air
through a soft bitumen under controlled temperature conditions. The chemical reaction results, in terms of
primary properties, in a reduction of penetration and in an increase of the softening point.
Blown or industrial grades are widely used in the manufacturing of roofing felts, waterproof papers, electrical
applications, etc.
Grades are normally specified by both their mid-softening point and by their mid-penetration value, prefaced
by “R”
Hard grades: They are manufactured in ways similar to those used for paving grades. In general
they have lower penetration values than the paving grades, and are used, for example, in the
preparation of bitumen-based paints and enamels. Hard grades are normally designed by their
softening point limits, prefaced by “H”.
E-grades or Bitumen Emulsion: Bitumen emulsions are usually prepared from bitumens with penetration of
100 dmm or softer dispersed in water with the aid of up to 1% of emulsifying agent. This process temporarily
reduces the viscosity and eases their handling and application. E-grades are mainly used for road surfacing, but
in some cases they have various industrial uses, including flooring..
2.7 Test Methods
There is a multitude of test methods used to establish the suitability of bitumens to specific applications; they
can be divided in two categories: 1) those applied to the asphalt as it is used (e.g. a mixture with aggregate for
paving grades or a combination with filler as roofing grades) and 2) those applied to the bitumen as such.

Asphaltenes6
Asphaltenes are the fraction of bitumen insoluble in low-boiling hydrocarbons (n-heptane). The
determination of asphaltenes is arbitrary and depends in the first place on the solvent used. The asphaltenes
should be denoted by a prefix indicating the solvent used. Normal-heptane is the standard. The resulting product
is called C7-asphaltene.
The asphaltene content of a bitumen provides an important insight into its constitutions particularly if
different solvents are used in conjunction. Even so, it has been found that the result of this test is not related to
performance in practice. For this purpose other data would be required.

Flash and fire Point7
The flash point is the minimum temperature at which bitumen creates a flammable vapour mixture with air,
covering all the liquid part, under controlled conditions.
The fire point is the minimum temperature at which bitumen creates flammable vapours mixed with air under
controlled conditions and undergoes combustion for 5 seconds.
23
These tests are usually required by customs and transport authorities.

Fraass breaking point8
The Fraass breaking point is used to determine the behaviour of bitumens at very low temperatures (as low as
-30°C).
In this method a steel plaque of 41*20 mm coated with 0.5mm of bitumen is slowly flexed and then released.
The temperature of the plaque is lowered by 1°C/min until the bitumen reaches a critical stiffness and cracks.
This temperature is called breaking point or Fraass temperature.
It has been found that at the Fraass temperature the bitumen has a stiffness of 2.1x10 9 Pa. This test is an
indicator of the low-temperature flexibility of bitumens. The results is the temperature at which a thin plaque
breaks under a certain strain, i.e. has a certain brittleness. The lower the breaking point, the longer the material
retains a certain flexibility when the temperature is lowered.

Rolling thin film oven test (RTFOT) 9
In this test a moving film of bituminous binder is heated in an oven up to a specified temperature (163°C), for
a given period of time (75 min) and with a constant supply of air.
The aim of this test is to simulate within the laboratory the effect of the heat and the air based on a change in
mass or on the change in the characteristics of the bitumen, such as penetration, softening point or dynamic
viscosity. The properties of this residue should be used to judge the performance of road bitumen. This method is
not applicable to some modified binders or to those whose viscosity is too high to provide a moving film.

Loss on heating (LOH)10
The loss on heating test is a milder alternative for the RTFOT.
In this analysis a sample of bitumen is positioned in a specific shelf rotating oven preheated at 163°C and
time recording is started. At the end of a 5h heating period, the product is cooled down to room temperature and
the loss in weight is registered.
The main disadvantage of the LOH is that surface-to-volume ratio is too low and a skin rapidly formed by
oxidation hampers further oxidation and evaporation.

Penetration11
The test is carried out in a sample container which is positioned in a water bath at 25°C. The needle is
lowered until its tip just makes contact with its image reflected by the surface of the test sample; this position is
called zero position. Afterwards, for a period of 5s and at a loading of 100.0g, the needle is inserted in the
sample and the penetration, expressed in dmm, is registered.
Possible variations within the scope of the test method are by changing:
1.
the load of the needle from 100 to 200g
2.
the test temperature from 25°C to 0, 5, 10, 15, 40°C
24
3.

the time of loading from 5 to 1 or 10s
Penetration Index (PI)5
The Penetration Index is not an analytical test method, but a calculation procedure to indicate the temperature
susceptibility of the penetration of a bitumen. The PI can be derived from actual penetration measurements
carried out at two different temperatures. Alternatively, as a good approximation, the PI can also be derived from
one actual penetration measurement and the R&B temperature (At the temperature of the softening point, the
penetration of a bitumen is 800 × 0,1 mm).
Ip 

20 xTR&B   500 x log P   1952
TR& B  50 x log P   120
2.6
Softening point (Ring & Ball temperature TR&B)12
Asphaltenic bitumens do not possess a real melting point: they gradually become softer upon being heated.
The determination of the softening point, therefore, is quite arbitrary.
In this test two horizontal disks, in brass rings, of bitumen supports a steel ball in a liquid bath and heated
under controlled temperature. The softening point will be the temperature at which the ball reaches the bottom of
the water bath. In the case the softening points will be above 80°C, the glycerol will replace water for the
execution of the analysis.

Solubility13
The solubility of bitumen is determined by dissolving a quantity of sample in toluene or xylene. The solution
is subsequently filtered through a layer of powdered glass in a sintered crucible. The insoluble material is then
washed, dried and weighed.
The final data is given in percentage of insoluble matter.

Viscosity
The viscosity of a liquid can be reported in two different ways: kinematic viscosity and dynamic viscosity.
Both are related to each other by the equation 2.5.
The dynamic viscosity is determined with a vacuum capillary. The time needed for a fixed volume of the
liquid to be drawn up through a capillary tube by means of a vacuum, under closely controlled conditions of
vacuum and temperature is registered. The viscosity is calculated by multiplying the flow time in seconds by the
viscometer calibration factor14.
The kinematic viscosity is also determined with a heated capillary. The time for a fixed volume of the liquid
to flow through the capillary of a calibrated glass capillary viscometer under an accurately reproducible head and
at a closely controlled temperature is determined (efflux time). The kinematic viscosity is calculated by
multiplying the efflux time in seconds by the viscometer calibration factor15.
25
3
Polycyclic Aromatic Hydrocarbons (PAHs)
3.1
Definition
The polycyclic aromatic hydrocarbons (PAHs) are a large group of over 100 organic compounds,
characterized by possessing two or more fused aromatic rings. They can be subdivided in low molecular weight
PAHs (LMW PAHs) that have two or three aromatic rings and are emitted in the gaseous phase and high
molecular weight PAHs (HMW PAHs), with five or more rings, that are emitted in the particulate phase16. PAHs
do not contain any heteroatoms in their structure and, by definition, they are cyclic compound that contain only
carbon and hydrogen.
PAHs are a large class of organic pollutants, that can undergo photodecomposition when exposed to solar
ultraviolet light17.
During the last century it was demonstrated that some of these compounds have carcinogenic, mutagenic, and
teratogenic properties1. The toxicity of PAHs is not due to the parent compound but rather to the formation of
electrophilic metabolites upon metabolic phase 1 oxidation.
Table 3.1 Structure of the PAHs studied
Naphtalene
1-methyl
Naphtalene
2-methyl
Naphtalene
Acenaphthylene
Acenaphthene
26
Fluorene
Phenantrene
Anthracene
Fluoranthene
Pyrene
Benzo[a]
anthracene
Chrysene
Benzo[b]
fluoranthene
27
Benzo[k]
fluoranthene
Benzo[a]pyrene
Indeno[1,2,3-c,d]
pyrene
Dibenzo[a,h]
anthracene
Benzo[g,h,i]
perylene
28
3.2
Sources of PAHs
There are several hundred PAHs which are originated in lower concentration from natural processes such as
forest fires and volcanic eruptions. However, most of them are produced by anthropogenic activities related to
incomplete combustion of organic materials or pyrolytic processes.
Anthropogenic PAH sources are related to the combustion of coal, oil and petrol, as well as to woodpreservation sites or processes involving the petrochemical industries 18.
In the last specific case PAHs are present in discrete amount in crude oils and generally in low concentrations
in bitumens due to the manufacturing processes. The main refinery process which removes the majority of PAHs
with three to seven fused rings is vacuum distillation 1. However, HMW PAHs are difficult to remove.
There is a continuous concern regarding the human exposure to PAHs. These compounds are pollutants
found in different matrix sources (air, water, soil, food) with variable concentrations.

PAHs in Air
When we talk about air and the exposure of humans to PAHs we have to subdivide it in Outdoor sources and
Domestic sources.
Outdoor sources: In this environment the sources of PAHs are multiple. Emissions may occur from
industries by burning fuels such as gas, oil, and coal, but also from the manufacturing of raw materials such as
aluminium. Common industrial sources of PAHs include emission in the primary aluminium industry (Søderberg
technology), in power generation (fossil fuel plants), coke production, petrochemical industries which produce
bitumen and asphalt, rubber tire and cement manufacturing, wood preservation, and waste incineration1,16.
The emission from various industrial stacks, has shown significantly high concentration of high molecular
weight PAHs from coke oven, electric furnace and heavy oil combustion. The total-PAH emission factors from
different industrial stacks (blast furnace, basic oxygen furnace., a coke oven, an electric furnace, heavy oil plant,
coal power plant and a cement plant) in the south of Taiwan were between 77.0 and 3970 μg/kg feedstock, while
Benzo[a]pyrene (the most studied model PAH) emission factors were between 1.87 and 15.5 μg/kg feedstock 19
Other major causes of PAH emissions, in urban areas, are exhaust fumes of vehicles, including automobiles,
ships, aircrafts, and other motor vehicles. This kind of sources are usually called mobile sources and the PAHs
are generally formed by synthesis from smaller molecules and aromatic compounds, pyrolysis of lubricants and
storage in engine deposits16.
Other sources come from agricultural operations and burning of brushwood and common heaters which
involve burning of organic materials.
Indoor sources: Within the households the main domestic sources of PAHs are influenced by the climate and
the domestic heating system used and cooking procedures.
It has been demonstrated that the total PAH contents range from 0.425 to 36.2 μg/m3 with the highest
concentrations generally in the kitchen areas, where low molecular weight PAH are predominant in residential
non-smoking air. However it was shown that the PAH concentrations in indoor air of smoking residences tend to
be higher than those of non-smoking residences20.

PAHs in Water
29
The presence of PAHs in water is strictly dependent upon the ability of suspended particles in water to adsorb
them, since PAHs have low solubility in water. The concentration of PAHs in water may include both dissolved
and extractable contributions. In drinking water the presence of PAHs is related to the environment from which
the water is taken.
PAH levels in uncontaminated groundwater are usually in the range of 0–5 ng/L, whereas the
concentrations of individual PAHs in coastal water and surface water are generally below 50 ng/L. However,
there are several exceptions to be found, for example, in highly industrially polluted rivers, or in those areas
which have been the recipients of atmospheric or urban depositions. In this case the concentration level can
21
reach 10 μg/L .
The collective concentration of six PAHs (fluoranthene, benzo[b]fluoranthene, benzo[k]fluoranthene,
benzo[a]pyrene, benzo[g,h,i]perylene and indeno[1,2,3-c,d]pyrene) in drinking-water indicated that generally it
did not exceed 0.1 μg/L. The concentrations of these six PAHs were between 0.001 and 0.01 μg/litre in 90% of
the samples and higher than 0.11 μg/L in 1%17.

PAHs in Soil
The soil is a storage place for PAHs due to a high concentration of organic matter, in which PAHs have a
high solubility. This gives a relative unavailability of PAHs for degradation processes with consequent
accumulation in soils. This long-term posing may lead to further potential contamination of vegetables and food
22
chains, and then cause direct or indirect exposure to humans .
PAH concentrations are strongly linked to the use of the land’s site, the environment and climate conditions.
A report by the US National Oceanic and Atmospheric Administration in the USA show indicated that typical
concentrations in forest soil range from 0.01 to 1.3 mg/kg with a medium concentration of 0.05 mg/kg. In rural
soil the concentration of PAHs is on average 0.07 mg/kg with a range from 0.01 to 1.01 mg/kg. Higher values of
PAHs were found in urban soil, with an average value of 1.10 mg/kg and a range of 0.06-5.8 mg/kg, and road
dust, with an average value of 137 mg/kg and a range of 8-336 mg/kg23.
The main sources of PAHs in soil come from vehicle and industrial emissions, forest fires, fertilizers but also
include sludge disposal from public sewage treatment plants 22.

PAHs in Food
PAHs are found in substantial quantities in some foods, which can be contaminated by the carcinogenic
pollution in the air (by deposition), soil (by transfer), or water (deposition and transfer). PAHs amounts are also
dependent on the mode of cooking, preservation and storage, and are detected in a wide range of meats, fishes,
vegetables and fruits17,24.
Food processing (drying and smoking) and cooking at high temperatures (grilling, roasting, frying) are major
sources of PAHs. A high level, 200 μg/kg, has been found for individual PAHs in smoked fish and meat; a peak
of 130 μg/kg has been reported in barbecued meat, whereas the average background is in the range of 0.01-1
μg/kg in uncooked foods. Technological processes can contaminate vegetable oils (including olive oil residues)
with PAHs. A common process is the direct fire drying, where combustion products may come into contact with
the oil seeds or oil 24.
30
3.3
Physico-chemical properties
PAHs are a group of several hundred individual organic compounds which contain two or more aromatics
rings and generally present themselves as complex mixtures rather than single compounds. PAHs are classified
by their melting and boiling point, vapour pressure, and water solubility, depending on their structure 16. Table
3.2 list some physico-chemical characteristics, of individual PAHs.
31
Table 3.2 Physico-chemical properties of selected PAHs a 298 K from Chemspider25.
PAH
No. ringsa
Mol Wtb
PLc
Log Kowd
Log Koae
Naphtalene
2
128
36.8
3.30
5.19
1-methylNaphtalene
2*
142
10.96
3.87
-
2-methylNaphtalene
2*
142
9.08
3.86
-
Acenaphthylene
2*
152
4.14
3.94
-
Acenaphthene
2*
153
1.35
3.92
6.31
Fluorene
2*
166
0.53
4.18
6.79
Phenantrene
3
178
0.087
4.46
7.57
Anthracene
3
178
0.065
4.45
7.55
Fluoranthene
3*
202
0.0081
5.16
8.88
Pyrene
4
202
0.011
4.88
8.80
Benzo[a]anthracene
4
228
1.07 × 10-4
5.76
-
Chrysene
4
228
1.68 × 10-4
5.81
-
Benzo[b]fluoranthene
4*
252
1.7 x 10-3
5.78
-
Benzo[k]fluoranthene
4*
252
1 × 10-5
6.11
-
Benzo[a]pyrene
5
252
3.9 × 10-3
5.99**
-
Benzo[g,h,i]perylene
5*
276
8 × 10-6
6.70**
-
Indeno[1,2,3-c,d] pyrene
5*
278
8.8 x 10-4
6.76**
-
Dibenzo[a,h]anthracene
5
276
3.3 × 10-5
6.75
-
a-Number of aromatic rings (* indicating the presence of a non-aromatic structure or substitution )
b-Molecular weight (a.m.u)
c-Sub-cooled liquid vapour pressure (Pa)
d-Natural logarithm of the octanol-water partition coefficient
e-Natural logarithm of the octanol-air partition coefficient
- Indicating data unavailable
** Estimated
32
The main characteristics of PAHs are the relatively low solubility in water and a higher solubility in organic
solvents, which is correlated with the lipophilicity. Most of the PAHs with low vapour pressure in the air (HMW
PAHs) are adsorbed on particles. As it is possible to observe from table 3.2, with the increase in size (more
aromatics rings) there is a concurrent decrease in volatility (P L) and increased attraction to environmental
matrices rich in lipid or organic matter (Kow/Koa). These characteristics are similar to those of other families of
persistent organic pollutants (POPs), and result in different partitioning of compound between particle and gas
phase in the atmosphere. The octanol/water partition coefficient (Kow), vapour pressure, and acqueous solubility
are chemical specific chemical properties that are of direct relevance in predicting the environmental fate of a
substance, including its multi-media partitioning behaviour, bioavailability, and resistance to biodegradation 26.
3.4
Toxicity
Several PAHs have long been recognized as having the potential to cause cancer in a wide variety of
vertebrate species as well as some invertebrates. Humans are exposed to PAHs, both outdoors and indoors and a
long-term exposure to high concentration of PAHs is associated with adverse health problems. PAHs are
transported into all tissues and the most common pathway of exposure is the inhalation of PAH vapours or dust
and other particulate matter containing PAHs, but they can also enter the body via food and water consumption
or skin contact16,26.
PAHs themselves are not carcinogenic but their electrophilic metabolites, which are more reactive, are the
main problematic compounds. PAHs are converted by isoforms of the cytochrome P450 (CYP) oxidase enzyme
complex to arene oxides, and this is the first step in mutagenesis/genotoxicity26.
O
P450
O
Reaction 3.1 Naphthalene conversation by Enzyme P45027
The metabolites formed can undergo two different metabolic pathways. In most of the cases. As described in
reaction 3.2, they can be primarily converted to nontoxic compounds by the enzyme Glutathione S-transferase
which catalyses a nucleophilic substitution reaction of glutathione, with epoxide ring opening. Secondary
pathways occur when the arene oxides escape destruction by this enzyme and toxicity may result. In these
pathways covalent bonds are formed with DNA, RNA, and proteins. DNA adduct formation is considered the
initial event of malignant cellular transformation (Reaction 3.3) because DNA adducts may lead to mutations if
they remain unrepaired.
33
OH
O
SG
+ Isomer
OH
O
SG
+ Isomer
Reaction 3.2 Formation of Glutathione adducts from Naphthalene oxide27
Epoxide
Hydrolase
HO
O
O
OH
HO
OH
O
O
N
NH
N
N
NH
HO
dR
OH
HO
HO
OH
Reaction 3.3 DNA adduct formation with benzo[a]pyrene
dR=2’-deoxyribosyl 27.
Multi-cellular organisms are characterized by mitotic division in which a cell tends to duplicate into two
identical daughter cells. This process happens only after there is adequate replication of critical cellular
components such as the replication of DNA. The changing of the cell line from the normative state is furthered
by the mutations negatively affecting the cell cycle and the cell division timing. This is because genetic stability,
which is the ability of cell replication and division of DNA without error, becomes compromised.
34
When cells are transformed several genetic aberrations might occur. Some cells may contain more than two
paired sets of chromosomes (polyploidy) or an erroneous multiplicity of chromosomes (aneuploidy). However,
some cells might also have missing chromosomes. Another commonly observed genetic aberration is the
presence of abnormal karyotypes and an increase in nuclear size26.
3.5
Toxicokinetics
The process of toxicokinetics is described by the ADME of compounds. ADME is the acronym of
Adsorption, Distribution, Metabolism and Excretion.

Absorption
PAHs are substances with high affinity to lipids. The absorption of the PAHs changes depending on their
source. They can be absorbed by inhalation in the respiratory tract, by gastrointestinal tract or by dermal
absorption.
Low molecular weight PAHs, like naphthalene, have a very low vapour pressure and they are mostly found
in the gaseous fraction, whereas high molecular weight PAHs with a greater value of vapour pressure, like
benzo[a]pyrene (BaP), are rapidly adsorbed on airborne particulate, and only very small particles can reach the
alveoli. The absorption is mediated by the epithelial barriers. In the case of highly lipophilic PAHs, before the
absorption process, which is diffusion-controlled, the compounds are retained for several hours1.
The biphasic clearance of the PAHs from lungs is well known. Test using radiolabelled benzo[a]pyrene, have
shown a rapid clearance of this compounds. The clearance of the intratracheal distribution of the benzo[a] pyrene
was of 15.3 mL/min28.
PAHs can also enter the body by oral absorption or dermal absorption, both in low concentration with respect
to the inhalation. In the former case the absorption depends highly on the aqueous solubility, the bile, and the
lipid content of ingested foods, whereas the latter case shows slow absorption and a tendency to pass through the
skin to reach the blood1.

Distribution
In vivo animal studies, mostly of the case in rats and mice, indicate that PAHs are rapidly and widely
distributed in the organism irrespectively of the route of administration. The pattern of distribution of
benzo[a]pyrene has been found to be similar in both species but differences in concentration were observed,
which is strictly related by depending on the route of exposure. In fact the trend of concentration of BaP in lung
showed a decrease of the concentration, in the intestine the concentration arise and after 360 minutes has the
higher value was found to increase up to 360 min, whereas the concentration trends in the liver showed an
increase in the first period time (5 min to 15min), and a successively subsequent decrease in the second period
time (30 min to 360 min)28.
35
Other factors that can influence the distribution of PAHs are the size and composition of the particulate
matter1.

Metabolism
The metabolism of PAHs follows the general scheme given in the Toxicity part of this document. PAHs are
aromatic compounds and the common pathways of metabolism is involve the oxidation of substances, PAHs are
first oxidized initial oxidation by microsomal CYP-dependent mono-oxygenases to form phase-I metabolites,
such as arene epoxides, phenols, and dihydrodiols. Phase-I metabolites are very reactive compounds and can
undergo two different types of metabolic reactions. For example arene epoxides react rapidly with nucleophiles,
and toxicity can result. But on the other way they can be conjugated with glutathione, sulphate, or glucuronic
acid to form phase-II metabolites, which are much more polar and water-soluble than the parent PAHs27.
A number of factors may affect the metabolism of PAHs, like the structure and also the ability of the
metabolic system to produce enough glutathione-S transferase.

Excretion and elimination
After the metabolism hepatobiliary excretion and elimination of PAHs occurs. The major route of excretion
is the urinary system in which the phase II metabolites are excreted as gluthatione, glucuronic acid or sulphate27.
36
4 Aim of the work
The present thesis work is the result of the collaboration between the research group of analytical chemistry
of UNICAM and the laboratory department of Api Raffineria di Ancona Spa, leader in the refinery of petroleum
products in Italy.
The aim of this thesis was to develop a new method for the extraction and quantification of Polycyclic
Aromatic Hydrocarbons from a bituminous matrix.
The extraction of PAHs has been carried out by taking advantage of differences in polarity and solubility of
the SARA components of bitumen (Saturates, Aromatics, Resins and Asphaltenes). They have been separated
using a chromatography column filled with silica gel and alumina.
For the concentration of PAHs a concentrator apparatus operated under nitrogen atmosphere has been used in
order to avoid the oxidation of the bitumen components.
The quantification of PAHs has been carried out using Gas-Chromatography coupled with mass spectrometry
(GC-MS) technique with a calibration curve prepared by using standard concentrations of selected PAHs from 5
to 100 ppb. The experiment can be divided in three parts. The first was the research of the best compromise
between different literature reports and the laboratory equipment. The second part of the work was the creation
of a calibration curve with the right conditions in which all the compounds were separated. The third part was
the study of the best conditions for carrying out the extraction looking at GC-MS responses.
37
5 Experimental Techniques
The method that I have developed can be divided in three macro parts: the extraction, the concentration and
the quantification of PAHs.
5.1
Extraction
The extraction is a multistep procedure in which a solubilization, a filtration and a column chromatography
are involved.
Solubilization: In a system formed by a solvent, an association colloid, and at least one other component
(the solubilizate), the incorporation of this latter component into or on micelles is called micellar solubilization,
or, briefly, solubilization29.
During the interaction, between the solid and liquid phases, interionic or intermolecular bonds are broken
and the molecules of the solvent are further apart one from the other in order to give more space to the solid
molecules. The solubilization is influenced by different factors such as:
-Temperature: by increasing the temperature of the solution, the solubility increases.
-Polarity: in most cases, solutes dissolve in solvents that have a similar polarity. Chemists use a popular
aphorism to describe this feature of solutes and solvents: "Like dissolves like". Nonpolar solutes do not
dissolve in polar solvents and the other way round.
-Molecular size: the larger the molecules of the solute, the higher their molecular weight. It is more
difficult for solvent molecules to surround bigger molecules.
The solubilization of 0.5g of bitumen was performed using 20 mL of mixture of n-heptane (purity
≥99.78%) purchased from Chevron Phillips Company, and toluene (purity ≥99.7%) purchased from SigmaAldrich with a ratio of 85:15 using two different solubilization techniques.
The first process was a simple stirring for 2h at ambient temperature, using a magnetic stirrer. In this
procedure, the speed of the stirring increases the speed of the process. The stirring increases the movement of
the solvent molecules, exposing the solute to fresh portions of solvent thus simplifying the solubilization. As
molecules in liquid substances are in constant movement, the process would take place anyway with the only
difference that it would take more time.
The second process was an ultrasonication (40 kHz) in a water bath at constant temperature (25°C) for 1h.
The sonication is a process in which sound waves are used to agitate particles in a solution. Wave properties of
sound create alternating regions of high and low pressure in the liquid, generally called gaseous cavitation.
This term describes the formation of microscopic vacuum bubbles and their collapse into solution.
38
These vibrations are able to disrupt molecular interactions (e.g. between molecules of water), break clumps
of particles apart, and lead to mixing. The ultrasonication technique increases the rate of solute solubilization
and at the end of the sonication process the solution appears as a suspension.
Filtration: This is the process of segregation of phases; e.g. the separation of suspended solids from a liquid
or gas, usually by forcing a carrier gas or liquid through a porous medium 29.
The fluid that passes through the filter is called filtrate, and the solid parts which are bigger with respect to
the pore size of the filter are retained. This separation has not been complete, in our case because, due to the
pore size and filter thickness, because some filtrate contains fine particles.
The filtration step was performed using ionic chromatography syringes and filters. The filters used were
made in Phenex with a porosity of 0.20μm. Through the filtration process, the asphaltenic and insoluble parts
of bitumen have been separated very efficiently.
Column Chromatography: Chromatography is a separating process that is achieved by distributing the
components of a mixture between two phases, a stationary phase and a mobile phase. Those components, held
preferentially in the stationary phase, are retained longer in the system than those that are distributed
selectively in the mobile phase. As a consequence, solutes are eluted from the system as local concentrations in
the mobile phase according to their increasing distribution coefficients with respect to the stationary phase; and
to a separation is achieved30.
During the chromatography, in each part of the column, the molecules are involved in dynamic processes.
These processes can be described as dynamic equilibriums in which molecules are transferred between the two
phases. These mechanisms, extraction-dragging- extraction…, occurs, in succession, in the whole
chromatography column, and determine the separation of the substances by affinity.
Figure 5.1 Example of the different affinities of two molecules.
The interactions between the molecules and the two phases are weak interactions such as hydrogen bonds,
dipole-dipole and Van der Waals interactions. In all these interactions the polarity of the two phases, is the
driving force for the separation.
39
The stationary phase here used was composed by two different materials: silica gel and alumina. The silica
gel used (60-200 mesh) was purchased from Grace Davison, while aluminum oxide (activated, basic,
Brockmann I) was purchased from Sigma-Aldrich.
To separate the mixture two different eluents, with different polarities were used. The first elution was
carried out using 50 mL of n-heptane, whereas the second 50 mL of a mixture toluene:n-heptane (80:20 v/v).
Then the eluate that contained the PAHs was collected and, at a later step, concentrated.
5.2
Concentration
The concentration process is a simple operation in which the solution is concentrated using temperature
and/or a gas flow (Air, N2). In this method a Techne Sample concentrator, combined with Techne DB-2A
heaters was used. The sample concentrator consists of a gas chamber mounted above a Dri-Block heater into
which vessels containing the samples were placed. Hypodermic needles carried the gas down from the
chamber into the test tubes. At the same time, the samples were heated from below, and the flow of the gas was
directed over the surface of the sample to displace the evaporated solvent.
5.3
Quantification
The quantification step of this method has been performed using the Gas Chromatography-Mass
Spectrometry (GC-MS) technique. In this analytical method the features of the gas chromatography and mass
spectrometry are combined to identify the target molecules with high precision.
Gas Chromatography: This is one of the most widely applied analytical separation techniques. It provides a
method whereby complex mixtures can, in one operation, be fractionated and analysed qualitatively and
quantitatively. In a gas chromatograph the mobile phase is generally an inert gas (N2,H2,He), called carrier gas,
and the stationary phase could be liquid or solid. Due to the inertness of the mobile phase, the performance and
the separation mechanism of a GC are determined by the physico-chemical characteristics of the stationary
phase.
There are two types of columns in commonly use in GC, the conventional packed column and the open
tubular column. The former is usually 2 to 4 mm I.D.,1 to 4 meters long and, packed with a suitable adsorbent,
is mostly used for gas analysis. As a result of the simpler injection procedure and the more precise sampling
method, the packed column tends to give greater quantitative accuracy and precision. However, despite its
problems with sample injection, the open tubular column is seen as the 'state of the art' column and is by far the
most popular column system generally used. The length of open tubular columns ranges from about 10 m to
100 m and can have internal diameters from 100 micron to 500 micron. The stationary phase is coated on the
internal wall of the column as a film 0.2 micron to 1 micron thick 31.
The sample is injected using a syringe and depending on the type of column, different quantities and
methods of injection are used. In the case of a packed column the common procedure is the injection through a
silicone rubber septum directly into the column packing or into a flash heater. Although the latter tends to
40
produce broader peaks, it also disperses the sample radially across the column 31. In the case of the capillary
column, due to the small size of the column, a small quantity of sample is injected using a split or splitless
system. In the former case, the split system, after the injection of the sample into the glass liner a portion of the
carrier gas sweeps past the column inlet to waste. As the sample passes the column opening, a small fraction is
split off and flows directly into the capillary column. In the latter case, the splitless system, the injection of the
sample follows the same procedure of the split mode, but in this case, the split valve is closed for a few
seconds. When the valve is opened again the sample will enter the column, resulting then in an increase of
sensitivity.
After the injection, the sample passes through the column and it is finally detected. The common detectors
are the Flame Ionisation Detector (FID), the Electron Capture Detector (ECD) or , as in the case of this thesis,
the Mass Spectrometer.
Mass Spectrometry: The concept of mass spectrometry is relatively simple: a compound is ionized, the ions
are separated on the basis of their mass/charge ratio, and the number of ions representing each mass/charge
“unit” is recorded as a spectrum32
Mass spectrometry is an analytical tool from which different information can be obtained such as molecular
weight, molecular formula, structure of molecules, isotopic patterns or characterization of proteins, often using
tandem MS (MS-MS).
Schematically, the MS instrument is divided into four parts, the Sample introduction, Ionization Chamber,
Ion Separation sector and the detector, all connected to a computer system that controls the operations. In the
case of the GC-MS, the sample arrived as a vapour from the chromatography column.
Generally, the method of ionization is independent on the method of ion separation. The most widely used
methods for generating ions from relatively volatile sampler are Electron Impact (EI) and Chemical Ionization
(CI). In the electron impact system, a vapour phase sample is bombarded with high energy electrons (generally
70 eV), which eject an electron from a sample molecule to produce a radical cation known as molecular ion.
Chemical ionization uses a soft ionization in which a reagent gas (usually methane isobutene or ammonia)
is introduced into the source, and ionized. The sample molecules collide with the ionized reagent and undergo
secondary ionizations by proton transfer32.
The mass analyzer, which separates the mixture of ions that are generated during the ionization step by m/z
in order to obtain a spectrum, is the heart of each mass spectrometer. The most used mass analyzers are the
Quadrupole, the Ion-Trap and the Time of Flight types.
A quadrupole mass analyzer consists of four cylindrical rods which are mounted parallel to each other, at
the corner of a square with a constant DC voltage, modified by an applied radio frequency voltage. The ions
are introduced into the quadrupole, in the centre, and only the ions with a certain m/z value will possess a
stable trajectory, whereas all the ions with different m/z values will travel unstable or erratic paths and collide
with the rods or pass outside.
The ion trap generally consists of three electrodes, one ring electrode (hyperbolic inner surface) and two
hyperbolic endcap electrodes.
The ion trap can work using three basic modes: i) with a fixed RF voltage and no DC between the
electrodes; ii) using a DC potential across the endcaps, and iii) using the DC potential coupled with an
oscillatory field between the endcaps.
41
The Time of Flight analyzer is simpler. The ions are accelerated through a potential (V) and are allowed to
drift down the tube to a detector. While entering the tube all the ions have the same energy and fly down the
tube. By knowing the length of it and the mass of the different ions which arrived to the detector in different
times, the mass of the ions is calculated.
The instrument used for the development of the method was an Agilent Technologies GC-MS. The 7890A
Gas Chromatograph was equipped with a VF-5ms (30m x 0.25mm, 0.25μm of ID Factor four) capillary
column the Varian Company. The column used is a non-polar column equivalent to a 5% phenyl, 95%
dimethylpolysiloxane low bleed phase. The 5975C VL Mass spectrometer used, an electron impact ion source
and a quadrupole, analyzer.
42
6 Development of the method
6.1
Preliminary tests
Unfortunately, a standardized method for the extraction and the quantification of PAHs from a bituminous
matrix has not developed,the procedure was based on literature reports for some different matrix methods,
adjusted to fit the bituminous material. To find the way and understand better how bitumen performs under
different procedure, some preliminary tests were conducted.
The starting point was the Zek method33, which was originally applied for a polymer matrix. The test
instructions state that 0.5g of the sample shall be mixed with toluene and internal standards. The internal
standards to be used are naphthalene-d8, pyrene-d10 or anthracene-d10, or phenanthrene-d10 and benzo(a)pyrened12 or perylene-d12 or triphenylbenzene.
The mixture is then placed in a ultrasonic bath for 1 hour at constant temperature of 60 °C for the
extraction, and reduced to 1 mL in a rotavapor. After the concentration, a chromatography column, using silica
gel as the stationary phase and petroleum ether as eluent, is performed and the eluate is spiked with 1 mL of
toluene and concentrated in a rotavapor under N2. The quantification is performed with a gas chromatograph
with a MS detector operating in SIM mode.
Due to the fact that this test was previously developed for polymers, the first approach was a simple
extraction in toluene with the ultrasonic bath at 60°C for 1 h. At the end of the extraction the solution was
cooled down and an aliquot of 900μL plus 100μL of a 1 ppm standard mixture were injected in the GC-MS
system. The standard mixture was composed by surrogate compounds (p-therphenyl-d14, 2-fluoro-1,1’biphenyl, 3-methylcholanthrene) and internal standards (naphthalene-d8, anthracene-d10, phenanthrene-d10,
pyrene-d10, chrysene-d12, perylene-d12).
The GC-MS method (IPA_SIMSCAN_0404) used for this injection was used also for all the preliminary
test. The gas chromatographic parameters of the method were:
Injection: 1μL
Inlets: Splitless
Table 6.1 Inlets Parameters of IPA_SIMSCAN_0404
Temperature (°C)
320
Pressure (psi)
9
Septum purge flow (mL/min)
3
Flow Column: 1.2mL/min
Table 6.2 Oven programmed temperature of the IPA_SIMSCAN_0404 method
Rate (°C/min)
Initial
1
2
3
50
10
50
Temperature (°C)
38
120
280
340
Hold time (minutes)
1
0.5
0.05
5
43
Mass spectrometry parameters of the method:
Mass instrument windows: 46.00 amu to 320.00 amu
Solvent delay: 5 minutes
Mass source (Electron Impact) temperature: 300°C
Mass Analyzer (Quadrupole) temperature: 150°C
Quantification done in SIM mode.
From the spectra obtained in the test (Spectrum 6.1) performed it is possible to observe that the bituminous
matrix is a very complex mixture of different components and that toluene at 60°C is able to dissolve a
significant number of compounds. Due to this matrix problem a second test always using the Zek method was
performed with the difference that in this case the complete method were performed, with some adjustment.
A series of tests was performed in which the sample was initially dissolved in 20mL of toluene with 100μL
of the standard mixture at 1 ppm, and ultrasonicated at 60°C for 1h. Due to the absence of a rotavapor, the
sample was directly added to a chromatography column and 50 mL of n-heptane were used as eluent. The
chromatographic purification step was then performed using n-heptane which is a non-polar solvent like
petroleum ether, which unfortunately, was not available in the laboratory. The sample was collected and spiked
with 1 mL of toluene and at a later step was concentrated at the sample concentrator, under nitrogen flow. The
temperature of the drying block was fixed at 60°C.
44
Spectrum 6.1 Bitumen extraction using the Zek method without chromatography purification
Total Ion
Scan Mode
Total Ion
Sim Mode
45
Spectrum 6.2 Bitumen extraction with toluene Zek method
Total Ion
Scan Mode
Total Ion
Sim Mode
46
From Spectrum 6.2 it is possible to observe two different spectra. Above is the total ion in SCAN mode,
whereas below is the total ion in SIM mode. At a first analysis it is possible to observe some differences
compared to Spectrum 6.1, obtained without the chromatographic purification. The first observation to be made
is the higher quality of the chromatogram in the SCAN mode of the second test performed. This indicate that the
chromatography purification is able to remove unwanted substances, which increases the signal intensities in
chromatogram but prevent crippling the GC-MS system. In fact, considering the different typical components of
bitumen, the asphaltenic part is retained from the chromatographiy column. From the Scan mode it is also
possible to observe some characteristic peaks corresponding to the saturated part of the bitumen. From the SIM
mode it is possible to observe the ability of the method to recognize and quantify the compounds.
From this series of tests important characteristics were taken into consideration, such as the future possibility
of a chromatographic purification, which helps in the separation of the bitumen components, and also the ability
of the extraction in toluene at 60°C is able to dissolve a significant number of compounds.
A different procedure34 was performed, from which other important information was obtained. In this case
the procedure steps included a solubilization of 0.3 g of bitumen plus the surrogate in 12 mL of n-hexane
standards for 2 hours on a bench stirrer. The resulting solution was vacuum filtered in the next step to remove the
solid particles not previously solubilized. After the filtration a Matrix Solid Phase Dispersion was performed
using 15 g of silica gel. The mixture plus 5.0 g of alumina and 0.5 g of copper powder, were added to a
chromatography column. The eluent was composed by four different mixtures. The first eluent was 50 mL of nhexane and the others three aliquots were mixtures of n-hexane-ethyl acetate in different proportions:
36.0mL n-hexane + 4.0 mL ethyl acetate
16.0mL n-hexane + 4.0 mL ethyl acetate
14.0mL n-hexane + 6.0 mL ethyl acetate
The eluted part was collected and concentrated. Subsequently, a GC-MS analysis is performed for the
characterization of the PAHs.
The first test performed using the guideline of this procedure was to solubile 0.3g of bitumen in 12 mL of
cyclohexane (purity >99.7% for HPLC) purchased from Sigma-Aldrich, plus 100μL of a standard mixture at 1
ppm. The choice of cyclohexane was made by considering that the n-hexane present in the laboratory had purity
lower than 95%, whereas the purity of cyclohexane was about 99.7%. The second observation for the choice of
cyclohexane is that the index polarities of the two solvents are comparable, 0 for n-hexane and 0.2 for
cyclohexane.
After 2-hour long solubilization on a bench stirrer the solution was filtered using a syringe and filters for the
ionic chromatography. At this point, the MSPD method was not performed due to the absence of concrete
instruments for the packaging of the column after the dispersion. The filtered solution was added to the column
chromatography prepared as the method explains: 5.0g of alumina were positioned below the 15.0 g of the silica
gel (the powder copper was not used in this test). The elution of the chromatography column was performed
using 50 mL of n-heptane as first eluent and a mixture of n-heptane:ethanol for the gradient polarity elution. Due
to the different polarity indeces of ethanol (5.2) and ethyl acetate (4.4) different proportions were used to be sure
to obtain the same gradient polarity, recommended in the MSPD method. Using the polarity of the solvent to be
replaced and the polarity of the solvent to use it is possible to obtain a solution with the same polarity changing
the percentage of the solvent by the equation 6.1:
47
% 𝒐𝒇 𝑬𝒕𝒉𝒂𝒏𝒐𝒍 =
𝑷𝒐𝒍𝒂𝒓𝒊𝒕𝒚 𝒐𝒇 𝑬𝒕𝒉𝒚𝒍 𝒂𝒄𝒆𝒕𝒂𝒕𝒆∗𝟏𝟎𝟎
𝑷𝒐𝒍𝒂𝒓𝒊𝒕𝒚 𝒐𝒇 𝑬𝒕𝒉𝒂𝒏𝒐𝒍
6.1
The three aliquot after the calculation was:
-
36.5 mL of n-heptane + 3.5 mL of ethanol
-
16.5 mL of n-heptane + 3.5 mL of ethanol
-
14.8 mL of n-heptane + 5.2 mL of ethanol
After the elution with the different polarity gradient the collected fractions were concentrated, to 1 mL, at the
sample concentrator under nitrogen flow and a temperature of 60°C. At the end of the concentration phase the
sample was then injected in the GC-MS using always the same method explained above.
48
Spectrum 6.3 Bitumen extraction using cyclohexane
Total Ion
Scan Mode
Total Ion
Sim Mode
49
From Spectrum 6.3 it is possible to observe a decrease in the signal compared to Spectrum 6.1 and Spectrum
6.2 in the SCAN mode. In this case the extraction also gave results due to the ability of the method to quantitate
the compounds in the SIM mode. The bigger difference between the SCAN mode of the two methods is the
decrease in peaks related to saturated compounds. This decrease of aliphatic compounds is due to the different
polarity gradients used for the elution in the chromatographic purification, but also to the use of the alumina,
which enhanced the separation between saturated and unsaturated compounds.
Another test was performed changing the dissolution and chromatography solvents. In this test 0.3g of
bitumen was dissolved in 12 mL of toluene plus the standard mixture and stirred for two hours. The solution was
filtered as before and alumina and silica gel were used as stationary phase in the column chromatography. The
first elution was done always using again 50 mL of n-heptane but the three aliquots, with increasing gradient
polarity in this case were prepared using n-heptane and toluene with a polarity index of 2.4:
-
32.8 mL of n-heptane + 7.2 mL of toluene
-
12.8 mL of n-heptane + 7.2 mL of toluene
-
10.3 mL of n-heptane + 9.3 mL of toluene
The eluate was collected and concentrated to 1 mL at 60°C, under nitrogen air in the sample concentrator.
After concentration the sample was injected in the GC-MS always using again the IPA_SIMSCAN_0404 as
quantification method.
From the Spectrum 6.4 it is possible to observe a decrease in the intensity of the peak in the SCAN mode
(above) and in the SIM mode (below) which was attributed to a decrease in the quantity of resins and saturated
compounds extracted.
50
Spectrum 6.4 Bitumen extraction using toluene and alumina
Total Ion
Scan Mode
Total Ion
Sim Mode
51
From this series of tests, important characteristics were taken into consideration. One was the importance of
the chromatographic purification, in this case performed using two different compounds for the stationary
phases, alumina and silica gel. The chromatographic purification performed using alumina helps in the
separation of the saturated components of bitumen from the aromatic fraction which contain the PAHs. Also the
use of a polarity gradient in the elution process enhanced this separation, which helped in the separation of resins
and asphaltenic residue present in the original mixture.
Due to the lengthy test duration, additional methods in which the PAHs are extracted from different matrices
were performed. The principal reason to conduct additional studies was the decrease of the time test duration and
also the attempt to find another methods which took advantages from other properties to increase the efficiency
of extraction of the PAHs from bitumen.
Thus, another test was performed in which the extraction was done by dichloromethane, the separation was
performed by solid phase extraction and the quantification of PAHs by HPLC with UV detection at 254 nm35.
The test instructions indicated that the sample is extracted in an ultrasonic bath using dichloromethane (2x 40
mL). After the extraction, the mixture is centrifuged and evaporated to dryness. The residue is then dissolved in a
mixture of 4 mL of acetonitrile:water (1:1 v/v) and purified by solid phase extraction (SPE) using C18 octadecyl
columns.
To perform this method some changes were made to provide the extraction of PAHs from bitumen. The
solubilization was done using 0.5 g of bitumen in 40 mL of dichloromethane plus 100 μL of the standard mixture
at 1ppm. The ultrasonic stirring was performed at 25°C for one hour, with a power of 40 kHz, and, in the next
step the sample was centrifuged at 4000 rpm for 10 minutes. The liquid fraction was collected and evaporated
under nitrogen air at ambient temperature. When the solution was dried, the dried part was then dissolved in
10mL of acetonitrile:water (1:1 v/v), and to achieve a better dissolution an ultrasonication for 15minutes at 25°C
was performed. The solution was purified by solid phase extraction using a filter of C18 octadecyl polimers, and
the extracted fraction was recovered with 15mL of dichloromethane. After concentration at the sample
concentrator under nitrogen air and ambient temperature, the sample was injected in the GC-MS system and
quantitated using the IPA_SIMSCAN_0404method.
52
Spectrum 6.5 Bitumen extraction with dichloromethane
Total Ion
Scan Mode
Total Ion
Sim Mode
53
From Spectrum 6.5 it is possible to observe a great decrease in the signal both in SCAN and SIM mode,
which is explained by an ineffective extraction for the bituminous matrix. From the SIM mode it is possible to
observe the absence of different internal standards and target compounds which were lost during the procedure
step.
Table 6.3 Average concentrations of target compounds obtained with the different test methods performed.
Compound Name
Bitumen
extraction
toluene Zek
Bitumen extraction
with Cyclohexane
Bitumen extraction
Toluene Alumina
Bitumen extraction
dichlorometane
Units
p-Terphenyl-d14
60.43
101.68
101.15
0.66
μg/L
Naphthalene
Naphthalene,
1-methylNaphthalene,
2-methyl1,1'-Biphenyl,
2-fluoroAcenaphthylene
152.21
98.07
149.75
521.69
μg/L
563.90
251.41
247.66
929.50
μg/L
555.22
245.62
198.96
617.80
μg/L
142.51
119.32
132.38
0
μg/L
18.38
10.11
9.69
0
μg/L
Acenaphthene
112.50
55.83
118.88
33.81
μg/L
Fluorene
209.85
117.05
99.60
47.86
μg/L
Phenanthrene
916.97
556.19
594.76
0
μg/L
Anthracene
224.87
124.59
135.51
0
μg/L
Fluoranthene
141.99
129.65
107.32
0
μg/L
Pyrene
Benz[a]
anthracene
Chrysene
Benz[b]
fluoranthene
Benz[k]
fluoranthene
Benzo[a]
pyrene
3-Methyl
cholanthrene
Indeno[1,2,3-cd]
pyrene
Dibenzo[a,h]
anthracene
Benzo[g,h,i]
perylene
678.27
766.10
592.28
0
μg/L
494.32
676.60
242.17
0
μg/L
4703.96
1095.78
554.28
206.94
μg/L
259.28
57.80
178.53
73.01
μg/L
280.08
66.61
1097.48
80.08
μg/L
1144.8
157.25
3167.31
242.21
μg/L
137.25
263.70
2694.21
239.30
μg/L
44.64
12.89
97.54
0
μg/L
124.20
286.20
915.91
0
μg/L
421.01
145.68
1019.54
48.43
μg/L
54
From the Table 6.3 it is possible to observe the different averages of the concentration found for the target
compounds during all the test methods performed. The extraction via dichloromethane was performed only once
due to the unsatisfactory results obtained. Looking at them and analyzing the spectra from the different tests
performed, some consideration were drawn.
The first important consideration is that the extraction with dichloromethane is not efficient. Therefore, was
rejected.
The second consideration was the greater concentration in low molecular weight PAHs in the Zek method
shows the aptitude of toluene for the extraction of these compounds. Toluene was also used in the method with
alumina as stationary phase and an increase in the high molecular weight PAHs was observed. This indicates
that toluene is a good solvent for the extraction but a disadvantage is the higher solubility of the resins and
asphaltenic fractions of the bitumen as shown in the Spectrum 6.2.
The third important consideration was the great enhancement of the separation of saturated and unsaturated
compounds observed using alumina, which is shown in Spectrum 6.3, in which the classical peaks related to
saturated compounds are absent.
All these considerations explain that for the extraction of the PAHs, which are contained in the aromatic
fraction of the bitumen, a separation of the bitumen components is needed. To perform this separation the
generally called SARA (Saturates, Aromatics, Resins, Asphaltenes) separation was used as reference36.
The method, used as reference, is a simple thin layer chromatography (TLC) in which the bitumen
components are separated by retention factors, depending on the polarity of the eluent.
The procedure indicates that 100 mg of bitumen is dissolved in dichloromethane, which is added till
complete dissolution of the components in ultrasonic bath. After solubilization different sequential TLC are
performed using different polarities of the eluent. The first eluent is n-heptane which is able to separate the
saturates with a retention factor above 0.6. The second eluent is a mixture of toluene:n-heptane (80:20 v/v),
which is able to separate the aromatic fraction with a retention time below 0.6. The third eluent is a mixture of
dichloromethane:methanol (95:05 v/v), which due to the higher polarity is able to elute the resin fraction of the
bitumen. The asphaltenes are not eluted and have a retention time of 0.
At the end of the three TLC the layer is positioned in an lactroscan Mark V Analyzer with a flame ionization
detector, which is able to quantitate the bitumen components (figure 6.1).
55
Resins
Aromatic
Saturate
Asphaltenes
Figure 6.1 Typical TLC/FID chromatogram of petroleum residue36
The first approach for the extraction of the aromatic fraction from bitumen using the SARA analysis was to
solubilize 0.5g of bitumen in 20 mL of n-heptane:toluene (85:15), plus 100μL of the standard mixture at 1 ppm,
and stir for two hours on a bench stirrer at ambient temperature. At the end of the stirring process the solution
was filtered using a syringe and filter for ionic chromatography to eliminate all the insoluble substances. The
filtered solution was added to a chromatography column which was packed using n-heptane and contained 5g of
alumina and 15 g of silica gel as stationary phase.
The first elution was performed using 50 mL of n-heptane, to eliminate all the saturates present in the
column. The second elution was performed using 50 mL of a mixture of toluene:n-heptane (80:20 v/v) and the
aromatic fraction was collected.
The eluted fraction was concentrated to 1 mL at the sample concentrator under nitrogen at a fixed
temperature of 60°C. At the end of the concentration the sample was injected at the GC-MS system using
IPA_SIMSCAN_0404 as method of quantification.
56
Spectrum 6.6 Bitumen extraction using the SARA method
Total Ion
Scan Mode
Total Ion
Sim Mode
57
From the top of Spectrum 6.6 it is possible to observe a good separation of the saturates and aromatic
components of bitumen, due to the absence of the classical peaks related to saturated compounds. From the SIM
mode, it is possible to observe that the target compounds could be quantitated. Also, it is possible to observe in
SCAN mode an increase in the signal in the region of the spectrum from 19.00 minutes to 22.00 minutes, which
can be explained by the presence of a significant quantity of high molecular weight aromatic compounds. Due to
the high temperature at which the column was operating in the same region, the bleeding effect increased the
signal and also decreased the sensitivity of the system to the target compound. To try to avoid this problem a
different programmed temperature was used and the result is shown in Spectrum 6.7.
For this analysis, the same sample used for the analysis of SARA shown in Spectrum 6.6 was used and the
temperature was controlled at a rate of 10°C/min from 120°C to 340°C with an hold time of 6 minutes at 340°C.
What it is possible to observe the decrease of the signal in the region after 19.00 minutes, which can also be
explained as the result of a greater separation of the aromatic components in the GC-MS system. This controlled
temperature was, in the next step of the work, used for the creation of the calibration curve which was then
adopted for the quantification of the PAHs.
To confirm the ability of the system to separate the compounds an injection of the first eluted fraction of the
column chromatography was performed.
From Spectrum 6.8 it is possible to establish that some target compounds were able to leave the column with
the saturates. The first reflection was that the column was not sufficiently well packed to block the aromatic
compounds, but other tests were performed and the results were always been the same. Then problem was
attributed to a polarity higher than that of the solubilization solution, which was able to increase the flowing of
the aromatic compounds trough the column. To avoid this problem in the stationary phase of the
chromatographic column another layer of alumina was added, which was used as a block system for all the
aromatic compounds.
58
Spectrum 6.7 SARA method with a different programmed temperature
Total Ion
Scan Mode
Total Ion
Sim Mode
59
Spectrum 6.8 First eluted part of the SARA method
Total Ion
Scan Mode
Total Ion
Sim Mode
60
At this point all the considerations discussed above were taken into account and used to develop the skeleton
of the method.
The starting point was the selection of the best solvent for the solubilization of bitumen either a solvent or a
mixture of solvents able to dissolve all components in the aromatic fraction of the bitumen. After the
solubilization, the selection of the best chromatographic purification to separate the bitumen components had to
be done. And, at the end, the best quantification method of the target compounds had to be selected.
Solubilization: To solubilize the components solubility properties of the bitumen were used. As indicated in
chapter 2.4 bitumen is composed by asphaltenes and maltenes (Saturates, Aromatics, and Resins). The
asphaltenic components of bitumen are not soluble in n-heptane while the maltenic fraction is soluble in this
solvent.
Taking advantage of these properties, the solvent used for the solubilization was a mixture of nheptane:toluene with a ratio of 85:15. The mixture ratio is the result of different consideration:
-
To attempt a prior separation of the bitumen components.
-
All the maltenes should be dissolved.
-
If a small amount of asphaltenes are present at the end of the solubilization, they will be
separated in the next step.
From the preliminary tests performed it was observed that the bitumen/solvent ratio was of 0.1 g of bitumen
per 4 mL of solvent. For this method the same ratio was used. The quantity of bitumen used was about 0.5 g and
the solvent mixture used was about 20 mL.
Filtration: For the filtration step the syringe and a filter (0.2μm) of ionic chromatography were always used,
due to the easiness of utilization.
Column chromatography: To purify the sample and try to obtain only the aromatic fraction from the bitumen,
a chromatographic purification was performed.
After the solubilization and the filtration steps, the mixture contained maltenes, composed by saturates,
aromatics and resins, and also a small amount of asphaltenes.
To perform the separation the physico-chemical properties of the mixture components present were
investigated. The most studied property was the polarity of the components. The results obtained shows
differences in the polarity of the components which increases in the sequence:
Saturates < Aromatic < Resins
These concepts were strongly used for the choice of the eluent in the chromatographic purification. Polarity
properties, combined with the affinity of the component for the stationary phase, were used to enhance the
purification of the aromatic fraction of the bitumen, which contained the PAHs.
The stationary phase was composed by two different materials, silica gel and alumina. The silica gel used
(60-200 mesh) was purchased from Grace Davison, while the aluminum oxide (activated, basic, Brockmann I)
was purchased from Sigma-Aldrich.
The aluminum oxide was used due to the strong interaction formed with aromatics, which enhances the
separation between aromatics and saturates in the bitumen. The column was packed using 5g of alumina, 15g of
61
silica gel34 and another 5 g of alumina (Figure 6.2). The second layer of alumina was added to ensure that no
aromatic compounds left the column during the first elution.
5.0g of Alumina
15.0g of Silica gel
5.0g of Alumina
Figure 6.2 Chromatography column packaging
To separate the mixtures two eluents with different polarities were used. The first elution was carried out
using 50 mL of n-heptane. With the first nonpolar eluent, all saturates left the column first, while the aromatic
and resins fractions remained in the column. The two fraction still present in the column where then eluted using
slightly polar eluent. The second elution was thus carried out using 50 mL of a mixture of toluene:n-heptane
(80:20 v/v), which was polar enough to interact with the aromatic fraction of the bitumen and separate it from
the resin fraction, which needed a more polar solvent for the elution. The eluate, which contained the PAHs, was
collected and, at a later step, concentrated.
Concentration: The concentration step is also a very important step. The selection of the temperature and the
gas used for the concentration can change radically the mixtures composition.
The bitumen components are easily oxidized, and if air instead of nitrogen is used, the PAH content
decreases. The temperature at which the drying block is set also plays an important role in the evaporation of the
solvent. Due to this consideration, the gas used for the concentration was nitrogen whereas the temperature of
operation has been the focus of further studies.
Quantification: For the quantitation the GC-MS technique was used. The calibration curve was created using
standards at 5, 10, 20, 50, 100 μg/kg.
62
6.2
Calibration Curves
Instrumental calibration is an essential step in the creation of a method for the quantification of compounds.
It is a set of operations in which the relationship between the output of the measurement system (in this case the
area of the peak) and the concentrations of the standards used is established. A typical operation involves the
preparation of a predetermined number of standards with a known concentration and the record of the response
of the instruments related to each standard, which establish the relationship between the instrument response and
the analyte concentration. This relationship is then used to quantitate the amount of analyte present in a test
sample.
The IPA_SIMSCAN_0404 method used for the preliminary tests showed some difficulty in the separation
and in the quantification of the high molecular weight PAHs. In most of the preliminary tests a manual
integration was used to better quantitate the peak of the compound and the harder compound to be identified was
always perylene-d12, which was used as internal standard.
From Spectrum 6.7 it is possible to observe that when the rate of the temperature change was decreased
(compared to Spectrum 6.6), a decrease in signal and a better separation in the region of high boiling compounds
was obtained. Due to this observation, during the creation of the calibration curve, three different methods were
tested in which the best compromise between time and accuracy of the analysis was studied.
In the first method, called IPA_SIMSCAN_RENO, all the GC-MS parameters were the same of the method
used in the preliminary test. The only difference was in the temperature program.
Table 6.4 Oven temperature program of the method IPA_SIMSCAN_RENO
Rate (°C/min)
Initial
1
2
50
10
Temperature (°C)
38
120
340
Hold time (minutes)
1
0.5
5
In the second method, called IPA_SIMSCAN_RENO1, all the GC-MS parameters were the same of the
method used in the preliminary test. The main difference was in the temperature program, also different from the
previous one and from the next methods.
Table 6.5 Oven temperature program of the method IPA_SIMSCAN_RENO1
Rate (°C/min)
Initial
1
2
50
5
Temperature (°C)
38
120
340
Hold time (minutes)
1
0.5
5
In the third method, called IPA_SIMSCAN_RENO2, all the GC-MS parameters were the same of the method
used in the preliminary test. The main difference was in the temperature program, which is different also from
both of the previous methods.
63
Table 6.6 Oven temperature program of the method IPA_SIMSCAN_RENO2
Rate (°C/min)
Initial
1
2
3
50
10
25
Temperature (°C)
38
120
280
340
Hold time (minutes)
1
0.5
0.05
5
To study the differences between the three different temperature programs, an injection of a standard solution
was performed using each program. The standards prepared had a concentration of 100 μg/L.
The results obtained are shown in the three spectra below.
64
Spectrum 6.9 Test using the IPA_SIMSCAN_RENO method
Total Ion
Scan Mode
Total Ion
Sim Mode
65
Spectrum 6.10 Test using the IPA_SIMSCAN_RENO1 method
Total Ion
Scan Mode
Total Ion
Sim Mode
66
Spectrum 6.11 Test using the IPA_SIMSCAN_RENO2 method
Total Ion
Scan Mode
Total Ion
Sim Mode
67
In all of the three spectra it is possible to observe the occurrence of bleeding which gave an increase of the
signal at higher temperatures.
Looking at the SCAN mode of Spectrum 6.9 several consideration can be done. First of all, the low
molecular weight PAHs present in the standard gave good response. Secondly, it is possible to identify the
presence of the high molecular weight PAHs at the beginning of the bleeding effect of the column.
By observing the Spectrum 6.10 it is possible to recognize a decrease in the bleeding process of the column
in the region of interest (20-40 minutes). This should be also translated in an increase of the signal for the higher
molecular weight PAHs.
Spectrum 6.11 showed the highest bleeding effect, which decreased the sensitivity of the instrument for the
compounds present in this chromatogram region.
To understand better which of the three methods could be used for the calibration curve and also for the
future quantification of PAHs, an attempt of auto quantification was done with the auto quant setup of the GCMS computer interface for each of the three samples.
In the first case during the auto quant setup the instrument was able to recognize most of the target
compounds, which confirmed the preliminary visual identification.
The second attempt of auto quantification, using the second method, showed more difficulty in the
identification of the standard compounds in the sample.
The last attempt confirmed the conclusion drawn before and this method was evaluated as not adequate for
the analysis of the PAHs in the bitumen.
The differences between the first and the second method are mainly two. One is the duration of the analysis
In fact the IPA_SIMSCAN_RENO method was carried out in half time required to perform
IPA_SIMSCAN_RENO1. The second difference is in the response obtained from the auto quantification, which
showed a decrease of sensitivity of the system if the time of the analysis was too long.
Due to all these considerations IPA_SIMSCAN_RENO which is the best compromise between the time of
analysis and the response of the instrument was chosen for the calibration curve.
In the choice of the parameters for the calibration curve the United States Environmental Protection Agency
(USEPA) 8270D method was used 37. Gas chromatography-Mass spectrometry parameters were:
Injection: 1μL
Inlets: Splitless
Column: Silicone-coated fused-silica capillary column VF-5ms 30m x 0.25mm, 0.25μm of ID Factor four of
the Varian Company equivalent to J&W Scientific DB-5.
Carrier gas: He
Column Flow: 1.2mL/min
Table 6.7 Inlet Parameters of IPA_SIMSCAN_RENO used for the calibration curve
Temperature (°C)
320
Pressure (psi)
9
Septum purge flow (mL/min)
3
68
Table 6.8 Oven programmed temperature of the method IPA_SIMSCAN_RENO used for the calibration curve
Rate (°C/min)
Initial
1
2
50
10
Temperature (°C)
38
120
340
Hold time (minutes)
1
0.5
5
Mass instrument windows: 46.00 amu to 290.00 amu
Solvent delay: 5 minutes
Mass source (Electron Impact) temperature: 300°C
Mass Analyzer (Quadrupole) temperature: 150°C
Quantification done in SCAN mode.
The solvent delay was chosen as 5 minutes, due to the high boiling point of toluene (111°C) and to avoid the
toluene peak in the gas-mass chromatogram. The splitless mode was chosen to increase the sensitivity of the
instrument. The windows of the mass spectrometer were chosen to be sure that all the primary and secondary
ions of the compounds in the calibration curve could be present. In fact the highest ion was at m/z 279
(dibenzo[a,h]anthracene) and the upper limits fixed was 290 amu. The value of 46.00 amu was chosen to be sure
that after the solvent delay time if the solvent was still present the system could recognize it. The temperatures of
the electron impact source and of the quadrupole analyzer were chosen to be as high as possible to avoid the
condensation of the target compounds, which would corresponds to a decrease in the sensitivity of the
instruments.
The compounds used for the calibration curve were chosen from the USEPA 8270D method, in which all the
primary and secondary ions are scheduled.
The calibration curve was created with the internal standards (IS) method, where the quantification of the
target compounds is done using as reference the response of internal standards; along this line reproducibility
problems are decreased.
The internal standard must be a molecule absent in the matrix, having chemical and physical behavior similar
as much as possible to the analytes in question, it must be added in an amount comparable to that expected for
the analyte, it must be discriminated with certainty from the analytical system. Therefore, the deuterated
compounds are the best choice, they are not present in nature, they are discriminable for mass by GC-MS
system, and are virtually identical to the native with small difference in polarity of the C-D bond with respect to
the C-H: the former is more polarized an higher overall dipole moment, and in the case studied the deuterated
comes out a bit 'before when non-polar column are used.
To create the calibration curve the GC-MS system uses the retention time of the compounds, but also
quantification ions or primary ions of the internal standards, in order to calculate the amounts of the target
compounds present. Secondary, ions are used for the identification of the peak, in other words the system
recognizes that a peak is the peak of a compound only if the ratio between the primary and secondary ions is
respected.
69
Table 6.9 Characteristic internal standard ions used for the calibration curve
Compound
Naphthalene-d8
Acenaphthene-d10
Phenanthrene-d10
Pyrene-d10
Chrysene-d12
Perylene-d12
Primary ions
136
162
188
212
240
264
Secondary ions
137
160;164
184;189
208
236
132;260
Surrogate compounds were used as a reference of the ability of the method to recover the target compounds.
Table 6.10 Characteristic surrogate compound ions used for the calibration curve
Compound
p-Therphenyl-d14
2-fluoro- 1,1’Biphenyl
3-Methylcholanthrene
Primary ions
244
172
268
Secondary ions
243;245
170;171
252;269
In order to develop the calibration curve, several standards at different concentrations should be prepared.
The first step in this case was the choice of the analytes to be identified. Due to the absence of a legislative list,
the idea was to try to quantitate the maximum number of PAHs possible. This decision was taken considering the
possibility of a future legislative number of analytes which could be already present in the quantification method
used. In a second step the number of standards and their concentrations were selected and five-point calibration
curves with concentrations of 5, 10, 20, 50 and 100 μg/L was chosen. The selected concentration were chosen
considering the initial amount of bitumen weighted 0.5 g since the amount of PAHs should in this small sample
was expected to be low.
Table 6.11 Characteristic target compound ions used for the calibration curve
Compound
Naphthalene
1-Methylnaphthalene
2-Methylnaphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenantrene
Anthracene
Fluoranthene
Pyrene
Benzo[a]anthracene
Chrysene
Benzo[b]fluoranthene
Benzo[k]fluoranthene
Benzo[a]pyrene
Benzo[g,h,i]perylene
Indeno[1,2,3-c,d] pyrene
Dibenzo[a,h]anthracene
Primary ions
128
142
142
152
153
166
178
178
202
202
228
228
252
252
252
276
276
278
Secondary ions
129;127
115;141;143
139;141;143
150;151
152;154
163;165
176;179
176;179
200;203
200;203
226;229
113;226;229
126;253
126;253
126;253
138;274;277
274
139;279;
70
To prepare the standards for the calibration curve a certificated standard solutions, purchased from Restek,
were used in which the concentrations of the analytes were:
-
Internal standard: 2000 μg/mL
-
Surrogate mix (p-Therphenyl-d14; 2-fluoro- 1,1’Biphenyl): 1000 μg/mL
-
3-Methylcholanthrene: 2000 μg/mL
-
Standard mix 5 (All the target compounds of Table 6.11): 2000 μg/mL
From the standard solutions two stock solutions were prepared. The first stock solution included the internal
standards, the surrogate mix and 3-methylcholanthrene at a concentration of 1 mg/L. The second stock solution,
prepared with a concentration of 200 μg/L contained only the standard mix 5.
From the stock solution the 5 standards for the calibration curve were prepared in which the concentrations of
the internal standards (naphthalene-d8; acenaphthene-d10; phenanthrene-d10; pyrene-d10; chrysene-d12; perylened12) and of the surrogate compounds (p-therphenyl-d14; 2-fluoro- 1,1’biphenyl; 3-methylcholanthrene) was
maintained constant at 100 μg/L for each standard.
The standards were injected and the calibration curve was created. In the setting of the deuterated internal
standard the mean time of the reference standard was selected and no plot was observed due to the constant
concentration (Figure 6.1). The instrument used the retention time of the internal standards as reference for the
quantification of a certain number of analytes. In other words for each internal standards a certain number of
analytes were calculated. For instance naphthalene-d8 was used for the quantification of the compounds from the
naphthalene to 2-fluoro- 1,1’biphenyl as shown in Figure 6.2.
Figure 6.1 Plot of the internal standard Naphthalene-d8
71
Figure 6.2 Retention time list of the analyte taken from the computer interface of the instruments
The setting of the surrogate standards used shows that p-therphenyl-d14 was used as external standard
whereas 2-fluoro- 1,1’biphenyl, 3-methylcholanthrene were used as common surrogates. For each of the three
surrogate standards the plot of the calibration curve was forced through the origin. In this manner the
instrumental response had a direct proportionality to the concentration of the surrogate compound (Figure 6.3).
Figure 6.3 Plot of the surrogate compounds p-Terphenyl-d14
72
For the target compounds a linear regression was used to calibrate curves. The first parameter determined
from the calibration curves of the target compounds was the correlation coefficient (r2), which is an expression
of the linearity of the calibration curve prepared. It gives the ratio of the fluctuation (variance) of a variable
which is detectable from another variables . It is a measure of the certainty of the graphic model. It is related to
the strength of the linear association between the response of the instrument and the concentration of the analyte.
The value of the coefficient r2 is in the range 0-1 and the value of 1 indicates that a perfect positive linearity is
present. For instance a value of 0.99 of r2 indicates a 99% probability that the instrument response has a linear
relationship with the concentration of the analyte.
The highest value of the correlation coefficient observed in the calibration curves created was for fluorene in
which r2 had a value of 0.999235, while the worst result in coefficient of determination was that of the
dibenzo[a,h]anthracene, with a value of 0.985498 (Figure 6.4,Figure 6.5).
The calibration curves performed were considered suitable for the quantification of the PAHs extracted from
the bitumen. The next step was the study of the best operating conditions for the method developed.
Figure 6.4 Calibration curve for fluorene.
73
Figure 6.5 Calibration curve for dibenzo[a,h]anthracene.
The analytical performance, detection limit (X DL), minimum detectable value (XMDV) and the limit of
quantification (XLQ) of the calibration curve was calculated.
The detection limit is the smallest concentration or absolute amount of analyte that has a signal which can be
detected, which l arise from a reagent blank 38.
The limit of quantification is the smallest concentration or absolute amount of analyte which can be
quantitated38.
𝑥𝐷𝐿 =
𝑥𝑀𝐷𝑉 = 𝑥𝐷𝐿 +
𝑆𝑦
𝑏
𝑆𝑦
𝑏
1
𝑥̅ 2
2
𝑖=1(𝑥𝑖 −𝑥̅ )
6.239
1
𝑥̅ 2
2
𝑖=1(𝑥𝑖 −𝑥̅ )
6.339
𝑡𝑓,𝛼 √1 + 𝑁 + ∑𝑁
𝑡𝑓,𝛼 √1 + 𝑁 + ∑𝑁
74
Table 6.12 Analytical performance of the calibration curve created.
Compound
Naphthalene
1-Methylnaphthalene
2-Methylnaphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenantrene
Anthracene
Fluoranthene
Pyrene
Benzo[a]anthracene
Chrysene
Benzo[b]fluoranthene
Benzo[k]fluoranthene
Benzo[a]pyrene
Benzo[g,h,i]perylene
Indeno[1,2,3-c,d] pyrene
Dibenzo[a,h]anthracene
XDL (μg/L)
4.30
4.39
4.65
5.07
2.21
3.85
3.46
3.10
3.58
7.77
3.08
9.97
8.22
7.14
5.83
6.72
10.63
4.53
XMDV (μg/L)
8.60
8.78
9.30
10.14
4.42
7.70
6.93
6.20
7.16
15.53
6.16
19.95
16.44
14.27411
11.65
13.43
21.26
9.05
XLQ =10* XDL (μg/L)
42.97
43.90
46.52
50.71
22.12
38.50
34.65
31.02
35.82
77.66
30.84
99.73
82.17
71.37
58.24
67.15
106.29
45.26
75
6.3
Study of the best operating conditions
Many factors can affect the success of an experiment, the last part of the development of the method was to
study how these factors affect the process to obtain optimized conditions. The principal factor studied was the
instrumental response in the GC-MS quantification while the secondary factor considered was the time needed
for the progression of the analysis. The latter factor was imposed by the working requirements. In an industrial
environment the time needed for the execution of different analysis must accommodate the duration of the
working day.
The general scheme of the procedure is divided in chronological steps. At each step a separation of one
fraction is achieved by using the physico-chemical properties of that fraction, with the goal to obtain at the end
only the aromatic fraction of the bitumen in which the PAH are presents.
In order to study the physico-chemical status of the work, the chronological order of the method has been
followed starting from with the solubilization and continued with the subsequent parts of the method.
The solubilization was carried out taking advantage of the solubility properties of bitumen’s components.
Bitumen was dissolved in a mixture of n-heptane, which dissolves the maltenic part, and toluene, which
increases the dissolution of the maltenes but also dissolves a lower quantity of the asphaltenic fraction. Two
different techniques of solubilization were compared: the stirring process, ruled by the stirring rate, and the
ultrasonication method in which the frequency of the ultrasound wave ruled the process.
The next step of the method was the filtration, in which the insoluble asphaltenic and waxy substances were
separated from the matrix. The study conducted at this step was to observe differences in the use of a common
filter paper or the use of ionic chromatography syringe and filters.
Due to the studies performed in the preliminary tests, the chromatographic purification was not changed and
the same quantity of stationary phase and mobile phase were used.
The next step was the concentration; The main concern was the low vapour pressure of low molecular weight
PAHs, such as naphthalene, which under nitrogen flow and higher temperature can evaporate. Due to the
instrumentation limits, the only parameter that could be changed was the temperature of the drying block. The
other parameter which regulates this process, the pressure of the nitrogen flow, cannot be controlled by a
pressure gauge and the only control was the visual monitoring. How, by observing the bubbles generated on the
surface of the liquid. In the tests conducted three different temperatures of the drying block were used, 60°C,
40°C and 20°C, always under nitrogen flow.
The last parameter studied was the moment of addition of the internal standards and the surrogates.
At this stage of the method, the GC-MS apparatus used began to show a warning regarding the lifetime of the
EI source, which was ending. In fact a decrease in sensitivity and an increase of baseline signal were observed.
Due to this problem few experiments were done in which only one parameter was changed and the results
obtained from the GC-MS method were compared.
At the initial stage, six different tests were carried out using always the same matrix. In the first experiment,
called SARA 5-6-2014, 0.5 g of bitumen were dissolved in 20 mL of n-heptane:toluene (85:15) plus 100 μL of
the mixture of internal standards and surrogates (1 ppm). The solubilization process was carried out on a bench
stirrer for two hours at ambient temperature. After the solubilization process a filtration with the ionic
76
chromatography syringe and filters was performed. The filtrate was added directly to the column to decrease the
experimental error of the method by adding a new passage. After the filtration process the chromatographic
purification was carried out and the eluted fraction was concentrated to 1 mL at constant temperature of 60°C
with nitrogen flow. The concentrated sample was subsequently injected in the GC-MS station.
In the second experiment, called SARA 9-6-2014, 0.5 g of bitumen were dissolved in 20 mL of nheptane:toluene (85:15) plus 100 μL of the mixture of internal standards and surrogates (1 ppm). The
solubilization process was carried out in an ultrasonic bath at 40 kHz at a constant temperature of 25 ± 2°C using
ice for cooling the solution; the temperature was chosen to be as similar as possible to the ambient conditions of
the bench stirring. After the solubilization process a filtration with the ionic chromatography syringe and filters
was done. The filtrate was added directly to the column and the chromatographic purification was carried out.
The eluted fraction was concentrated to 1 mL at constant temperature of 60°C with nitrogen flow and
subsequently injected in the GC-MS station.
77
Spectrum 6.12 Bitumen extraction SARA 5-6-2014
Total Ion
Scan Mode
Total Ion
Sim Mode
78
Spectrum 6.13 Bitumen extraction SARA 9-6-2014
Total Ion
Scan Mode
Total Ion
Sim Mode
79
The results of the two experiments are shown in spectra 6.12 and 6.13. In both the spectra it is possible to
observe that the methods used in the GC-MS was able to separate, recognize and quantify the PAHs. The main
difference between the two spectra is the higher abundance obtained when the solubilization process is the bench
stirring shown in the SCAN mode of Spectrum 6.12, compared with the solubilization process that gave
Spectrum 6.13. In order to further compare the two solubilization techniques, the quantity of the target
compound and of the surrogate were taken into consideration. The general idea was that due to its properties the
ultrasonication technique should be the best one.
Table 6.13 Concentration of the surrogate and target compounds in SARA 5-6-2014 and SARA 9-6-2014
Compound Name
p-Terphenyl-d14
Naphthalene
1-Methylnaphthalene
2-Methylnaphthalene 2-fluoro-1,1’Biphenyl
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo[a]anthracene
Chrysene
Benzo[b]fluoranthene
Benzo[k]fluoranthene
Benzo[a]pyrene
3-Methylcholanthrene
Indeno[1,2,3-c,d]pyrene
Dibenzo[a,h]anthracene
Benzo[g,h,i]perylene
Amount
SARA 5-6-2014
83.88
54.66
113.13
84.35
3.77
43.56
179.14
612.43
5798.51
948.09
75.62
402.45
1052.18
2339.51
1226.47
1439.23
3767.92
565.83
293.86
296.01
1173.85
Amount
SARA 9-6-2014
97.38
815.52
2189.47
1614.53
98.09
54.30
92.23
684.24
5080.32
952.24
131.948
644.586
1354.65
3607.33
1140.56
1339.27
3927.73
446.66
289.66
249.48
1341.01
Units
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
In both cases a manual integration was used to ensure that the system in each tests recognized always the
same peak and that the retention time of the compounds were comparable.
Table 6.13 shows the results obtained from the two analyses. The first observed parameter was the quantity
of the surrogate compounds recovered by the system. The p-therphenyl-d14 and the 2-fluoro-1,1’biphenyl show a
very good result and only 2-3% of the total amount was lost. The third surrogate compound 3methylcholanthrene showed a higher than expected value in both cases.
The second factor considered in the analysis was the concentration of the target compounds. At first look it
was observed that the order of magnitude for the concentration obtained in the two different analyses is the same,
which suggests a great ability of the system to give reproducible result. Looking at the table it is possible to
observe an increase in the concentration of the compounds extracted in the experiments SARA 9-6-2014. This
suggest a greater ability of the ultrasonication process to increase the amount of solubilized PAHs.
Due to the results obtained from this test, two other experiments were performed in which the temperature of
the drying block was changed.
80
In the third experiment, called SARA 11-6-2014, 0.5 g of bitumen were dissolved in 20 mL of nheptane:toluene (85:15) plus 100 μL of the mixture of internal standards and surrogates (1 ppm). The
solubilization process was carried out on a bench stirrer for two hours at ambient temperature. After the
solubilization process a filtration with the ionic chromatography syringe and filters was performed. The filtrate
was added directly to the chromatography column and the chromatographic purification was carried out. The
eluted fraction was concentrated to 1 mL at constant temperature of 40°C with nitrogen flow and subsequently
injected in the GC-MS station.
In the fourth experiment, called SARA 12-6-2014, 0.5 g of bitumen were dissolved in 20 mL of nheptane:toluene (85:15) plus 100 μL of the mixture of internal standards and surrogates (1 ppm). The
solubilization process was carried out in an ultrasonic bath at 40 kHz at a constant temperature of 25 ± 2°C using
ice for cooling the solution. The temperature was chosen to be as similar as possible to the ambient conditions of
the bench stirring. After the solubilization process a filtration with the ionic chromatography syringe and filters
was performed. The filtrate was added directly to the chromatography column and the chromatographic
purification was carried out. The eluted fraction was concentrated to 1 mL at constant temperature of 40°C with
nitrogen flow and subsequently injected in the GC-MS station.
81
Spectrum 6.14 Bitumen extraction SARA 11-6-2014
Total Ion
Scan Mode
Total Ion
Sim Mode
82
Spectrum 6.15 Bitumen extraction SARA 12-6-2014
Total Ion
Scan Mode
Total Ion
Sim Mode
83
From the two spectra of the tests performed at 40°C (Spectrum 6.14 and Spectrum 6.15) it is possible to
observe that in this case the methods were able to separate, recognize and quantify the PAHs. These new two
tests were used to study two different factors: the solubilization process and the temperature of the drying block
at the sample concentrator.
For the solubilization process studies, the spectra were analyzed and it is possible to deduce that contrary to
previous test, in this case no big differences between the intensities of the peaks in the SCAN mode of the two
spectra were observed. To continue the comparison of the two solubilization processes, here too, the amount of
the components of the two spectra was calculated.
Table 6.14 Concentration of the surrogate and target compound in SARA 11-6-2014 and SARA 12-6-2014
Compound Name
p-Terphenyl-d14
Naphthalene
1-Methylnaphthalene
2-Methylnaphthalene 2-fluoro-1,1’Biphenyl
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo[a]anthracene
Chrysene
Benzo[b]fluoranthene
Benzo[k]fluoranthene
Benzo[a]pyrene
3-Methylcholanthrene
Indeno[1,2,3-c,d]pyrene
Dibenzo[a,h]anthracene
Benzo[g,h,i]perylene
Amount
SARA 11-6-2014
35.62
335.26
636.12
317.96
37.10
38.45
156.20
968.67
7294.35
4238.45
74.93
360.52
7657.77
3094.67
332.28
1601.3
5158.67
395.98
102.29
204.28
1557.36
Amount
SARA 12-6-2014
85.44
242.25
522.70
365.65
51.66
32.95
97.08
626.97
2659.5
514.72
135.12
686.46
311.83
1594.55
740.67
2067.65
5772.53
519.06
530.41
233.33
1596.36
Units
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
As before, also in this case a manual correction of the integration was done. Table 6.14 shows the
quantification reports of the SARA 11-6-2014 and SARA 12-6-2014 tests. As before, the first result observed
was the amount of the surrogate compounds, which when compared to the previous test, confirmed an increase
of the recovery when ultrasonication was used. In this case, 3-methylcholantrene showed again a higher response
than expected, which at this step of the developing process was associated with two different possibilities: the
presence of interferences or the presence of the compound itself.
To confirm a higher ability of the ultrasonic process to dissolve bitumen, the quantity of the target
compounds were taken as reference. In this case it is possible to observe the same general decrease in the
concentration when a stirring process is performed, but some analytes show a different tendency with respect to
the process developed at a concentration temperature of 60°C.
84
After all these considerations, the ultrasonic technique was preferred to the stirring technique due to a higher
response of the system monitoring compounds (surrogatess). The other factor considered, the time of execution,
showed a decrease by one hour in the complete process when the ultrasonic bath was used.
To study the differences obtained with the temperature of the concentrator step, the main factor compared
was the amount of the low molecular weight PAHs from naphthalene to anthracene. In order to study these
differences by changing only one parameter each time the result obtained from Spectrum 6.12 and respective
quantification reports were compared to those from Spectrum 6.13 with the respective quantification reports.
In each of the two tests the solubilization method used was magnetic stirring and the only difference was the
concentration temperature, in the first case 60°C and in the second case 40°C. The general idea was that the
lower temperature process should increase the amount of the LMW PAH. From the two SCAN modes in the
spectra a higher abundance was obtained for the test conducted with a higher temperature of concentration. From
the quantitation reports listed in Table 6.13 and Table 6.14 it is possible to observe that the surrogate compound,
2-fluoro-1,1’biphenyl had a relevant decrease in concentration when it is concentrated at 60°C. The other target
compounds show the same trend, in which significant differences were observed from the concentration
temperature of 40°C.
From this comparison, it was concluded that the concentrator at 60°C save a very low quantity of the
compounds with lower vapour pressure. The same trend was expected when Spectrum 6.13 and Spectrum 6.15
were compared.
The two tests compared were the tests performed using ultrasonication as solubilization technique and the
two different temperatures of concentration (40°C and 60°C). As before; also in this case the SCAN mode of
Spectrum 6.13 showed the same trend of higher abundance compared to Spectrum 6.15 in which the temperature
used was of 40°C. From the quantitation reports listed in Table 6.13 and Table 6.14 it is possible to observe an
opposite trend in the concentration of the surrogate and target compounds. The test in which the concentration
temperature used was of 60°C shows a higher response in concentration of the LMW PAHs. This result raised
questions about the previous results.
To continue the study of the best compromise between the time of execution and the ability of the method to
recover and obtain the greatest results other two tests were performed.
In the former experiment, SARA 16-6-2014, 0.5 g of bitumen were dissolved in 20 mL of n-heptane:toluene
(85:15) plus 100 μL of the mixture of internal standards and surrogates (1 ppm). The solubilization process was
carried out on a bench stirrer for two hours at ambient temperature. After the solubilization process, a filtration
with the ionic chromatography syringe and filters was done. The filtrate was added directly to the
chromatography column and the chromatographic purification was carried out. The eluted fraction was
concentrated to 1 mL at constant temperature of 20°C with nitrogen flow and subsequently injected in the GCMS station.
In the latter experiment, called SARA 17-6-2014, 0.5 g of bitumen were dissolved in 20 mL of nheptane:toluene (85:15) plus 100 μL of the mixture of internal standards and surrogates (1 ppm). The
solubilization process was carried out in an ultrasonic bath at 40 kHz at a constant temperature of 25 ± 2°C.
After the solubilization process a filtration with the ionic chromatography syringe and filters was performed. The
filtrate was added directly to the chromatography column and the chromatographic purification was carried out.
The eluted part was concentrated to 1 mL at constant temperature of 20°C with nitrogen flow and subsequently
injected in the GC-MS station.
85
Spectrum 6.16 Bitumen extraction SARA 16-6-2014
Total Ion
Scan Mode
Total Ion
Sim Mode
86
The quantitation results obtained from the extraction called SARA 16-6-2014 are reported in the table below.
Table 6.15 Concentration of the surrogate and target compounds in SARA 16-6-2014
Compound Name
p-Terphenyl-d14
Naphthalene
1-Methylnaphthalene
2-Methylnaphthalene 2-fluoro-1,1’Biphenyl
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo[a]anthracene
Chrysene
Benzo[b]fluoranthene
Benzo[k]fluoranthene
Benzo[a]pyrene
3-Methylcholanthrene
Indeno[1,2,3-c,d]pyrene
Dibenzo[a,h]anthracene
Benzo[g,h,i]perylene
Amount
SARA 16-6-2014
101.73
298.99
842.27
676.10
90.54
24.88
107.46
510.58
2740.54
493.48
113.14
590.90
273.01
691.71
503.22
667.67
1671.98
186.199
149.62
121.14
485.69
Units
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
The result expected by this test is that the amount of LMW PAHs should be the highest. However, as listed
in Table 6.15, the concentration of the lowest vapour pressure PAHs does not match the expected results. The
explanation behind the un-matching results was that nine hours for the concentration step were needed, the
LMW PAHs were exposed to a prolonged nitrogen flow which causes the evaporation of these compounds.
The experimental results obtained from the test SARA 17-6-2014 are not reported. In fact the concentration
temperature of 20°C was rejected due to the fact that the amount of time needed for the operation and the amount
of nitrogen gas used were very high.
Other tests were performed in which the differences were in the use of a common paper filter or of an ionic
chromatography syringe and filter. The main results obtained from these tests were that the paper filter used was
not able to separate all the particulates present in solution. In fact on the glass of the chromatography column it
was possible to observe the presence of little amounts of particulate matter. Extra results were that the amount of
liquid retained in the paper filter was greater than with the ionic chromatography filter apparatus; also the latter
operation had a greater manageability.
At this point two factors were discovered to be the best compromise between the duration of the experiment
and the result obtained at the GC-MS. The first is the use of an ultrasonication bath at 40 kHz for 1 hour with a
temperature of 25 ± 2°C and the second is the use of ionic chromatography filter and syringe.
From the tests performed, the concentration temperatures were the most difficult to optimize. To avoid this
problem a new test was performed in which, 0.5 g of bitumen were dissolved in 20 mL of n-heptane:toluene
87
(85:15 ). The solubilization process was carried out in an ultrasonic bath at 40 kHz at a constant temperature of
25 ± 2°C using ice for cooling the solution down. After the solubilization process a filtration with the ionic
chromatography syringe and filters was done. The filtrate was added directly to the chromatography column and
the chromatographic purification was carried out. At this point the mixture was rinsed to 50 mL with toluene and
ultrasonicated for 15 minutes to increase homogenization of the solution. The collected fraction was split in two
aliquots of 25 mL and 100 μL of the standards was added to each solution. The former solution was concentrated
at constant temperature of 40°C (SARA 19-6-2014 a), while the latter was concentrated at constant temperature
of 60°C (SARA 19-6-2014 b). The concentrated samples was subsequently injected in the GC-MS station.
These tests were performed to study only concentration step. As a matter of fact, the idea was to eliminate
any error attributable to the solubilization, filtration and chromatography purification steps. The internal standard
mixture was added before the concentration step to verify which was the best temperature.
88
Spectrum 6.17 Bitumen extraction SARA 19-6-2014 a
Total Ion
Scan Mode
Total Ion
Sim Mode
89
Spectrum 6.18 Bitumen extraction SARA 19-6-2014 b
Total Ion
Scan Mode
Total Ion
Sim Mode
90
Table 6.16 Concentration of the surrogate and target compounds in SARA 19-6-2014 a and SARA 19-6-2014 b
Compound Name
p-Terphenyl-d14
Naphthalene
1-Methylnaphthalene
2-Methylnaphthalene 2-fluoro-1,1’Biphenyl
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo[a]anthracene
Chrysene
Benzo[b]fluoranthene
Benzo[k]fluoranthene
Benzo[a]pyrene
3-Methylcholanthrene
Indeno[1,2,3-c,d]pyrene
Dibenzo[a,h]anthracene
Benzo[g,h,i]perylene
Amount
SARA 19-6-2014 a
21.70
118.74
624.69
432.09
137.92
16.90
73.65
334.85
1566.6
195.16
41.40
803.58
281.18
508.98
642.33
773.12
1583.68
139.65
148.86
633.94
549.01
Amount
SARA 19-6-2014 b
86.10
117.86
657.41
452.80
136.20
16.76
75.55
422.32
1582.6
322.50
44.01
852.37
322.47
754.23
798.38
954.98
1983.69
207.78
152.09
370.71
565.08
Units
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
The results obtained from these tests reject the general idea that at a lower temperature the amount of the low
vapour pressure PAHs should be greater than at higher temperature. In fact from both spectra and from both
quantitation reports it is possible to observe that the order of magnitude of the results is the same and big
differences are not present. The major difference is the amount of p-terphenyl-d14 that at highest temperature has
a concentration four times higher with compared to the lower temperature. The other surrogate compounds are
both overestimated.
In both cases it is possible to observe that the amount of the target compounds is comparable and no great
differences are observed.
The results obtained from these analyses show that no big differences are present between the two
concentration temperatures however considering the duration of the process, 2 hours or 2 hours and half
differences were observed. The fastest method is the concentration performed at 60°C, which gives a good result
in terms of recovery of the sample. By the analysis of the surrogates and an increase in the rate of the process;
due to these observations it was concluded that.
From all the different analyses performed, the concentration that did not fit the general trend of the recovery
system was that of 3-methylcholanthrene. Due to this irregular result, a test was performed with in presence of
the internal standard but without the surrogate compounds.
The test was performed considering all the observations discussed above: 0.5 g of bitumen was dissolved in
20 m L of n-heptane:toluene (85:15) containing 100 μL of internal standards (naphthalene-d8, anthracene-d10,
phenanthrene-d10, pyrene-d10, chrysene-d12, perylene-d12) at 1 ppm. The solubilization process was carried out in
an ultrasonic bath at 40 kHz at a constant temperature of 25 ± 2°C. After the solubilization process a filtration
with the ionic chromatography syringe and filters was performed. The filtrate was added directly to the
91
chromatography column and the chromatographic purification was carried out. The eluted fraction was
concentrated to 1 mL at constant temperature of 60°C with nitrogen flow and subsequently injected in the GCMS station.
From the quantitation results listed in Table 6.17 it is possible to observe that the surrogate compounds were
not quantitated. This means that the system is able to recognize the compounds but some interference
compounds increase in amount during the quantitation process. The system uses the quantitation ions for the
quantification of the compound and the secondary ions as a confirmation of the presence of those compounds. In
this case the system was able to recognize the absence of the secondary ions of 3-methylcholanthrene, which is
translated with a zero response for the presence of this compound. When a compound is present the secondary
ions are present and the system recognizes them by giving a positive response to the presence of this compound.
In the quantification of 3-methylcholanthrene probably an interfering ion, corresponds to the quantitation ion of
this surrogate (m/z 268), increased the response of the instrument which gives as a result an amount of this
surrogate compound higher than the quantity expected.
92
Table 6.17 Concentration of the surrogate and target compounds in SARA 23-6-2014
Compound Name
p-Terphenyl-d14
Naphthalene
1-Methylnaphthalene
2-Methylnaphthalene 2-fluoro-1,1’Biphenyl
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo[a]anthracene
Chrysene
Benzo[b]fluoranthene
Benzo[k]fluoranthene
Benzo[a]pyrene
3-Methylcholanthrene
Indeno[1,2,3-c,d]pyrene
Dibenzo[a,h]anthracene
Benzo[g,h,i]perylene
Amount
SARA 23-6-2014
0
181.72
1025.96
727.71
0
51.53
215.01
585.95
2393.73
406.40
45.68
302.50
318.11
704.44
256.67
353.02
1535.45
0
152.86
132.15
542.70
Units
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
Studying this method and increasing experience with the GC_MS the study of the addition of the internal
standards was performed. The idea was to study the different response of the system when the internal standards
were added at the end before the injection.
The test was carried out adding 0.5 g of bitumen to 20 mL of n-heptane:toluene (85:15), a this point 100 μL
of the surrogate mix (p-terphenyl-d14; 2-fluoro-1,1’biphenyl; 3-methylcholanthrene) were added to the solution
and the extraction process took place. After the entire step and before injection in the GC-MS system 100 μL of
the internal standards (naphthalene-d8, anthracene-d10, phenanthrene-d10, pyrene-d10, chrysene-d12, and perylened12) were added.
From Spectrum 6.19 it is possible to observe that the system was able to quantify all target compounds but in
this case some inconsistencies were observed in the quantitation reports (Table 6.18). In all the experiments
carried out the same matrix was always used and it was observed that the order of magnitude of all the
quantitation reports followed the same trends consistently. However, in this case a decrease in the amount of
target compound quantified was observed.
The decreasing of the target compounds concentration was discovered to be related to the internal standards.
The central point in the use of IS is that the sample is spiked at the beginning, in order to monitor the whole
process present systematic errors. If the internal standard has an area of 100, and the system reads 50, it means
that during the process 50% of the analyte were lost. The system corrects the results obtained by multiplying the
final results by two.
The response factor calculated by the system is
93
RF 
 Ax Cis 
 Ais Cx 
6.4
Where:
Ax= Area of the compound
Cx= Concentration of the compound
Ais= Area of the internal standard
Cis= Concentration of the internal standard
If the internal standard is added just before the injection, the system reads the area as 100 and the correction
factor will be equal to one. In this last case the system will read as if the entire process gives 100% of recovery
of the internal standard and the result obtained is underestimated.
Due to this consideration and in view of the results obtained, the internal standard was chosen to be added at
the beginning of the process to avoid the problem of underestimation.
94
Spectrum 6.19 Bitumen extraction SARA 25-6-2014
Total Ion
Scan Mode
Total Ion
Sim Mode
95
Table 6.18 Concentration of the surrogate and target compounds in SARA 25-6-2014
Compound Name
p-Terphenyl-d14
Naphthalene
1-Methylnaphthalene
2-Methylnaphthalene 2-fluoro-1,1’Biphenyl
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo[a]anthracene
Chrysene
Benzo[b]fluoranthene
Benzo[k]fluoranthene
Benzo[a]pyrene
3-Methylcholanthrene
Indeno[1,2,3-c,d]pyrene
Dibenzo[a,h]anthracene
Benzo[g,h,i]perylene
Amount
SARA 25-6-2014
43.8583
152.423
942.127
646.74
90.8359
38.2453
185.57
765.481
903.09
322.212
71.3025
267.957
318.344
509.013
218.757
272.042
687.077
62.9434
84.8623
82.3827
270.049
Units
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
μg/L
96
7 Conclusions and future developments
This thesis work focused on the development of a method for the extraction and quantification of Polycyclic
Aromatic Hydrocarbons from a bituminous matrix.
Many experiments were performed to understand the physico-chemical properties of bitumen and the
response of this matrix to different solvents and extraction procedures.
The extraction of PAHs has been calculated by taking advantages of the properties of bitumen. From the
results obtained in the experiments SARA 5-6-2014, SARA 9-6-2014, SARA 11-6-2014 and SARA 12-6-2014 ,the
ultrasonication process was chosen to be the best compromise between the amount of PAHs solubilized and the
time needed for the solubilization process.
The filtration step chosen involved the use of a ionic chromatography syringe and a filter for the with a
porosity of 0.2 μm. The stationary phase used for the chromatographic purification was composed by alumina,
which enhanced the ability of the system to separate the aromatic fraction from the saturated fraction, and silica
gel. The mobile phase used consisted of n-heptane for the recovery of the saturates, and a mixture of toluene:nheptane (80:20) for the recovery of the PAHs.
From the results obtained, and especially from SARA 19-6-2014 a and SARA 19-6-2014 b experiments, the
concentration temperature adopted for this method was 60°C. This temperature which gave fast results also gives
very good results, comparable to the temperature of 40°C, in the quantification of the surrogate compounds.
From the results of the internal standards study shown in experiment SARA 25-6-2014, it was discovered that
in order to avoid the problem of underestimation, the deuterated internal standards should be added at the
beginning of the process.
The calibration curves obtained had very good correlation coefficients and also it showed a great ability of
the system in the separation and quantification of PAHs.
Unfortunately,, due to insufficient time, I was not able to determine the degree of precision of the method
(repeatability and reproducibility), this work is only the first step to the development of a new method for the
extraction and quantification of PAHs. With this method the basis for the improvements are created and only
future works with more specific equipment could help the extraction.
A possibility will be to rebuild a bitumen with a known concentration of PAHs, and make diluitions by
adding a similar matrix, not containing PAHs, to obtain always a more diluted sample. Unfortunately, this
approach is not very simple due to the lack of homogeneity of the bitumen, in which oxidation processes
decrease the amount of PAHs.
Further adjustments of the method could include the possibility of taking advantage of a solid phase
extraction (SPE) using a pre-prepared syringe for less amount of solvents and working time is needed. By using
SPE, the reproducibility compared to column chromatography should be obtained.
Another improvements could include the use of rotary-vapour equipment, in which less time is needed for the
evaporation of solvents compared to the sample concentrator used. This technique could be also useful to
decrease the amount of sample added to the chromatographic column.
The calibration curves used for this thesis were created with concentrations of 5,10, 20, 50 and 100 ppb but
during the studies it was observed that the amount of PAHs vary too much and more points for the calibration
curves were necessary. Other changes in the GC-MS instrument could include the use of a high temperature
97
column which enhances the ability of the system to recognised and quantify the PAHs present. Finally, a guard
column could be used to avoid compounds which irreversibly bond to the stationary phase of the column.
98
References
1 International Agency for Research on Cancer Bitumens and Bitumen emissions, and some N- and SHeterocyclic Polycyclic Aromatic Hydrocarbons Volume 103 IARC Monographs on the Evaluation of
Carcinogenic Risks to Humans, IARC Lyons 2013.
2 Asphalt Institute Inc. and European Bitumen Association–Eurobitume. The Bitumen Industry - A global
perspective - Second edition. 2011
3 Shell International Exploration and Production B.V.-The Hague. Bitumen Process Guide. Report MF 950500. 1995.
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