Sulfuric acid as an extractionmedium of lipids
Selene Cannelli
In part fulfilment of the requirements for the degreeof Bachelor of Science
In Archaeological Sciences, University of Bradford 2015
Word Count:
10,194
This dissertation is an unrevised examination copy forconsultation only and it should not be quoted or cited
without the permission of the Head of Division
Division of AGES, School of Life Sciences
UNIVERSITY OF BRADFORD
Abstract
Recovering lipids from potsherds has always been
important to archaeologists because of the data that
can be recovered . With the advance of science, even
lipid extraction is starting to improve by finding new
methods that need fewer samples and less time to
collect results. In 2000 York University developed a
more efficient method to extract lipids from
archaeological potsherds with sulfuric acid. This
project compared this new technique with conventional
solvent extraction by BSTFA and saponification methods
to test which technique is the more appropriate to
recover residues from ceramic walls. The solvent
extraction proved to be the procedure that recovers
every main molecule of the compounds tested, while the
saponification and sulphuric acid techniques
derivatized the molecules. The sulfuric acid method was
long to conduct, increasing the chances to
contaminate the samples. This novel method proved to
yield higher recoveries of lipids, but at the same time
there has been compositional information loss due to
hydrolysis of lipids, in fact no wax esters or TAG
molecules were recovered. The only molecule that this
method was not able to derivatize is cholesterol from
the pistacia spp resin. It is recommended to proceed with
this method if the conditions of the sample are in a
poor conservation state, to make sure that more lipids
are recovered from the ceramic walls. It is also useful
to further analyse molecules obtained with other
extraction methods to conduct more studies on them.
Table of Contents
LIST OF ILLUSTRATIONS............................................................................I
LIST OF TABLES.......................................................................................III
LIST OF ABBREVIATION...........................................................................IV
ACKNOWLEDGMENT.................................................................................V
PART 1: LITERATURE REVIEW - WHY LIPIDS RECOVERY IS IMPORTANT?. 1
Chapter 1 – Introduction........................................................................1
Chapter 2 - Conventional solvent extraction and saponification, the
problems.................................................................................................. 3
Chapter 3 - Revision of solvent extraction, derivatization and
saponification methods.........................................................................5
Chapter 4 - A novel method...................................................................8
PART 2: RESEARCH..................................................................................10
Chapter 1 – Introduction......................................................................10
1.1 AIMS AND OBJECTIVES...........................................................................................11
Chapter 2 – Methods............................................................................13
2.1 STANDARD SAMPLE PREPARATION............................................................................13
2.2 CONVENTIONAL SOLVENT EXTRACTION BY BSTFA.....................................................13
2.3 SAPONIFICATION................................................................................................... 15
2.4 SULFURIC ACID..................................................................................................... 16
2.5 GC-MS ANALYSIS.................................................................................................. 18
Chapter 3 – Results...............................................................................19
3.1 Modern beeswax............................................................................19
3.2 MODERN OLIVE OIL...............................................................................................23
3.3 MODERN PISTACIA SPP. RESIN................................................................................27
3.4 MODERN FINGER PRINTS........................................................................................32
3.5 SULFURIC ACID BLANK TESTS...................................................................................35
Chapter 4 – Discussions.......................................................................38
4.1 METHODS............................................................................................................ 38
4.2 MODERN BEESWAX................................................................................................ 42
4.3 MODERN OLIVE OIL...............................................................................................47
4.4 MODERN PISTACIA SPP RESIN.................................................................................51
4.5 MODERN FINGER PRINTS........................................................................................59
Chapter 5 – Conclusions.......................................................................63
SUGGESTIONS FOR FURTHER WORK.......................................................65
REFERENCES............................................................................................66
List of illustrations
Chapter 2:Figure 1. Flow chart illustrating the sequential stages
for the solvent extraction methodology by BSTFA
Figure 2. Flow chart illustrating the sequential stages
for the saponification extraction methodology
Figure 3. Flow chart illustrating the sequential stages
for the sulfuric acid extraction methodology
Chapter 3:Figure 4. Chromatogram produced by beeswax with a)
solvent extraction methodology; b) saponification
extraction methodology; c) sulfuric acid extraction
methodology.
Key: n-alkane; n-TMS; n-alcohol; n-
alkene; WE wax ester; FAME; underivatized
fatty acid; P phthalate
Figure 5. a) mass spectrum at 28.11 min of alcohol
molecule, C30H62O, extracted from beeswax with
saponification methodology; b) mass spectrum at 18 min
of underivatized molecule, C16H32O2 n-Hexadecanoic acid,
extracted from beeswax with saponification methodology;
c) mass spectrum at 17.7 min of FAME molecule methyl
i
palmitate, C17H34O2, extracted from beeswax with sulfuric
acid methodology; d) mass spectrum at 18.98 min of TMS
molecule, C19H40O2Si, extracted from beeswax with solvent
extraction methodology by BSTFA
Figure 6. Chromatogram produced by modern olive oil
with a) solvent exctraction methodology by BSTFA; b)
saponification extraction methodology; c) sulfuric acid
extraction methodology.
Key: TAG; n-TMS; FAME; underivatized fatty
acid; n-alcohol; P phthalate.
ii
Figure 7. a) mass spectrum at 24.83 min of TAG
molecule, C27H56O4Si2, extracted from olive oil with
solvent extraction methodology by BSTFA; b) mass
spectrum at 19.32 min of FAME molecule, C19H36O2 ,
extracted from olive oil with saponification
methodology.
Figure 8. Chromatogram produced by modern pistacia spp.
resin with a) solvent exctraction methodology by BSTFA;
b) saponification extraction methodology; c) sulfuric
acid extraction methodology.
Key: n-TMS; FAME; underivatized fatty acid;
n-alcohol; n-alkene; n-alkane; P phthalate.
Figure 9. Chromatogram produced by modern pistacia spp.
resin between minutes 25-30 with a) solvent exctraction
methodology by BSTFA; b) saponification extraction
methodology; c) sulfuric acid extraction methodology.
Figure 10. Mass spectra from pistacia spp. resin: a) mass
spectrum at 29.53 min of β-sitosterol, extracted with
saponification methodology; b) mass spectrum at 27.99
min of lanosterol molecule extracted with
saponification methodology; c) mass spectrum at 27.38
min of lup-20(29)-en-3-one extracted with sulfuric acidi
methodology; d) mass spectrum at 28.82 min of betulin
extracted with sulfuric acid methodology.
Figure 11. Chromatogram produced by modern finger
prints with a) solvent extraction methodology by
BSTFA; b) saponification extraction methodology; c)
sulfuric acid extraction methodology.
Key: n-TMS; FAME; n-alcohol; n-alkene; P
phthalate.
Figure 12. a) mass spectrum at 25.24 min of squalene
molecule, C30H50, extracted modern finger print with
solvent extraction methodology by BSTFA; b) mass
spectrum at 27.02 min of cholesterol molecule, C27H46O,
extracted from modern finger print with sulphuric acid
methodology.
Figure 13. Chromatogram produced by blank test for
sulfuric acid method with a) blank test 1; b) blank
test 2.
Key: FAME; P phthalate.
Figure 14. a) mass spectrum at 23.03 min of phthalic
acid, mono-(2-ethylhexyl) ester, C16H22O4, from test
blank 1 with sulphuric acid methodology by BSTFA; b)
mass spectrum at 17.62 min of the FAME moleculeii
hexadecanoic acid, methyl ester, C17H34O2, from blank
test 2 with sulphuric acid methodology.
Chapter 4:Figure 15. Comparison of amount of time spent for
solvent extraction methodology by BSTFA,
saponification extraction methodology and sulfuric acid
extraction methodology.
Figure 16. Reactions of beeswax molecule with three
different reactants.
Figure 17. Reactions of triglyceride molecule with
three different reactants.
Figure 18. From Skeletons of penta- and tetra-cyclic
triterpene derivatives identified in pistacia spp resin
(from Assimopoulou and Papageorgiou 2005, 588-589).
iii
List of tables
Chapter 4:
Table 1. Comparison of solvent extraction by BSTFA,
saponification and sulfuric acid procedures.
Table 2. Material required for one sample for each
method.
.
Table 3. Table 3. Significant mass spectra of modern
beeswax identified in this study.
Table 4. Significant mass spectra of modern olive oil
identified in this study.
Table 5. Significant mass spectra of modern pistacia spp.
resin identified in this study.
Table 6. Significant mass spectra of modern finger
prints identified in this study.
iv
List of abbreviation
BSTFA: N,O-Bis(trimethylsilyl)trifluoroacetamide
DAG: diacyglycerols
DCM: dichloromethane
FAME: fatty acid methyl esters
H2SO4: sulphuric acid
MAG: monoacyglycerols
MeOH: methanol
TAG: triacyglycerols
TMAH: tetraalkylammonium hydroxide
TMS: trimethylsilylether
TMTFTH: (m-trifluoromethylpheny1) trimethylammonium
hydroxidev
WE: wax ester
Acknowledgment
I would like to thank myself for finishing what I
started and be able to present this thesis. I want to
thank my parents to let me go to do a degree abroad. I
would like to thank my mum for helping with the grammar
with this thesis, but also with other coursework. I
want to thank the friends that I spent my universities
years together. A more than deserve thank you goes to
my supervisor Dr. Benjamin Stern for his support and
advice and to have being patient with me during the lab
and while writhing this dissertation.
vi
Thank you for letting me be part of Bradford University
and giving me the chance to do this incredible course.
vii
1
PART 1: Literature Review - Why lipids recoveryis important?
Chapter 1 – Introduction
In the subject of archaeology, recovering and
identification of organic material is important to
reconstruct not only the life of an individual, but the
lives of a population, their trades, and costumes. The
scientific aspect of archaeology is the part that helps
to confirm theories and what has been written, but can
also help to discover more relevant evidence about our
ancient past and the one of the planet.
This project is about testing a novel method of lipid
extraction from archaeological potsherds. This chapter
will talk about the importance of lipids, the methods
that are mostly used to extract and analyse them, what
their disadvantage is and why archaeologists are in
need of a new improved method that is more rapid,
requires less material to analyse and that gives more
precise recovered information.
2
Vessels have different uses, whether it is utilitarian
or decorative. This was the case also in the past, but
it is more difficult to understand what vessels were
used for, especially when there is no written record
about them or what is left is just a sherd. Lipids are
able to help us to have a better understanding of the
purpose of a pot (Correa-Ascencio and Evershed 2014).
In modern times pots are most of the time made by
machine and their materials are almost always the same
and they are very resistant. In the past people did not
have specific oven to fire the pots, they also used
more material to make their vessels more resistant.
Those materials are the matrix of the pots (Stern et al
2000; Evershed 2008b; Correa-Ascencio and Evershed
2014). When the pottery is fired, small pores, almost
invisible to the human eye, will be formed (Powis et al
2002). If a substance is placed inside this vessel, in
most cases some molecules will settle in those pores
(Correa-Ascencio and Evershed 2014). This happens
especially with liquid and viscous materials (Copley et
al 2005; Evershed 2008a and 2008b). The pores of the
ceramic wall will conserve the organic residues by
3
entrapping them, thus “limiting their loss by water
leaching and microbial degradation” (Correa-Ascencio
and Evershed 2014: 1330). The lipids chemical structure
does not make them soluble in water, letting them
survive for centuries and making them the best
preserved biomolecules. It is for this characteristic
that lipids have been greatly studied and why they are
often used as archaeological biomarkers (Stern et al.
2000; Craig et al 2004; Evershed 2008b; Correa-Ascencio
and Evershed 2014). Scientists are able to identify a
wide range of organic materials e.g., beeswax, resins,
bitumen, waxes, wine, etc. and to distinguish between
milk, terrestrial and marine animal fats and vegetable
oils, (Stern et al 2000).
4
Chapter 2 - Conventional solvent extraction and
saponification, the problems
It is important to use the right techniques to extract
and analyse lipids. Until now two main methods have
been used: conventional solvent extraction and
saponification. Both of them use a solvent mixture
(dichloromethane–methanol 2:1 v/v for conventional
solvent extraction and sodium hydroxide for
saponification) and ultra-sonication of the ground pot
(Stern et al 2000; Pollard and Heron 2008). The solvent
mixtures, with the ultra-sonication, allow the lipids
to dissolve, thus to break down their fatty acid chain
with the functional groups, if any are present, and
later to be identified (Pollard and Heron 2008; Correa-
Ascencio and Evershed 2014). With those methods it is
possible to recover common classes of lipids, such wax
esters (WE), n-alkenes, n-alkanes, fatty acids,
acylglycerols and long-chain ketones (Regert et al 1998;
Correa-Ascencio and Evershed 2014). Nonetheless, when
using these two techniques, the lipid recovery can be
incomplete, especially because some of the molecules
will be still trapped in the potsherds (Stern et al 2000;
Craig et al 2004). The amount of lipid extracted is not
solely dependent on the extraction method, but by
several factors that are all linked to the material of
the pot and their use (Correa-Ascencio and Evershed
5
2014). The vessel fabrication, for example, will
affect since the amount of organic residue absorbed
because this absorption depends on the size and shape
of the ceramic pores, which in turn depends on how the
surface has been treated and fired. It is also more
common to obtain higher lipid concentrations on pots
that were containing viscous or liquid material, or
even used as cooking objects (Rice 1987; Pollard and
Heron 2008). When cooking, heat will help convert fats
in liquid forms, in solution or colloidal form, into
the ceramic walls. Indeed the cooking pots contain the
higher concentration of lipids over other vessel forms
6
(Charters et al 1997; Pollard and Heron 2008). Cooking
pots will therefore release higher concentrations even
than vessels that were carrying viscous or liquid
material because to let a high concentration of those
to be absorbed, they need to be inside of the container
for a long time, and also for better results the
container should always carry the same substance (Rice
1987; Correa-Ascencio and Evershed 2014). The
environmental conditions of burial play an important
role as well because here it is where degradation
happens. The major factors that affect degradation and
the time until complete degradation are waterlogging,
temperature, light exposure and burial place. This will
result in the lipids to be the first to completely
degraded (Evershed 2008a). The perfect climate that
preserve and showed the highest concentrations of
organic residues, but also other material such mummies,
is in very arid geographical areas. One of the main
factors that contribute to degradation is water. Its
absence limits microbial degradation and preventing the
dissolution of organic residues (Correa-Ascencio and
Evershed 2014). However, scientists still not fully
understand the dynamics that determine the presence of
lipid residues on vessels (Evershed 2008a).
It is because of the complexity not just of some
lipids, but also of the ceramic itself, how it was used
and the burial place, that lipids extraction can be
7
complicated. A range of diagnostic polar lipids are
inside the sherds, captured by the pores, and they
cannot be extracted without the help of a strong
extractant, i.e. dichloromethane (DCM)–methanol 2:1 or
methanolic sodium hydroxide (Pollard and Heron 2008;
Correa-Ascencio and Evershed 2014). At times, better
results can be gained by doing a multi-stage extraction
protocol. Multi-stage extraction, however, can be
impracticable with the time frame of most projects
because they will take long time to complete,
particularly in case large numbers of potsherds need to
be investigated to answer important archaeological
questions (Correa-Ascencio and Evershed 2014).
8
Chapter 3 - Revision of solvent extraction,
derivatization and saponification methods
The ceramic anyway it is not the only problem. The
techniques that will be used also need to be chosen
carefully. Ideally, it is needed a procedure that can
extract all the lipids out of the matrix without
destroying them, that does not contaminate the samples
and is able to recognize every detected molecule. The
performance of conventional solvent extraction and
derivatization protocols can take a long time and there
are high chances that it will contaminate or create
analytical artefacts. Those risks are high particularly
because of the manipulative stages needed (Stern et al
2000). Those stages can be avoided by methylate, the
samples directly done with the gas chromatograph (GC)
using a tetraalkylammonium hydroxide (TMAH) derivative.
The use of the derivative TMAH abled Kossa et al (1979)
and Stern et al (2000) to identify diterpene and
triterpene acids, triacylglycerols, fatty acids,
proteins, waxes and kerogen. However, TMAH’s basicity
leads to the isomerization of polyunsaturated fatty
9
acids to conjugated polyenes, thus altering chemical
structures (Stern et al 2000). A different derivatization
molecule is (m-trifluoromethylpheny1) trimethylammonium
hydroxide (TMTFTH). Even if this can be used easier
than TMAH, the results will show lower yields of fatty
acid methyl esters (FAMEs) because TMTFTH has a lower
basicity than TMAH (Kossa et al 1979; Stern et al 2000).
There is another derivatization molecule as well that
is more commonly used because of the faster procedure
and the good results, which are better than TMAH and
TMTFTH: BSTFA. BSTFA can derivatize labile, instable,
groups, such as hydroxyl, on other chemicals. This can
happen because of the more stable trimethylsilyl group
that protects the labile group. This compound differs
from the other two because instead of being added in
the GC to derivatize, it is added at the end of an
extraction prior to analysis (Stalling et al 1968;
Correa-Ascencio and Evershed 2014). The more volatile
physic of siloxane groups than the corresponding
hydroxyl compounds, allow the new form compound to be
analysed with gas chromatography better than the parent
compound (Stalling et al 1968). Then again, the
10
conventional solvent extraction even with BSTFA still
has a problem: it is still not strong enough to extrude
all the lipids from the ceramic matrix. Even if not all
the lipids are released from the potsherd, this method
is still able to procure researchers with enough
information (Craig et al 2004). The molecules that can be
detected are waxes, resins, n-alkene and n-alkanoic
acids, diacyglycerols (DAG), triacyglycerols (TAG),
monounsaturated n-alkenoic acids and some amounts of
monoacyglycerols (MAG) (Craig et al 2004; Correa-Ascencio
and Evershed 2014). Some of those components are part
of degraded animal fat, but this technique cannot
distinguish if they do belong to an animal fat or to a
vegetable oil and also understanding the provenience of
resin can be challenging (Kimpea et al 2001; Craig et al
2004; Evershed, RP. 2008b; Gregg and Slater 2010). This
technique is really useful because all the basic
information, most of the time, will be obtained without
drilling a great amount of potsherd, usually 0.10g is
satisfactory, and also the laboratory procedures are
easy and fast (Craig et al 2004).
11
As an alternative there is saponification technique.
This method is usually able to release greater
abundance of lipids out of the ceramic vessel matrix
through alkaline hydrolysis, or treatment with aqueous
TMTFTH (Adams et al 1986; Craig et al 2004). Analysis after
saponification will have a greater yield of fatty acids
than using the conventional solvent extraction method,
even if the solvent extraction method will mostly have
a greater yield of wax esters or TAG (Gregg and Slater
2010). If saponification is able to extract more lipids
out of the vessels, there is a chance that some
information will be lost in the process because this
technique is using a stronger base that can result in
degradation of some molecules (WE and TAG are
hydrolyzed to their constituent fatty acids and
alcohols). With this method it will be easier to
distinguish between animal fat and vegetable oil and it
can help distinguish the different kind of resins.
What would be preferred is using an extraction
technique that requires less potsherd and that is
faster to complete, in this case conventional
extraction with BSTFA. The researcher could stop at
12
this point if the information acquired are satisfying
enough, or proceed with another technique if more
information about a certain molecule has to be
confirmed or analysed more deeply. Techniques need to
be also chosen in consideration of the conditions and
state of degradation of the material (Craig et al 2004).
Degradation will affect the result by not showing some
molecules, because completely degraded, or can mistake
a compound for another one. Also result could be not
perfectly clear because not enough information can be
deducted, thus the entire organic residue on a pot
could go lost if the chosen method consists of a high
basic concentration that most likely will decompose the
already low abundance molecules (this could happen by
using saponification) (Craig et al 2004; Evershed 2008b;
Gregg and Slater 2010; Correa-Ascencio and Evershed
2014).
13
Chapter 4 - A novel method
One fact is known: the necessity of a new efficient and
rapid analytical protocol. It needs to be fast, because
of the necessity to process large collections of
sherds, and be capable to give a solution to important
archaeological questions. Nevertheless it is not just a
method that will give higher yield of lipids, but also
a better identification of the unknown molecules.
Correa-Ascencio and Evershed (2014) explain how they
think they found this new improved analytical protocol:
sulfuric acid method. They tested this procedure on 33
subsamples analysed in previous works to be sure of the
results. In general their result showed, in most cases,
four times more organic residues extraction from the
vessel pores respect to the conventional solvent
extraction, therefore giving a higher lipid
concentration. Even sherds that give little yield with
other methods, gave higher yield with acidified
methanol extraction. An important aspect that was
observed is that more samples can be prepared at one
time than with other methods. The major advantage of
this protocol is that “free” and “bound” fatty acids
14
are simultaneously extracted and methylated yielding
FAMEs” (Correa-Ascencio and Evershed 2014, 1338). This
means that there is the simultaneous extraction and
derivatization of lipid residues. The only flaw they
found on this method is the loss of information due to
the hydrolysis of complex lipids (e.g. wax esters).
What is not clear is the amount of material needed to
complete this method, which should be compared with the
material needed for other methods. Another unclear
point is the amount of information that will be lost.
Correa-Ascencio and Evershed explain what might get
lost in the process, but justify this loss by saying
that other methods can be performed to investigate more
on the unknown molecules found. The main point of a new
technique is also to reduce time to obtain good results
and in what they are saying it is seems like they are
using more time than with other techniques to run
subsequent analysis to know what the previous could not
tell. This will not just be time consuming, but it also
means spending more money. This was even the case of
the saponification and conventional solvent extraction,
but they do not require much time to be finished and in
15
most cases the results are good enough to understand
which kind of component/s was placed on the vessel.
16
PART 2: Research
Chapter 1 – Introduction
Archaeologists have a great interest in lipids because
they are the best preserved biomolecules, thanks to the
lipids chemical structure. Lipids are medium-sized
molecules composed by linear, branched or cyclic
hydrocarbon skeletons. It is the lipids skeleton that
makes them insoluble in water, but in organic solvents
(Pollard and Heron 2008). Because lipids do not degrade
easily, they are used in archaeology as biomarkers
(Evershed 2008a; Evershed 2008b; Correa-Ascencio and
Evershed 2014). An example of the importance of lipid
analysis is in Maya archaeology. Powis et al (2002),
through lipid extraction and analysis, understood that
some Maya ceremony involved the use of alcoholic
chocolate by the priest, and that this civilization was
one of the first to drink chocolate. Lipids do not just
help to identify what a vessel contained or its use,
they help scholars to understand the diet of a
population and also any trades between populations as
well (Ambrose et al 2003).
17
In 2000 York University improved a new and more
efficient method to extract lipids from archaeological
potsherds (SOP, acid extraction of organic residues
from archaeological ceramics, direct contact with York
University). They were investigating the extraction of
lipids through sulfuric acid. The results of their
experiment are not published yet, however researchers
from Bristol University, Correa-Ascencio and Evershed
(2014), have published this new extraction method. In
their paper, Correa-Ascencio and Evershed state that
this technique is giving results faster than previous
methods and the yield of lipids is greater. Although
this novel method has been applied to fatty acid, it
has not previously been used to extract other lipid
material; therefore this dissertation, in addition to
the extraction of fatty acids (from olive oil) will
also focus on the extraction of other lipid material,
as resins and beeswax.
Waxes and resins are complicated molecules and
knowledge of their molecular behavior might help to
predict how they are going to react with sulfuric acid
18
and if an improvement in their recovery could be made
(Pollard and Heron 2008).
1.1 Aims and Objectives
The aim of this project is to determine if sulfuric
acid is the best and fastest way to extract lipids from
archaeological sherds The experiments will be conducted
by using known lipid profiles from modern samples
(olive oil, beeswax and resin). This project will be
able:
1) to confirm that the sulfuric acid method results in
higher yields of fatty acid;
2) to compare the sulfuric acid method to conventional
solvent extraction and saponification;
3) to examine any analytical artefacts created by the
sulfuric acid method (such as degradation of some
lipids).
To answer those questions, the sulfuric acid procedure
will be compared with alkaline hydrolysis, performed
with saponification, and solvent extract by BSTFA
method. The experiments will provide data about how
19
much lipids sulfuric acid technique is able to extract
with 0.10mg. After this, it will be possible to know if
the previous step could allow researchers to identify
between the different organic groups (ester, alkane,
etc.). If this last step is possible, a more advanced
examination can be made to try to identify the organic
material within the same organic group, for example to
recognize waxes, which have esters group in their
molecules. If those steps are carried out correctly,
the researcher could be able to understand what the
examined vessel was once containing and used for (Orna
1996).
The laboratory procedures that will be followed will be
the standard operating procedures (SOP) used by both
York and Bristol Universities. By following the SOP it
will be established if York method is the best above
all or not, and if some changes are needed. All the
samples, consumables and analytical costs that will be
used in this project will be provided by the
archaeological department of the University of
Bradford.
20
The researcher of this project is hoping that this
study could lay the foundations of a better
understanding of sulfuric acid to extract lipids as to
do more researches with molecules that are not fats.
21
Chapter 2 – Methods
2.1 Standard sample preparation
An unused modern potsherd (previously Soxhlet solvent
washed) was divided into four sub-samples. Three sub-
samples were separately coated with beeswax, olive oil
and pistacia spp. resin. A final sub-sample was untreated
and will serve as the method blank.
Each sub-sample was then separately powdered using a
Dremel drill fitted with an abrasive tungsten bit. All
the samples and drilled potsherds were stored in glass
containers that were earlier washed with
dichloromethane (DCM).
Further sub-samples (0.10g) of each powder were taken
to compare the different extraction methods described
below.
Before being used, all glassware was solvent rinsed
with DCM to prevent eventual contamination in case the
sample touches it. Every tool was also rinsed with DCM.
2.2 Conventional solvent extraction by BSTFA
To each (0.10g) sample (ceramic powder plus either
beeswax, olive oil, pistacia spp. resin or method blank)
22
was added 2ml of DCM:methanol (2:1, v:v). To aid
dissolution, the samples were placed for five minutes
in the sonicator. Separate and insoluble materials were
centrifuged for five minutes at 2000 r.p.m. The solvent
was then transferred to a clean auto sampler vial and
combined with two further repeats of the solvent
extraction. Once the solvent has been obtained, the
samples were placed on the nitrogen blowdown machine
for 10 minutes which evaporates the liquid solvent and
leaves just the lipids. 10 drops of BSTFA were added to
each sample and they were left at room temperature for
one day. The day after the samples were analyzed by the
gas chromatography-mass spectrometer (GC-MS) machine
(figure 1).
23
24
2.3 Saponification
3ml of 0.5M methanolic NaOH were added to each sample.
These were heated at 70°C for three hours in a sealed
glass vial. After cooling, the solution was acidified
with 10 drops of HCl (tested with pH paper). 3ml of
hexane were added and the top layer was extracted with
a pipette and placed in a new container. This procedure
was repeated other two times to obtain about 6ml of
supernatant. The hexane was left to evaporate and 1 ml
of DCM added to dilute the sample prior to analysis by
GC-MS. This procedure is shown in figure 2.
25
26
2.4 Sulfuric acid
Once the samples have been placed in the hach tubes,
4ml of MeOH (methanol) was added to each sample with a
Pasteur pipette. In total there were six hach tubes
with two blanks. Each hach tube was ultrasonicated for
15 minutes. Then 800µ of concentrated sulfuric acid was
added with a Pasteur pipette. This procedure was
performed in the fume cupboard. The sulfuric acid was
added carefully, drop by drop, and observing for any
reactions with sample, because in case a sample is
calcareous the carbon dioxide will be released and it
can result in the loss of sample. The samples were then
placed on a heating block at 70°C for four hours.
Simultaneously, six Pasteur pipette were cleaned with
DCM and prepared for the next steps. At the bottom of
each pipette was placed about 1cm of glass wool, enough
to plug the pipette. Then about 5mm of prepared
potassium carbonate were added. The potassium carbonate
was previously prepared by extracting it three times
with DCM and baked at 350°C. After the four hours, the
hach tubes were cooled and the pH was tested. To
precede, the acidity parameter had to be below or equal
to three, if not the pH needs to be adjusted by gently
adding drops of sulfuric acid. The samples were then
centrifuged for five minutes at 3000 rpm. The liquid
was extracted and placed into a new clean hach tube and
27
2ml of hexane were added and mixed using the vortex
machine. When using the machine the liquid will touch
the cap, this is why they were previously cleaned with
solvent. The (upper) hexane layer was allowed to
separate out, and carefully pipetted off and passed
through the previously prepared Pasteur pipette with
potassium carbonate. Further hexane is used to rinse
the sample through the potassium carbonate. Now it is
necessary to remove sulphur from the samples to prevent
the corrosion of the GS-MS column. To do so activated
copper was added to each sample and left overnight. The
next day each sample was transferred to another
scintillation vial, to separate them from the activated
copper. The samples were evaporated to almost dryness
under a gentle flow of nitrogen at 40°C. To dilute the
samples, to be analysed by GC-MS, 20 drops of DCM were
added. In case analyses are not carried out straight
away, the extracts must be placed in a freezer at -20°C
until analysis (figure 3).
28
2.5 GC-MS analysis
Analysis was carried out by combined GC-MS using an
Agilent 7890A Series GC connected to a 5975C Inert XL
mass selective detector. The splitless injector and
29
interface were maintained at 300°C and 340°C
respectively. Helium was the carrier gas at constant
flow. The temperature of the oven was programmed from
50°C (2 min) to 350°C (10 min) at 10°C/min. The GC was
fitted with a 15m X 0.25mm, 0.25µm film thickness HP-
5MS 5% Phenyl Methyl Siloxane phase fused silica column
(Agilent J&W). The column was directly inserted into
the ion source where electron impact (EI) spectra were
obtained at 70 eV with full scan from m/z 50 to 800.
30
Chapter 3 – Results
3.1 Modern beeswax
The chromatograms of the three different extraction
methods (figure 4) show the main components of a
beeswax molecule recorded by Tulloch (1971). It is
common that when wax esters, alcohol, alkenes and
trimethylsilylether (TMS) groups are present, the
molecule can be identified as from beeswax (Nelson &
Blomquist 1995; Tulloch 1980; Garnier et al 2002; Correa-
Ascencio Evershed 2014). Anyway the concentration of
those components varies depending on the method of
extraction (figure 4).
The results from extraction with BSTFA identified all
the typical beeswax components (figure 4a). TMS
derivatized molecules start at retention time 18.97
with C16:0 (figure 5d). The molecules are increasing in
size through the chromatogram until C26:0 at 27.3
minutes. The same happened to the alcohol molecules:
the first molecule is a fatty acid chain of C25:0 and the
last one identified is C44:0 at 36 minutes. The most
abundant molecules found here are the n-alkanes, while
31
there is just one alkene, double bonded between carbon
atoms in a chain, pentatriacontene (C35H70), shown by
the arrow symbol in figure 4a. The first identified
alkane, at minute 21.17, is C23:0. Even these molecules
are increasing in size with the last found alkane being
C46:0. Only one wax ester molecule has been identified
at minute 32.97, C40:0. The first 18 minutes and from
minute 30, the chromatogram shows just different TMS
molecules that are not relevant to the sample because
were part of the used solvent. Identifying the
different peaks was easy since the solvent did not
destruct important information during the process of
extraction.
The lipid extraction with saponification gave different
results (figure 4b and 5b). All the molecules that were
identified with the previous method were present; but
two other types of molecules appeared: FAMEs (fatty
acid methyl esters) and underivatized molecules. FAME
molecules were identified between 17.65 and 24.36
minutes. They have an increased pattern in size through
the retention time. Two underivatized molecules were
32
present at 18 and 19.3 minutes (figure 4b). Their
presence means that those molecules, C16:1 (figure 6b)
and C18:1 (figure 5b) were not completely broken down
during the saponification process. By looking at the
chromatogram it is possible to see that the compounds
are in high abundance, which means that not all the
reactions took place. A WE molecule was present as
well, as alcohol (figure 6a), alkene and alkane. No TMS
molecules were detected. The molecules that were
detected, before the first high peak and after minute
30, were different TMS, as for the previous method.
The sulfuric acid methodology did not recover any
alkanes, TMS or WE molecules. Underivatized molecules
were not present as well, which means that every
molecule had been broken down. FAMEs are the
predominant molecules found here. Two important
molecules recognized are C17H34O2, palmitic acid (figure
6c), and C19H36O2, oleic acid. Those two fatty acids are
important because they are component of beeswax (Regert
et al 2001; Garnier et al 2002; Kimpea et al 2002). The
chromatogram does not show more information than the
33
previous two methods, which means that information were
lost in the process. In the case of the sulfuric acid
and saponification extraction methodology a phthalate
molecule was found. This means that the samples were
contaminated with plastic. Because of this the molecule
will not be taken in consideration.
34
Figure 4. Chromatogram produced by beeswax with a) solventextraction methodology; b) saponification extraction methodology; c) sulfuric acid extraction methodology.Key: n-alkane; n-TMS; n-alcohol; n-alkene; WE wax ester; FAME; underivatized fatty acid;
35
Figure 5. a) mass spectrum at 28.11 min of alcohol molecule,
C30H62O, extracted from beeswax with saponification
methodology; b) mass spectrum at 18 min of underivatized
molecule, C16H32O2 n-Hexadecanoic acid, extracted from beeswax
with saponification methodology; c) mass spectrum at 17.7 min
of FAME molecule methyl palmitate, C17H34O2, extracted from
beeswax with sulfuric acid methodology; d) mass spectrum at
36
3.2 Modern olive oil
Lipid extraction by BSTFA showed the principle
components of olive oil (figure 6a). C18:1 was the
highest peak at 20.55 minutes and one of the lowest
peaks at 20.57 minutes. TGA, C18:1 (figure 7a) was
identified as well with squalene and β-sitosterol,
which are components of oils (Kimpe et al 2001). TMS C16:0
and C18:0 were also found. With this information it is
clear that the analysed compound was plant oil. Since
the presence of the two C18:1 molecules, which are oleic
acids, it can be deducted that the oil compound derive
from olives.
Extraction by saponification seemed to have detected
more molecules than the previous method and to have
produced higher peaks, however the sulfuric acid have
detected the highest peak out of all the methods
(figure 6). In this case an alcohol molecule was
detected, C21:1 at the beginning of the chromatogram and
three underivatized compounds. FAMEs compounds were
found with the following fatty acid chains: C16:0 and and
C18:1. Those two compounds are the result of the
37
reaction between the sodium hydroxide (NaOH) and TGA
groups. When NaOH was introduced in the sample it broke
the bonds of every CH2 atoms forming FAMEs and glycerol.
Because of this we can deduct that oleic acid, giving
the FAME C18:1 is present as well as palmitic acid, FAME
C16:0 (table 4). With this method it is possible to detect
that the compound came from olive oil, but it is not
straight forward as in the previous case. This is due
to different reactions that take place in the sample.
The sulfuric acid extraction method is different
because it shows more peaks at the beginning of the
chromatogram and less by going towards the end (figure
6c). The main peak is the FAME molecule methyl oleate,
C18:1. As in the case of the sulfuric acid the TGAs went
through a different process were the molecules, in this
case, have been added a methyl group. FAME
heptadecanoic acid, C16:0 is possibly the palmitic acid.
Isopropyl palmitate, C18:0 was also found. This is the
ester of the palmitic acid. Another derivative of the
palmitic acid, C15:0: methyl palmitate.
38
With this technique not all the molecules could be
easily identified, or with accuracy, especially the
ones at the beginning and end of the chromatogram. All
the main molecules anyway have been recognized. With
the molecules listed above it is possible to accomplish
that this compound is from olive oil.
39
Figure 6. Chromatogram produced by modern olive oil with
a) solvent exctraction methodology by BSTFA; b)
saponification extraction methodology; c) sulfuric acid
extraction methodology.
40
Figure 7. a) mass spectrum at 24.83 min of TAG
molecule, C27H56O4Si2, extracted from olive oil
with solvent extraction methodology by BSTFA;
b) mass spectrum at 19.32 min of FAME
molecule, C19H36O2 , extracted from olive oil
with saponification methodology.
41
3.3 Modern pistacia spp. resin
Figure 8a shows the chromatogram of modern pistacia spp
resin through solvent extraction methodology. In this
case it was possible to identify with certainty only
two TMS molecules (C16:0 and C18:0). From minute 20, the
GC-MS library is starting to recognize molecules with
at least one cyclic carbon ring. Between minute 28 and
31.20 the chromatogram shows high peaks close together
(figure 9a). Those peaks are all penta- or tetra-
cyclic triterpenes that will help identify if the
studied molecule is resin and also could help to trace
back the resin origins, thus identifying which kind of
resin it is.
The results achieved with saponification and sulphuric
acid methods are different from the result with solvent
extraction and more similar to each other. In both
cases were found FAMEs molecules, but in the
saponification technique also underivatized compounds
were found (C15:0, C16:1 and C16:0) (figure 8b). This method
was also the only one able to extract and recognise an
alcohol molecule that could help identify the
42
provenience of this resin: β-sitosterol (figure 10a).
An important component found in the underivatized
molecule is the oleic acid (C16:0) which is a component
of two species of pistacia spp. resin (Colombini et al
2000). This time the cyclic triterpenes start to be
seen from minute 23.20 until minute 31 (figure 10b). To
identify those peaks it was easier for the GC-MS
library, which gave more accurate results. Three peaks
were recognised as lanosterol (figure 10b), while the
last one is β-sitosterol. The sulfuric acid methodology
(figure 9c) identified FAME compounds and at 24.13
minutes it recognises an alkane molecule (C27:0). The
FAME compounds were not big, the first has just a fatty
acid chain of 15 carbon atoms, and the biggest has just
23 carbon atoms. With this extraction technique,
between 23.42 and 31 minutes cyclic triterpenes were
detected. The most peaks traceable to cyclic
triterpenes are between 27.35 and 30.24. Most of those
peaks belong to botulin molecules (figure 10d) and to
lup-20(29)-en-3-one (figure 10c), which should be
lupene molecule after going through extraction process.
43
The saponification and sulfuric acid technique were
able to identify the peaks belonging to cyclic
triterpenes compounds with more accuracy.
44
45
46
47
3.4 Modern finger prints
The extraction with conventional solvent extraction by
BSTFA is the only method that identified both squalene
(figure 12a) and cholesterol molecules (figure 11).
Other than those molecules four TMS compounds were
found: C14:0, C15:0, C16:1, C16:0 (figure 11a). TMS are
compounds that are formed as a result of the used
method. When the reactant was introduced on the
samples, in this case BSTFA, substitution reaction took
place. In substitution reactions two molecules exchange
parts to give two new products; thus TMS are products
that, for example, are formed bysubstituting the
alcohol group (-OH) at the end of a hydrocarbon chain
with a O- trimethylsilyl group (O-C3H9Si). Thus the TMS
C16:1, palmitelaidic acid, trimethylsilyl ester is the
derivatized silyl esters of the original fatty acid
palmitoleic acid, which has been recognised as a
component of skin (Bernier et al 1999).
The saponification methodology (figure 11b) was able to
extract just the squalene molecule, no cholesterol was
found. Phthalate contamination was present with other
48
two FAME molecules (C16:0 and C18:0). The other peaks were
difficult to identify, in fact the MS library could not
give accurate results from the mass spectra. The
reactions probably were too strong and destroyed the
main lipids that help identify finger prints and
establish the contamination in archaeological
potsherds.
The sulfuric acid technique extracted more information
from the sample than the saponification method (figure
11c). In this case just cholesterol (figure 12b) was
found and no squalene was detected. The other molecules
that were in the chromatogram belong to the FAMEs
class, and one of those was oleic acid.
49
50
51
3.5 Sulfuric acid blank tests
The chromatograms of both tests show that they have the
same components in the same concentration, with peaks
detected at the same retention time. In both
cases phthalate plasticiser molecules (figure 14a) and
three FAMEs (C15:0, C17:1, C17:0) (figure 13) were found.
Other peaks are present, but they are below 5 µm and
because of this they will not be considered, since also
Correa-Ascencio and Evershed (2014) do not assumed
peaks below this value because
considered contamination.
The identification of FAME compounds (figure 14b shows
FAME C15:0) could mean two possibilities: either the
preparation of the solution was carried out correctly,
which should assure the correct procedure of the
solvent, or they come from contamination and in this
case could help to identify contamination on the other
samples. Since an internal standard was not added,
making it not possible to quantify the FAME compounds,
it is difficult to be sure if they are contamination
molecules or not. The only molecule that is sure to be
the outcome of contamination is the phthalate molecule,
52
which was present in all the sulfuric acid
chromatograms. The results show FAMEs because they
could have already been present on the modern
potsherds. So even in this case, these organic
materials could be part of the ceramic walls or
contamination. If those molecules are part of the
ceramic walls, this will demonstrate how strong this
method is and that it is able to recuperate not just
organic residues, but also component of the ceramic
material. Further studies are needed to affirm this
hypothesis, but this could help understand the
technology that different population had, as
well as how their techniques developed.
These results will not be discussed on the discussion
chapter since everything has already been covered.
53
54
55
Chapter 4 – Discussions
4.1 MethodsAll the methods were easy to execute, although the
conventional solvent extraction was the easiest and
fastest of all (table 1 and figure 15). This method did
not require more than 10 minutes wait between each step
and it was necessary to wait until next day only to
place the samples on the GC-MS machine. Thus results
will be ready to analyse the day after the method is
performed, anyway this apply for the other two methods
as well.
Saponification method required more steps and more
material than the solvent extraction technique (table 1
and table 2), but even in this case the procedure was
easy to follow and achieve.
56
Conventional solvent
extraction by BSTFA
SaponificationSulfuric acid
24.4
2728.3
1
14.35
Figure 15. Comparison of amount of time spent for solvent extraction methodology by BSTFA, saponification extraction methodology and sulfuric acid extraction methodology.
Tot hoursWaiting time Working time
The method that required more hours in the laboratory
and more material is the sulphuric acid method (figure
15 and table 2). It could be still thought that even
with all those stages, the method is still fast because
some stages do not take long to be performed. If some
steps are fast to do, there is still a wait of four
hours after the sulphuric acid is introduced in the
sample. Anyway, in this four hours window it can be
prepared the Pasteur pipette with inside the glass woo
and potassium carbonate (if this last has been prepared
in advance, since it takes around three hours to be
57
ready), as well as the activated copper. This method
can be long to conduct and multiple stages are needed,
which means that it is easier for the sample to be
contaminated and lipids to be lost.
Before starting the experiment, it was only known how
saponification and the conventional solvent extraction
methodologies would have react with the samples,
causing certain reaction with known results, but it was
not sure which reactions would take place with the
sulfuric acid as a reactant (figure 16 and figure 17).
In the case of beeswax, sulfuric acid reacted as
expected: derivatizing molecules to their FAMEs
corresponding, but also an alcohol and an alkene
molecule were detected. The alcohol molecule is
probably the result of the separation reaction, where
the other formed molecule is FAME. FAME molecules were
formed also on the other samples, except that finger
print sample is the only other that shows an alcohol
molecule. The olive oil sample produced, other than
FAMEs, an underivatized molecule, which could still be
a product of a separation reaction. The pistacia spp
58
sample was the only one where an alkane molecule was
identified.
59
Table 1. Comparison of solvent extraction by BSTFA, saponificationand sulfuric acid procedures.
60
61
4.2 Modern beeswax
Beeswax is a complex mixture that includes different
combinations of molecules (Nelson and Blomquist 1995).
Aichholz and Lorbeer (2000) identified 80 separate
lipid components, while Tulloch (1980) and Nelson and
Blomquist (1995) identified more than 300. Even though
it has this many components, it is possible to identify
it by its major components. Tulloch (1971) identified
the percentage of the main common molecules that
compose it: hydrocarbons, predominantly n-alkanes
(c.14%), monoesters, mostly of palmitic (C16:0
hexadecanoic) acid, (c.35%), hydroxy monoesters (c.
4%), diesters (c.14%), free fatty acids (c.12%),
62
triesters (c. 3%), hydroxyl polyesters (c.8%), acid
monoesters and acid polyesters (c.1% and c.2%
respectively), free alcohols (c.1%) and unidentified
material (c.6%) (Nelson and Blomquist 1995).
The conventional solvent method was able to identify
all those molecules. At 18.98 minutes a peak was
detected and its mass spectrum showed it to be the
derivatized TMS of palmitic acid (figure 5d and table
3) (Tulloch 1980; Aichholza and Lorbeer 2000; Regert et
al 2001; Evershed et al 2002; Garnier et al 2002). The
saponification process gave almost the same results,
but the differences of those results are the
underivatized molecules. These molecules can be present
because the material is fresh and new, thus all the
molecules did not break or react, because the NaOH
already had a high amount of compounds to break, so it
was not sufficient to break all the organic material.
This means that all the equilibria in the reactions
were achieved before all the molecules could break
(McMurry 2012, 186). As stated in the results, all the
molecules that characterized beeswax were found with
63
this process (figure 5b), especially the most
important: wax ester. Just in the sulfuric acid method
this molecule was not found. Anyhow, all other main
compounds were seen: palmitic acid, oleic acid, 1-
ecosanol and 1-hexacosanol (alcohols). In this case
just FAMEs were present in the chromatogram (figure 5c)
because of the different reagent introduced on the
sample.
When comparing the three chromatograms (figure 5), the
first noticeable thing is the few amount of peaks in
the sulfuric acid than in the other two methods.
Because this is a modern compound, which did not go
through deterioration or much contamination, it is
expected to detect every molecule, since most of them
did not go through any changes. It is with
archaeological potsherd that the outcomes will not be
guaranteed because it is not known how much residue is
left and, most important, their state of preservation.
Thus, just by looking at this the sulfuric extraction
method is not recommended. When analyzing fresh
beeswax, the majority of monoesters contain an even
64
number hydrocarbon chain with values ranging between 38
and 52. Also long chain alcohols have been recognized
(Tulloch 1980; Nelson and Blomquist 1995; Aichholz &
Lorbeer 2000; Regert et al 2001; Garnier et al 2002).
Sulfuric acid is a strong acid that could risk to
disintegrate some molecules and not letting the MS
identify them (Correa-Ascencio and Evershed 2014). This
is probably why there are fewer peaks, and it could
also be because of the weak forces that bond beeswax
molecule, which will be easy to unboned. If used with
archaeological beeswax it could be risked to lose the
residue if the conservation is not good. Correa-
Ascencio and Evershed (2014) study showed that this
method was extracting more residues from the ceramic
wall than any other, thus if the preservation is good
but not much organic residue is present this method
should still be able to identified some molecules, but
from this research it cannot be known if the analysis
could be positive in identifying at least three of the
main components or not. Since here WE were not
identified, there is a high probability that they will
65
not be detected neither on archaeological samples.
Garnier et al (2002) says that C24:0 fatty acids, which
are generally the most abundant in fresh samples, will
degrade in ancient samples showing instead abundance of
C16:0, palmitic acid, but it is not the case of any
method because there is just one peak identified as
C24:0. Just saponification method recognised two
molecules of C16:0.
All three methods showed that they could identify
beeswax, but it is not suggested to use the sulfuric
acid method because it could be too aggressive on
ancient material, which preservation most of the time
is not known. The conventional extraction method with
BSTFA should be the most reliable because it showed to
be able to derivative all the necessary molecules that
will lead to beeswax. Even saponification could be
used, but it probably will require more time to analyse
the results as most molecules have been derivatized to
their FAMEs or were underivatized, and in
archaeological samples it could degrade some molecules
since there are strong reagents.
66
Abbreviated name Assignment and structure Formula Basepeak
M+ Characteristic ions
TMS C16:0
Palmitic acid,trimethylsilylester
C19H40O2Si 73, 117
328 55, 313
TMS c18:0
Stearic acid, trimethylsilylester
C21H44O2Si 73, 117
356 55, 82
Alkane C23:0
TricosaneC23H48 57 324 71, 85, 113
Alkane C27:0
HeptacosaneC27H56 57 380 71, 85, 99
Alkane C29:0
NonacosaneC29H60 57 408 71, 85, 99
Alkane C34:0 C34H70 57 436 71, 85, 99, 127, 155, 183, 211,
67
Tetratriacontane
239, 267, 309
FAME C16:0
Palmitic acid,methyl ester
C17H34O2 74 270 55, 87
Underivatized C15:0
Palmitic acid
C16H32O2 73 256 55, 60
FAME C18:1
Oleic acid, methyl ester
C19H36O2 55 296 69, 83
WE C33:0
Hexadecanoic acid, octadecyl
C34H68O2 57 592 57,207
68
ester
Alcohol C19:0
1-EicosanolC20H42O 57 280 69,83
Table 3. Significant mass spectra of modern beeswax identified in this study.
69
4.3 Modern olive oil
Vegetables oils are predominantly composed by a mixture
of triglycerides (TGA) (Gunstone 2004, 23-32). TGA will
have some mono- and diacyglycerols, but the majority
will be monounsaturated fatty acids esterified to
glycerol (Dimitrios et al 2006, 41-42). Other
components are free fatty acids and a variety of minor
components. Those constituents can include
phospholipids, tocopherols, chlorophyll and
hydrocarbons including alkanes, squalene, carotenes and
polycyclic aromatic hydrocarbons, sterols (cholesterol
and, β-sitosterol), fat soluble vitamins (e.g. vitamins
A, D, E and K) (Gunstone, 2004, 23-32). The most common
component of olive oil, which can distinguish it from
other oils, is the monounsaturated fatty acid oleic
acid (C18:1). Other characteristic components are the
two polyunsaturated fatty acids: oil are linoleic
(C18:2) and alpha-linolenic (C18:3) acid. Two common
saturated fatty acids are palmitic (C16:0) and stearic
acid (C18:0) (Dimitrios et al 2006, 41-42).
70
The identification was hard to conduct on the sulfuric
acid method because the MS library could not match most
peaks with known biomarkers. However, three of the main
components of olive oil were recognised: FAME C18:1,
oleic acid methyl ester, FAME C18:2, linoleic acid
methyl ester and derivatized FAME of palmitic acid
C16:0. Between 55-83% olive oil is composed by C18:1 and
4-21% by C18:2 (Gunstone 2004, 6). Anyway there is no
trace of TAG (Gunstone 2004, 23-32; Dimitrios et al
2006, 41-42). Even in this case it is possible that
this type of extraction was too strong that let
dissolve or broke down the organic sample molecules too
much. The saponification process had the same problem,
even if it yielded two more C18:1 than sulfuric acid. As
shown by the chromatograms in figure 6, there are again
not that many peaks, thus it is crucial that at least
50% can be identified by the machine and match with a
biomarker from the library. With the saponification
method it is possible to be more certain that it is
olive oil, anyway this researched showed again that the
conventional extraction technique was the most
71
appropriate to achieve the desired results. On this
last technique the identification of the peaks was easy
and with good results. There were not C18:2 fatty acids,
but two C18:1 were present together with TAG C18:1 (figure
7a and table 4), squalene and β- sitosterol
trimethylsilyl ether (figure 6a). In all cases not many
C18:0 were found, usually just one or two molecules,
because it is more abundant in old plants, while in
fresh oil C18:1 is dominant (Steele et al 2010). β-
sitosterol is another component that is typical of
vegetable plants and together with squalene can help
identify them; nevertheless sometime these two
molecules together can confuse the researcher that the
studied molecule is an animal fat (Steele et al 2010;
Evershed et al 2002; Copley et al 2005; Kanthilatha et
al 2014). Thus it is easy to identify olive oil with
the conventional extraction, this is modern material
and with archaeological sherds the results will be
different. Even if sulfuric acid was not able to
extract as many molecules as the conventional solvent
extraction, it is still possible to see that the
72
organic compound is olive oil because the known peaks
are corresponding to molecules that put together form
olive oil. This method might be more useful when
working with archaeological material because it will be
able to extract more residues from the ceramic, anyway
it is not known if those residues could be identified
or they could be damaged during the extraction process
as well. If this would be the case saponification
method is the suggested choice, even because it can be
carried out in a lower amount of time and with less
hazard of contamination.
73
Abbreviated name Assignment and structure Formula Basepeak
M+ Characteristic ions
TMS C16:0
Palmitic acid,trimethylsilylester
C19H40O2Si 73, 117
328 55, 313
TMS C18:1
Oleic acid, trimethylsilylester
C21H42O2Si 73 354 96,129
TAG C18:1
1-Monooleoylglycerol trimethylsilylether
C27H56O4Si2 73 500 129,203
Squalene C24:6 C30H50 69 410 95
74
TMSβ-Sitosterol trimethylsilylether
C32H58OSi 73 486 129, 207, 486
FAME C16:0
Palmitic acid,methyl ester
C17H34O2 74 270 55, 87
Underivatized C15:0
Palmitic acid
C16H32O2 73 256 55, 60
FAME C18:1
Oleic acid, methyl ester
C19H36O2 55 296 69, 83,264
Table 4. Significant mass spectra of modern olive
75
76
4.4 Modern pistacia spp resin
The chemistry of resins is complex, more than beeswax,
but most resins are composed of compounds of the
chemical class terpenoids. Natural resins can contain
mono-, sesqui-, di- and triterpenoids; although di- and
triterpenoids are not found together in the same resin,
allowing researchers to identify and classify the
studied resins (Mills and White 1999, 95-96; Charrié-
Duhauta et al 2007). In this research pistacia spp
resin was used to represent the triterpenoids and to
test their extraction and degradation under different
extraction methods.
Pistacia spp resin is characterized by triterpenoids.
When the resin is methylated, the main derivatives
found are methyl moronate, methyl oleanonate, methyl
isomasticadienonate and methyl masticadienonate (Stern
et al 2003). Because there are about 20 different
species of pistacia resin, in a chromatogram it needs
to be looked at the following skeletons to identify
this molecule (figure 18): ∆12-oleanene (I; R1, R2
varying), ∆18-oleanene (II; R1, R2 varying), 28-nor-
77
∆17-oleanene (III; R1 varying), ∆7-tirucallene (IV; R1,
R2 varying), 24,25-dehydro-∆7-tirucallene (V; R1, R2
varying), ∆8-tirucallene (VI; R1, R2 varying), 24,25-
dehydro-∆8-tirucallene (VII; R1, R2 varying), dammarane
(VIII; R1, R2, R3 varying), lupane (IX; R1, R2
varying), lupene (X; R1, R2 varying) and ∆12-lupene
(XI; R1, R2 varying). Since some R chain are subject to
variation, this will result on the mass spectrum on
different results for m/z fragments, which are also due
to the diverse fragmentation pattern of each triterpene
skeleton (Assimopoulou and Papageorgiou 2005).
78
Figure 18. From Skeletons of penta- and tetra-cyclic triterpene derivatives identified in pistacia spp resin (from Assimopoulou and Papageorgiou 2005, 588-589).
79
For all the three methods, it was difficult to analyse
the results because even if resins have been greatly
studied (Evershed 2008a), the MS library could not
identify the mass spectra with more than 80% accuracy,
in most of the cases the accuracy was even lower than
70%. This could have been expected in the case of
triterpenoids, anyway also TMS were not accurately
identified.
With the conventional solvent extraction, the first TMS
detected is the palmitic acid trimethylsilyl ester,
which is a component found in plants (Assimopoulou and
Papageorgiou 2005; Evershed 2008b). An interesting
component that recurred in few peaks is the α-Pinene
(table 5). This molecule was found to be one of the
major components of the Pistacia lentiscus var. chia
(Koutsoudaki et al 2005). This was not expected, since
this component is supposed to be more common and
abundant in pine resin, but pine resin also contains β-
Pinene, and in this study this molecule has not been
found. Thus, it can be possible to distinguish these
two types of resins especially from the lack of one of
80
these components, or by the presence of both (Colombini
et al 2000). From 20.70 minutes the chromatogram (figure
8) starts to show an increase of penta- and tetracyclic
triterpenes, and less hydrocarbon chain. Between
minutes 28 and 31.60 there is a high amount of peaks
next to each other. By looking at figure 8, it is seen
that the solvent extraction is the only method to show
high abundance of those peaks. The abundance in
saponification method is really low compared to the
other techniques, while sulphuric acid method shows two
main peaks with high abundance while the other are
still lower than the solvent extraction, but higher
than saponification method. Even if those peaks were
hard to identify, it was possible to know the
components of some of them. The solvent extraction
extracted lup-20(29)-en-3-one, with formula C30H48O.
This molecule it is the derivatized of lupine (figure
18 X). The identification of this cyclic molecule has
been easier with the saponification method. In this
case, the reaction inside the sample made most of the
molecules derivatized in their alcohols. Two
81
lanosterol, C30H50O, were identified. The presence of
this compound is important because it means that
extraction and diretivatasion were carried out
correctly. This tetracyclic triterpenoid is the
compound from which all steroids are derived (Abe et al
1993). Lanosterol is formed through a long pathway and
squalene is one of the last components that help to
form triterpenoid. If lanosterol goes through enzyme
catalysis, the result is the yield of cholesterol (Abe
et al 1993; Cholesterol is a component of resin
(Assimopoulou et al 2005), and it is possible that it is
not shown here because the lanosterol did not go
through enough processes to yield cholesterol. What
could be able to identify instead is squalene, but a
strong reaction needs to occur to break the cyclic
structure and give the linear unsaturated, one or more
carbon-carbon double bonds, hydrocarbon chain of
squalene. Anyway squalene has not been found in any of
the results from the three methods. Saponification
method was also the only one to detect β-Sitosterol,
which is a component of Pistacia spp resin (Stern et al
82
2003; Assimopoulou et al 2005). The derivatized of lupine
(figure 18X) has also been found: lupeol, one of the
functional group is an alcohol –OH.
The sulfuric acid results were not easy to study as
with the saponification method. Even in this case
lanosterol molecules are present, even if they are more
abundant in the previous method. Only two other
compounds were positively identified, one is lup-
20(29)-en-3-one, also present in the solvent
extraction, and betulin. Both of them are derivatives
of lupine. FAMEs were also identified as palmitic acid
methyl ester, C16:0, oleic acid methyl ester, C16:1,
stearic acid methyl ester, C17:0 and an alkane, C27:0. Even
if the first two are components of resins (Assimopoulou
et al 2005), it could be that they are a result of
contamination, since they are present, with stearic
acid, in both of the sulphuric acid balk tests, and, as
stated before, they could be contamination or material
that was already present in the ceramic. Instead, if we
would adopt the method of Correa-Ascencio and Evershed
(2014), not considering peaks lower than 5µm because
83
they are most likely contamination, in this
chromatogram (figure 8c) just the phthalic acid will be
considered since it is the only peak over 5µm. This
means that even all the peaks between 27 and 31.50
minutes, which represents cyclic molecules, will not be
analysed because most likely are an effect of
contamination. The oleic acid and palmitic acid were
found in their FAMEs derivatized compound in the
saponification. Because of this they should be
considered not as contamination, but either part of the
ceramic or a successful extraction and derivatization
of molecules by the extraction technique.
In conclusion saponification and sulphuric acid methods
were the best ones to detect and identify the mass
spectra. The saponification process acted as a strong
extraction since all the molecules found were component
of the triterpene class, one of those that was not
mentioned before is α-amyrin, natural chemical compound
of triterpenes, also important because a neutr
(Assimopoulou et al 2005). Thus, in this research it is
possible to identify the residue as resins, but
84
probably it could not be possible to assign the residue
to a specific class of resins. Anyway, in
archaeological material it could be better to use the
sulphuric acid method since it extracted more residues,
even if the abundance was lower than 5 µm. More studies
need to be done about which is the desirable abundance
for peaks to be considered with this method. Another
question that arise is why the peaks of what is
believed to be residues, from the pistacia spp resin, have
a lower abundance of the peak that represents phthalate
molecule, which is a component of plastic and
considered contamination (Correa-Ascencio and Evershed
2014; Kanthilatha et al 2014). It could be possible that
some plastic was used to fabricate the vessel and the
saponification and sulphuric acid methods retrieved
some of this component. The use of the conventional
extraction technique is not suggested in an
archaeological contest because it might not be able to
recover enough residues and might not be strong enough
to derivatize the molecules (Correa-Ascencio and
Evershed 2014).
85
86
Abbreviated name Assignment and structure Formula Basepeak
M+ Characteristic ions
TMS C16:0
Palmitic acid,methylsilyl ester
C17H34O2 73, 117
328 55, 313
TMSβ-Sitosterol trimethylsilylether
C32H58OSi 73 486 129, 207, 486
FAME C16:0
Palmitic acid,methyl ester
C17H34O2 74 270 55, 87, 143
Underivatized C15:0
Palmitic acid
C16H32O2 73 256 55, 60
87
FAME C18:1
Oleic acid, methyl ester
C19H36O2 55 296 69, 83,264
TMSα-Pinene, 3-trimethylsilyloxy-
C13H24OSi 73, 108
224 209, 299
AlcoholLanosterol
C30H50O 69 411 95, 393
AlcoholLupeol
C30H50O 68,93
426 207, 411
88
Table 5. Significant mass spectra of modern pistacia spp.
resin identified in this study.
89
4.5 Modern finger printsLittle is known about the chemical composition of human
fingerprints. This is due to two causes: the final
composition is affected by a high number of parameters
and not much research has been done about chemical
composition, because most of the studies are focusing
on how to identify the fingerprints and find the
corresponding person only (Asano et al 2002). What is
known is that sebaceous gland and the epidermis are the
origin of the skin surface lipids. On the palm and
fingerprints there are primarily eccrine glands;
however those parts are contaminated with sebaceous
gland secretions by the contact with regions rich in
this gland, such as the face (Nicolaides 1974; Bernier
et al 1999; Asano et al 2002). Sebaceous secretions,
eccrine sweat and apocrine sweat are believed to be the
main components of fingerprints because they are the
most abundant prior to the transfer onto a surface
(Nicolaides 1974). In this case it is expected to not
find much eccrine gland compounds because other than
amino acids, they mostly secrete inorganic components
90
(chlorides, metal and phosphate). Instead, sebaceous
glands produce just organic components, such as
cholesterol, alcohols, fatty acids and squalene
(Nicolaides 1974; Asano et al 2002). In conclusion, the
main component that should be found is squalene
followed by cholesterol, in fact if those two compounds
are found together it is usually modern fingerprint
contamination (Nicolaides 1974; Bernier et al 1999;
Asano et al 2002). Through the different reactions,
during the extraction methods, the molecules that
should be detected by the GC-MS should be the
following: wax esters, oleic acid, stearic acid,
palmitic acid, palmitoleic acid, methyl palmitate,
methyl palmitoleate and methyl stearate (Bernier et al
1999; Asano et al 2002).
The best method to pursue the extraction of those
lipids is either the conventional solvent or the
sulfuric acid methods. Saponification technique showed
poor analysis results; even the MS had difficulties to
find the relative mass spectra. Conventional solvent
extraction is the only that identify both squalene,
91
C30H50, and TMS molecule of cholesterol, C30H50OSi. When
those two compounds are found together they are
connected to contamination from modern finger prints
(Bernier et al 1999). Other identified main components
were palmitic acid methyl ester, C16:0 and the palmitel
acid trimethylsilyester, C16:1. Sulfuric acid method
identified just cholesterol, C27H46O, but was also able
to identify the other main component of finger prints:
methyl palmitoleate, palmitic acid methyl ester and
oleic acid methyl ester (table 6). If an archaeologist
is looking for finger prints in a vessel it would be
better to use the solvent extraction because it will
derivatized the main molecules. Anyway an archaeologist
is rarely looking for finger prints in archaeological
potsherds, because they disintegrate easily (Nicolaides
1974; Asano et al 2002). Thus an archaeologist only
needs to know the molecules that are found on finger
prints. The method to extract lipid should not be
decided on the best if is the best method to extract
lipids belonging to finger prints, researchers should
just know which molecules each method is most likely to
92
derivatize from human skin and in this way it can be
seen if contamination happened when the pot was
touched. Probably saponification extraction will be the
best method to use because is the most likely to not
trace any type of skin contamination, since it will
presumably destroy any molecules belonging to finger
prints.
93
Abbreviated name Assignment and structure Formula Basepeak
M+ Characteristic ions
TMS C16:0
Palmitic acid,trimethylsilylester
C19H40O2Si 73, 117
328 55, 313
TMS C18:1
Oleic acid, trimethylsilylester
C21H42O2Si 73 354 96,129
Squalene C24:6 C30H50 69 410 95
FAME C16:0
Palmitic acid,methyl ester
C17H34O2 74 270 55, 87, 143
94
FAME C18:1
Oleic acid, methyl ester
C19H36O2 55 296 69, 83,264
TMSCholesterol trimethylsilylether
C30H54OSi 73, 129
458 329, 443
Table 6. Significant mass spectra of modern finger prints identified in this study.
95
Chapter 5 – Conclusions
This research investigated the novel method of sulfuric
acid to extract lipids residues from archaeological
vessels. This technique has been compared to other two
commonly used extraction protocols: conventional
solvent extraction by BSTFA and saponification. Those
methods have been tested on modern potsherd samples,
where were applied pistacia spp resin, beeswax, olive oil
and finger prints.
As for carrying out the method procedures, the sulfuric
acid was the method that took more time to complete,
which means there are more chances for the samples to
be contaminated. Anyway, the different chromatograms
showed a higher recovery of absorbed lipids for the
resin and finger prints, while the solvent extraction
had a higher yield for the other two materials.
Chromatograms are also showing that the novel method
had more abundance of the extracted molecule, except
for the resin where all the peaks except one were lower
than 5µm. However, sulfuric acid method proved its
ability to hydrolyze complex moieties (functional
96
groups as wax esters and triglycerides). At the same
time it produced derivatives of FAMEs, which could be
used in further analyses (Correa-Ascencio and Evershed
2014), thus functional groups, as esters and alcohols,
were well recognised. But if those molecules are
gained, others are lost. During the hydrolysis of
complex lipids, as resins and beeswax, there will be a
loss of compositional information. In fact no WE or
TAGs were identified. Anyway it could be possible to
understand which type of residue was analysed by
looking at the rest of the compounds in the
chromatogram. The solvent extraction was the only one
that did not destroy this information, but it is
possible that this technique it is not strong enough
with archaeological residues (Stern et al 2000; Correa-
Ascencio and Evershed 2014). This new protocol showed
strong lipid recovery from ceramic walls, so it can be
supposed that even the material used to make the vessel
can be recovered as said in the blank tests results
chapter. Figure 16 and 17 show how it was thought the
sulfuric acid would have interacted with the other
97
molecules. From this experiment, it seems that it
interacts as those pictures shows, but more studies are
needed about the interaction with triglycerides and
especially with molecules containing cyclic rings, such
terpenoids. This is important to understand for the
recovery of resins and how is going to react, since in
this case the cholesterol molecule was not submitted to
any chemical changes.
In conclusion, this novel method could be able to
extract more lipids and maybe could extract the same
amount of lipids with less potsherd required,
decreasing the damage to the analyzed object. It could
be the best method to analyze archaeological material
because it will yield higher fatty acids even with poor
conservation of the vessel (Correa-Ascencio and
Evershed 2014) and this is why this method is
important. If some objects are found in good
preservation state, conventional solvent extraction is
preferable and if more answers are needed, this new
technique could be used to look at the molecules more
deeply. This method is also important because it
98
identifies more fatty acids even if the object is
degrading, so it has more chances to extract lipids
than the other methods.
Suggestions for further work As seen throughout this research, the sulfuric acid
technique can be used depending on your question, what
you already know of your sample and the information you
want to obtain. Anyway this research was based on
modern material that was not, or had just a little
contamination and that was conserved in a good
environment without going through degradation. The next
major two steps to be taken are first to repeat this
research with known archaeological samples. If the
results are satisfying it needs to be repeated another
time, it will not be necessary to compare with other
methods, but with less potsherd, instead of using 0.10g
it could be used 0.5 or 0.7g. It cannot be sure how
much potsherd is needed to have the wanted outcomes, it
could even be that if more than 0.10g are taken the
results could be better, more detailed, which means a
better knowledge of the compounds with more probability
99
to categorize them, for example distinguish between
resins or waxes.
Another interesting thing that could be done is to
analyze more intensely the chromatograms of the resins
molecules from 25 to 32 minutes (figure 10). The GC-MS
library does not have that many references to compare
and identifying resins compounds and if in the future
those peaks will be looked at, it will be possible to
know to which molecule they belong to, which technique
identifies them better and maybe, if there are good
outcomes, to update the GC-MS library with this new
information.
A last research could be conducted on archaeological
potsherds, preferably with no residues, to study if
this novel procedure could as well extract material
that was used to make the vessel.
100
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