FAT CRYSTALLIZATION – FRACTIONATION BY
ENTRAINMENT AND CHARACTERIZATION USING
REFRACTOMETRY

vorgelegt von
M.Sc.
Mi chaela Horn
g eb . in Sindelfingen

von der Fakultät III – Prozesswissenschaften
der Technischen Universität Berlin
zu Erlangung des akademischen Grades

Doktor der Ingenieurwissenschaften
- Dr.-Ing. –

genehmigte Dissertation

Promotionsausschuss:

Vorsitzender: Prof. Dr. rer. nat. habil. Lothar W. Kroh
Gutachter: Prof. Dr.-Ing. Eckhard Flöter
Gutachter: Prof. Dr. Stephan Drusch
Gutachterin: Dr. Elke Scholten

Tag der wissenschaftlichen Aussprache: 01. Juni 2018

Berlin 2018

The elegant pathw ay in theory too often turne d out to be
a troublesome applica tion in practice.
(Marc Kellens & Gijs Calliauw)

I
A B S T R A C T
In food industry , products conta ining fats and oil s are mainly structure d by a
functional mixture of high and low melting fats and oil s. The provided structuring
needs to be controlled b ecause it influe nces texture, stability, taste , and storage life of a
product. The mentioned fats and oils are mainly composed of a mixture of
triacylglycerides (TAGs). Since this mixture is not a binary system, the phase behavior
is quite complex. A possible distinction of the various fractions in these mixtures i s
their melting/crys tallization temperature .
The source of a fat/ oil determines its compo sition. TAGs from animal fats are
high in saturated fatty acid residues (except of e.g. aquatic organisms) while plan t-base d
oils are mainly high in unsaturated fatty acid moieties (except of tropical oils e.g.
coconut oil). The consumption of unsaturated fatty acids is known to be healthy for
humans and, thus, oils containing high concentrations of these are recommended with
respect to nutritional aspect s. If these unsaturated fatty acids are polyu nsaturated they
are so-called essential fatty acids. Due to processing or the nature of the fats, so -called
trans -fatty acids occur. These are known to ha ve detrimental effe cts for the health.
In fat technology, speci fic demands are made for different produc ts to achieve the
desired properties (e.g. shelf life, health, processing). This requires the functionalization
of naturally occu rring fats and oils, historically done usin g fractionation,
interesterification, and/or hydrogenation. During fractionation, which is performe d
batch-wise, the fractions are generate d due to their di stinct melting points.
Interesterificat ion exchanges the fatty acid residues at the glycerol backbone of the
TAGs randomly. For chemical catalysts, the randomization is complete. Enzymatic
catalysts are selective and yield lower react ion rates which makes the resulting
composition difficult to predict. The process of hydrogenation is used to achieve
different degrees of saturation independent of the raw material. However, during this
process, if not conducted complete ly, trans -fatty acids are formed which are known to
increase the risk of c ardiovascular dise ases.

II
The fractionation process is the only function alization which does not change the
molecular structure of the TAGs. Two commonly applied technologies are outlined
shortly. The dry fractionation is the cheapest and most applied process but also the
least selective and efficient one. The solvent fractionation is more efficient but
expensive and the used solvents can cause problems due to hazardous working
conditions.
A continuous fractionation process would be de sirable to decrease production time
and costs. Hence, we examined the application of a new emulsion fractionation process
which is based on a process applied for margari ne produc tion. It aims for a continuous
process and specific fractionation of the desired TAGs. The idea is that cold water
droplets are injected into a warm oil mixture initiating crystallization of the high melting
TAGs at the droplet surface due to local supercooling. These crystals stabilize the water
droplet, forming a so-called Pickering emulsion. The water droplets stabilized by fat
crystals have a higher density than the surrounding liqui d oil which makes a separation
by centrifugal force possible. This separation step was performed in a lab scale decanter
centrifuge achieving a c ontinuous process.
Preliminary tests were conducted using a mixture of rapeseed oil and a
predetermined amount of fully hyd rogenated fat (hardstock) as a model system to know
the exact amount of hardstock before and after emulsion fractionation. In addition,
experiments with palm oil as a model system were carried out. It was shown th at the
two processes of crystallization and separation need to be harmonized well to achieve
the best separation efficiency. In general, the separation was po ssible, but the efficiency
was very low. Therefore, a better understanding of the influencing parameters used to
control the process in the decanter need to be obtained. An accurate knowledge o f the
phase equilibrium and t he kinetics during the continuous process is crucial to establish
the window of the apparatus paramete rs for a successful applicati on.
To study the phase equilibrium of the applied materials , an analytical method is
required to charact erize fats and oils. This method should be re liable, fast, and easily
applicable for a large number of experiments. Therefore, the application of the new
temperature modulated optical refractomet ry was evaluate d.
Fat crystallization is usually investigated by distinct methods to determine phase
transitions, the amount of solids, and polymorphic crystal forms. Differential scanning

III
calorimetry is an established method to dete rmine melting and crystallization in fats
and oils. Pulsed nuclear magnetic resonance is normally applied to obtain the solid fat
content of a material, which is important as a quality paramete r an d determines the
application range of a f at. Powder X-ray diffraction is a well-known technique to
differentiate polymorphic forms in f ats which is important for products like chocolate
where only one polym orph of cocoa butte r delivers the desired product properties.
All of the se enumerated methods are quite expensive and partially complex in
sample handling. A more convenient and chea per method proofed to be the
temperature modulated optical refractometry (TMOR). It determines the refractive
index whi le a temper ature modulatio n is conducted di rectly on the prism. This yields
beside the mean refractive index a thermal volume expansion coefficient  . The
method can be carried out in an isothermal and a dynamic way. Both modes are
interesting for the application in fat technology and therefore the applicability of
TMOR for the investigation of fats and oils was part of thi s thesis.
We found that it is possible to determine ph ase transitions of aliphatic chain
components as well as of more complex systems like fats. Additionally, the device was
used to obtain the solid fat content by determining the apparent refractive index of
various fats such as coconut oil a nd applying the lever rule . So far, only the potentia l to
determine polymorphic forms using TMOR was shown. In future work this application
of TMOR needs to be further investigated .
The appl icability of TMOR was shown in this work. In the next step this technique
is applied to gain better knowledge of the phase behavior and kinetics so that the
process window of the continuous emulsion fr actionation can be ident ified.
In summary, bo th, the new emulsion fractionation technology and the temperature
modulated optical refractome try, could be combined. TMOR could be use d as analytic
method to determine the me lting behavior and the solid fat content of the fractionated
material. Thereby, important information about the separation efficiency and the
resulting TAG fraction s would be obtai ned su pporting the op timization of the process
design.

IV
Z U S A M M E N F A S S U N G
In der Lebensmitt elindustrie w erden Produkte, die Fette und Öl enthalten,
hauptsächlich durch eine funktionelle Mischung aus ho ch- und niedrigschmelzenden
Fetten und Ölen strukturiert. Die Strukturierung muss kontrolliert werden, da sie di e
Textur, Stabilität, Geschmack und Haltbarkeit eines Produkts beeinflusst. Die
genannten Fette und Öle bestehen hauptsächlich aus einer Mischung von
Triacylglyceriden (TAGs). Da diese Mischung kein binäres System ist , ergibt sich ein
komplexes Phasenverhalten. Eine mögliche Unterscheidung der verschiedenen
Fraktionen in diesen Ge mischen ist ihre Sc hmelz- bzw. Kristallisati onstemperat ur.
Die Quelle eines Fe ttes/ Öl es bestimmt seine Zusammensetzung . TAGs au s
tierischen Fetten sind reich an gesättigten Fettsäureresten (mit Ausnahme von z.B.
Wasserorganismen), während Öle auf pflanzlicher Basis hauptsächlich ungesättigte
Fettsäurereste enthalten (mit Ausnahme von tropischen Ölen, z.B. Kokosnussöl). Es
ist bekannt, dass der Verzehr von ungesättigten Fettsäuren gesund für den Menschen
ist. Daher werden Öle, die hohe Konzentrat ionen an ungesättigten Fettsäuren
enthalten, in Bezug auf Ernährungsaspekte empfohlen. Wenn diese ungesättigte n
Fettsäuren mehrfach ungesättigt sind, werden sie als sog . essentielle Fettsäur en
bezeichnet. Aufgrund der Verarbeitung oder der Art der Fette treten sog.
Transfettsäuren auf, die dafür bekannt sind, dass sie schädliche Auswirkungen auf die
Gesundheit haben.
In der Fetttechnologi e werden spezifische Anforderungen an verschiedene
Produkte gestellt, um die gewünschten Eigens chaften (z. B. Haltbarkeit,
Ernährungsphysiologie, Verarbeitung) zu erreichen. Dies erfordert die
Funktionalisieru ng von natürlich vorkommenden Fetten und Ölen, die historisch durch
Fraktionierung, Umesterung und/ oder Hydrierung durchgeführt wurden. Während
der Fraktionierung, die chargenweise durchg eführt wird, werden die Fraktionen
aufgrund ihrer Schmelzpunkte erzeugt. Die Umesterung tauscht die Fettsäure reste am
Glycerol-Rückgrat der TAGs zuf ällig aus. Bei chemischen Katalysatoren erfolgt eine
vollständige Zufallssteuerung. Enzymatische Katalysatoren sind dagegen selektiv und
zeigen niedrigere Reaktionsgeschwindigkeiten, was die Vorhersage der resultierenden

V
Zusammensetzung erschwert. Der Prozess der Hydrierung wird verwendet, um
unterschiedliche Sättigungsgrade unabhängig vom Rohmaterial zu erreichen. Bei
diesem Prozess werden jedoch, wenn er nicht vollständig durchgeführt wird ,
Transfettsäuren gebildet, von denen bekannt ist, dass sie das Risiko von Herz-
Kreislauf-Erkrankunge n erhöhen.
Die Fraktionierung ist der einzige Funktionalisierungsprozess, der die molekulare
Struktur der TAGs nicht verändert. Zwei gängige Technologien werden in Kürze
vorgestellt. Die Trockenfraktionierung ist der billigste und am meisten angewendete
Prozess, aber auch der am wenigsten selektive und effiziente. Die
Lösungsmitte lfraktionierung ist effizienter, aber teuer. Zudem können die verwendeten
Lösungsmitte l aufgrund von gefährlichen Arbei tsbedingungen Problem e verursac hen.
Ein kontinuierlicher Fraktionierungsprozess wäre wünschenswert, um
Produktionszeit und -kosten zu verringern. Daher haben wir die Anwendung eines
neuen Emulsionsfraktionierungs verfahrens untersu cht, das auf einem Verfahren zur
Margarineherstellung basiert. Die Ziele sind ein kontinuierlicher Prozess und eine
spezifische Fraktionierung der gewünschten TAGs. Die Idee ist, dass kalte
Wassertropfen in ein warmes Ölgemisch injiziert werden, di e die Kristallisation der
hochschmelzenden TAGs an der Tropfenoberfläche aufgrun d lokaler Unterkühlung
initiieren. Diese Kristalle stabilisieren den Wassertropfen und b ilden eine sog.
Pickering-Emulsion. Die durch Fettkristalle stabilisierten Wassertropfen haben eine
höhere Dichte als das sie umgebende flüssige Öl, was eine Trennung durch
Zentrifugalkraft ermöglicht. Dieser Tr ennschritt wurde in einer Dekanterzentrifug e im
Labormaßstab durchgef ührt, womit ein kontinu ierlicher Prozess e rreicht wurde.
Vorversuche wurden unter Verwendung einer Mischung aus Rapsöl und einer
vorbestimmten Menge an vollständig hydriertem Fett (Hardstock) als Modellsystem
durchgeführt, um di e genaue Menge an Hardstock vor und nach der
Emulsionsfraktionierung zu kennen. Darüber hin aus w urden Versuche mit Palmöl als
Modellsystem durchgeführt. Es wurde gezeigt, dass die beiden Kristallisations- und
Trennprozesse gut aufeinander abgestimmt werden müssen, um di e beste
Trennleistung zu erzielen. Im Allgemeinen war die Trennung möglich, aber d ie
Effizienz sehr gering. Daher muss ein besseres Verständnis der Einflussparameter, die
zur Steuerung des Prozesses im D ekanter verwendet werden, entwickelt werden. Eine

VI
genaue Kenntnis des Phasengleichgewichts und der Kinetik während des
kontinuierlichen Prozesses ist entscheidend, um das Prozessfenster der
Geräteparameter für eine erfolgreiche Anwen dung zu ermitteln.
Um das Phasengleichge wicht der ve rwendeten Materialien zu untersu chen, ist eine
analytische Methode zur Charakterisierung von Fetten un d Ölen erf orderlich. Diese
Methode sollte zuverlässig, schnell und leicht für eine große Anzahl von Experimenten
anwendbar sein. Daher wurde die Anw endung der neuen temperatu rmodulierten
optischen Refraktome trie evaluiert.
Die Fettkristallisation wird üblicherweise durch verschiedene Verfahren zur
Bestimmung von Phasenübergänge n, der Menge an Feststoffen und polymorpher
Kristallformen untersucht. Die dynamische Differenzkalorimetrie ist eine etablierte
Methode zur Bestimmung des Schmelzens und Kristallisierens in Fetten und Ölen.
Gepulste Kernspinresonanz wird normalerweise angewendet, um den Festfettgehalt
eines Materials zu ermitteln, welcher als Qualitätsparameter wichtig ist und den
Anwendungsber eich eines Fettes bestimmt. Die Röntgenbeugungsanalyse ist eine weit
verbreitete Technik, um polymorphe Kristallf ormen in Fetten zu unterscheide n. Dies
ist für Produkte wie Schokolade wichtig, in denen nur ein Polymorph der Kakaob utter
die gewünschten Pr odukteigenschafte n liefert.
Alle oben genannten Verfahren sind ziemlich teuer und teilweise komplex in der
Probenbehandlung. Eine praktische und billigere Methode ist di e
temperaturmodulierte op tische Refraktometrie (TMOR). Die Methode bestimmt den
Brechungsindex, während eine Temperatu rmodulation direkt am Prisma durchgeführt
wird. Dies ergibt neben dem mittleren Brechungsindex einen thermisc hen
Volumenausdehnungsk oeffizienten  . Das Verfahren kann isotherm und dynamisch
durchgeführt werden. Beide Modi sind interessant für die Anwendung in der
Fetttechnologie. Daher war die Anwendbarkeit von TMOR für die Untersuchung von
Fetten und Ölen Tei l dieser Arbeit.
Wir zeigten, dass es möglich ist, Ph asenübergänge von aliphatischen Kett en sowie
von komplexeren Systemen wie Fetten zu bestimmen. Zusätzlich wurde das Gerät
verwendet, um den Festfettge halt zu ermitteln, indem der scheinbare Brechungsindex
für verschiedene Fette wie Kokosnussöl bestimmt und das Hebelgese tz angewendet
wurde. Bisher wurde nur das Potenzial zur Bestimmung polymorpher Formen mit

VII
TMOR gezeigt. In zukünftigen Arbeiten muss diese Anwendung von TMOR weiter
untersucht werden.
Die Anwendbarkeit von TMOR wurde in dieser Arbeit gezeigt. Im nächsten Schritt
wird diese Technik verwendet, um das Phasenverhalten und die Kinetik besser zu
verstehen, s o dass das Prozessfenster der kontinuierlichen Emulsionsfraktionierung
identifiziert werden ka nn.
Zusammenfassend kön nen sowohl die neue Emulsionsfraktionierung stechnologie
als auch die temperaturmodulierte optische Refraktometrie kombi niert werden. TMOR
kö nnte als analytische Methode verwendet werden, um das Schmelzverhalten und den
Feststoffgehalt der verschiedenen Fraktionen zu bestimmen. Dadurch könnten
wichtige Informationen zur Trenneffizienz und den gewonnenen TAG-Fraktionen
generiert werden, die wiederum die Optim ierung des Prozessdesigns u nterstützen.

VIII
A C K N O W L E D G E M E N T S
This thesis was written at the department of Food Process Engineering at the
Technical University of Berlin and financially supported by the Elsa -Neumann
Scholarship of the fe deral state of Berlin.
First of all, I would like to than k Prof. Dr.-Ing. Eckhard Flöter for the supervision
of my thesis, the familiar work atmosphere he provides , and his supporting charac ter.
He was always open for questions, discussions, and gave helpful advices throughout
the thesis.
I owe thanks to Prof. Dr. Stephan Drusch and Dr. Elke Scholten for taking the
time to assess my thesis.
I want to especially thank Susanne, who spent lots of time reading and discussing
my research. T hank you for being always s upportive, helpful, and empowering .
A special t hanks goes to the people who were the reason why work did not always
feel like work: Karl Schlumbach, Maria Scharfe, and Miro Kirimlidou. Thank you for
being more than col leagues, thank you for bein g friends .
I also want to thank my Bachelor students (Dewi Dewanga Suprana, Frederike
Deckwerth, Wiebke Wilms Schulze-Kump, Valeska Hutschenreut er) and Master
students (Philipp Martin, Ratna Ayu Savitri) who worked with me throughout the last
years and helped me to develop.
A big thanks also goes to all colleagues at the department of Food Process
Engineering : Karin , Martha, Ruth, Julia, Evelyn, Alexandra, Gabi, Valentina, Simone,
Marco, Tim, Juliane, Melina, Jonathan, Stepha n, Susanne.
I´m grat eful for having parents who are proud of me , support me at every stage of
my life, and lov e me unconditionally. I also thank my friends all over the world who
care about me and make my life brighter e very day.
Last, I want to thank my husband Philipp for his advice, gratitude, and support.
You cheer me up whe n I´m down and help m e to be the best version of myself.

IX
L I S T O F P U B L I C A T I O N S
Parts of the st udy were presented as follows

Publications in journals
1. Häupler, M., Flöter, E. (2018) De termination of the Crystallization B ehavior of
Lipids by Temperature Modulated Optical Refractometry ; Food Analytical
Methods , do i . o r g/ 1 0 . 10 0 7 / s 1 21 6 1 - 018 - 1217 - y

2. Häupler, M., Savitri, R.A., Hutschenreuter, V., Flöter, E. (2018) Applicat ion of
Temperature Modulated Optical Refractometry for the Character ization of the
Crystallization Behavior of Palm Oil; European Journal of Lipid Science and
Technol og y , doi.org/10.1002/ej lt.201700511

3. Häupler, M., Kirimlidou, M., Wilms Schulze-Kump, W., Parisi, A., Wagner, L.,
Flöter, E. (2018) On the feasibility of continuous emulsion fractionation ,
submitted to the European Journal of Lipid Science and Technology .

4. Häupler, M., Hutschenreuter, V., R udolph, S., Flöter, E. (2018) Feasibility study
on the determination of the solid fat conte nt of fats using temperature
modulated optical refractometry , submitted to the European Journal of Lipid Science
and Technology .

Oral and poster prese ntations
1. Häupler, M., Schwarz G. Henriques, S., Flöter, E., (2015 ) Determination of
Melting Point and Polymorphic Form of Edible Fats via Temperature
Modulated Optical Refractometry (TMOR), Euro Fed Lipids Congress , Florence,
Italy.

2. Häupler, M., Flöter, E. (2016) Continuous Emulsion Fractionation of Fats, Euro
Fed Lipids Congress , Ghent, Belgium.

3. Häupler, M., Kirimlidou, M., Flöter, E. (2017) Lab Scale Continuous Emulsion
Fractionation of Edible Fa ts, Euro Fed Lipids Congress , Uppsala, Sweden.

4. Häupler, M. Kiriml idou, M., Flöter , E. (2018) Kontinuierli che
Emulsionsfraktionierung von Speisefette n im Labormaßstab, ProcessNet , Berlin,
Germany.

X
C O - A U T H O R S H I P
The work presented in this thesis was partially conducted with other scientists of
the Department of Food Process Engineering, TU Berlin. Dr. Susanne Rudolph was
involved in outlining and writing the publications as well as in scientific discussions
concerning all chapters. Dr. Fernanda Peyronel and Dr. Alejandro G. Marangoni
provided scientific input and the op portunity to use di fferent devices at the Food
Science Department, University of Guelph. With Prof. Dr.-Ing. Eckhard Flöter, who
supervised the wh ole PhD thesis, all chapter s were discussed scient ifically .

o Chapter 4
Michaela Häupler designed, planned, cond ucte d, supervised, and analyzed the
experimental work . The experimental work was partially carried out by Wiebke
Wilms-Schulze Kump, Miro Kirimlidou, Aless andra Parisi, and Le onie Wagner

o Chapter 5
Michaela Häupler designed, planned, conducted, and analyzed the experimental
work.

o Chapter 6
Michaela Häupler designed, planned, conducted, and analyzed the experimental
work. Parts of the ex perimental work were c arried out by Frederike D eckwerth.

o Chapter 7
Michaela Häupler designed, planned, supervise d, and analyzed the experimental
work. The experimental work was partially carried out by Ratna Ayu Savitri and
Valeska Hutsc henreuter.

o Chapter 8
Michaela Häupler designed, planned, conducted, and analyzed the experimental
work. Parts of the experimental work were car ried out by Valeska Hutschenre uter.
The results were discussed with Susanne Ru dolph.

o Chapter 9
Michaela Häupler designed, planned, conducted, and analyzed the experimental
work.

XI
S Y M B O L S A N D A B B R E V I A T I O N S
Symbols
 thermal expansion coef ficient -
c 0 speed of lig ht in vacuum m s -1
c m speed of light in mat erial m s -1
d diameter mm
  phase shift at angular f requency -
g acceleration of gravity m s -2
G Gibbs free energy J
 H m specific melting enthal py J g -1
Im(  ) imaginary part of  -
 wave length nm
m mass g
 chemical potential -
n re fractive index -
 dynamic viscosity mPas
 mass density g cm -3
r specific refractivity cm 3 g -1
R gas constant J mol -1 K -1
Re(  ) real part of  -
 angle °
t time s
T te mperature °C
T c crystallization temper ature °C
T m melting temperatu re °C
V
󰇗 volume flow L h -1
x i mole fraction mol mol -1
 angular frequency s -1

XII
Abbreviations

AOCS American Oil Chemist s Society
CBE cocoa butter equ ivalent
CO coconut oil
DAG diacylglyceride
DSC differentia l scanning calorimetry
FA fatty acid
FAME fatty acid methyl e ster
FFA free fatty acid
FHPO fully hydrogenated palm oil
FHRO fully hydrogenated rapese ed oil
FID flame ionization detec tor
fps frames per second
GC gas chromatography
HLB hydrophilic-lipophilic b alance
IV iodine value
LRT linear response theory
MAG monoacylglyc erides
MCT medium-chain length triac ylglyceride
MUFA monounsaturated fat ty acid
MTBE methyl- tert -butylether
NMR nucle ar magnetic resonance
OOO triolein
O/W oil - in -water e mulsion
PHPO partially hydrogenated palm oil
PLM polarized light microsco py
PMF palm mid fraction
PO palm oil
POP sn -1,3-palmitoyl-2-ole oyl triacylglyceride
PPP tripalmitate

XIII
PUFA polyunsaturate d fatty acid
rpm rounds per min ute
SFC solid fat content
SFI solid fat index
SSS tristearate
TAG triacylglyceride
TMOR temperature modulated optical refractomet ry
TMSH trimethylsulfoniumhydr oxide
W/O water- in -oil emulsion
XRD X-ray diffraction

XIV
T A B L E O F C O N T E N T S
1 INTRODUCTION 1
2 FUNDAMENTALS 7
2.1 F ATS & O ILS 8
2.1.1 C OMPOSITION OF F ATS & O ILS 8
2.1.2 P ALM O IL 12
2.1.3 P HASE B EHAVIOR 18
2.2 F AT C RYSTALLIZATION 24
2.2.1 N UCLEATION 25
2.2.2 C RYSTAL GROWTH 28
2.2.3 P OLYMORPHISM 30
2.3 F AT M ODIFICATION 36
2.3.1 H YDROGENATION 36
2.3.2 I NTERESTERIFICATION 37
2.3.3 F RACTIONATION T ECHNOLOGIES 39
2.4 E MULSION F RACTIONATION 50
2.4.1 E MULSIONS AND P ICKERING S TABILIZATION 50
2.4.2 D ECANTER C ENTRIFUGE 53
2.4.3 C ONCEPT OF E MULSION F RACTIONATION 56
3 EXPERIMENTAL METHODS 58
3.1 D IFFERENTIAL S CANNING C ALORIMETR Y 59
3.1.1 P RINCIPLE OF DETERMINATION 59
3.1.2 A PPLICATION IN THE FIELD OF FAT TECHNOLOGY 63
3.1.3 P ROCEDURES 64
3.2 T EMPERATURE M ODULATED O PTICAL R EFRACTOMETRY 65
3.2.1 R EFRACTOMETRY 65
3.2.2 F UNDAMENTALS OF THE T EMPERATURE M ODULATED O PTICAL R EFRACTOMETRY 67
3.2.3 R EFRACTOMETRY IN F AT T ECHNOLOGY 71
3.2.4 P ROCEDURES 73

XV
3.3 P ULSED N UCLEAR M AGNETIC R ESONANCE 76
3.3.1 P RINCIPLE OF P NMR 76
3.3.2 A PPLICATION OF P NMR IN FAT TECHNOLOGY 77
3.3.3 SFC D ETERMINATION P ROCEDURE 81
3.4 G AS C HROMATOGRAPHY 81
3.4.1 P RINCIPLE OF G AS C HROMATOGRAPHY 82
3.4.2 P ROCEDURE 83
3.5 P OLARIZED L IGHT M ICROSCOPY 84
3.5.1 P RINCIPLE OF PLM 84
3.5.2 PLM FOR THE INVESTIGATION OF FAT CRYSTALS 85
3.6 P OWDER X-R AY D IFFRACTION 86
3.6.1 P RINCIPLE OF XRD 86
3.6.2 A PPLICATION OF XRD IN F AT T ECHNOLOGY 87
3.6.3 P ROCEDURE 88
4 ON THE FEASIBILITY OF C ONTINUOUS EMULSION F RACTIONATION 89
A BSTRACT 90
4.1 I NTRODUCTION 91
4.2 M ATERIALS AND M ETHODS 94
4. 3 R ESULTS & D ISCUSSION 96
4.3.1 P RELIMINARY STUDY ON SURFACE CRYSTALLIZATION 96
4.3.2 E MULSION S TABILITY AND F LOCCULATION 99
4.4 C ONCLUSION 108
5 DETERMINATION OF THE C RYSTALLIZATION BEHAVIOR OF LIPIDS BY TEMPERATURE
MODULATED OPTICAL RE FRACTOMETRY 109
A BSTRACT 110
5.1 I NTRODUCTION 111
5.2 M ATERIALS AND M ETHODS 117
5. 3 R ESULTS AND D ISCUSSION 119
5.3.1 N -H EXADECANE 122
5.3.2 P ALMITIC ACID 125
5.3.3 T RIPALMITATE 128
5.3.4 C OMPARISON OF TMOR AND DSC 131
5.4 C ONCLUSION 134

XVI
6 EVALUATION OF TMOR PARAMETERS FOR THE IN VESTIGATION OF FATS 136
6.1 T HEORETICAL BACKGROUND 137
6.2 M ATERIALS AND M ETHODS 139
6. 2.1 M ATERIAL PROPERTIES 139
6.2.2 D EVICES 141
6.3 I NFLUENCE OF FAT COMPOSITION AND SCAN RATE 142
6.4 I NFLUENCE OF AMPLITUDE AND PERIOD 154
6.4.1 A MPLITUDE 154
6.4.2 P ERIOD 158
6.5 C ONCLUSION 161
7 APPLICATION OF TEMPERATURE MODULATED OPT ICAL REFRACTOMETRY F OR THE
CHARACTERIZATION OF T HE CRYSTALLIZATION BEH AVIOR OF PALM OIL 164
A BSTRACT 165
7.1 I NTRODUCTION 167
7.2 M ATERIALS AND M ETHODS 169
7.3 R ESULTS AND D ISCUSSION 171
7.4 C ONCLUSIONS 185
8 FEASIBILITY STUDY ON THE DETERMIN ATION OF THE SOLID FAT CONTENT OF FATS USING
TEMPERATURE MODULATE D OPTICAL REFRACTOME TRY 187
A BSTRACT 188
8.1 I NTRODUCTION 189
8.2 M ATERIALS AND M ETHODS 192
8.3 R ESULTS AND D ISCUSSION 194
8.3.1 T EMPERATURE D EPENDENCY OF D ENSITY AND R EFRACTIVE I NDEX 194
8.3.2 S OLID F AT C ONTENT C ALCULATION 196
8.3.3 D IFFERENT STABILIZATION PROCEDURES 201
8.4 C ONCLUSION 203
9 FEASIBILITY STUDY TO INVEST IGATE POLYMORPHIC TRANSITIONS USING TMOR 205
9.1 M EASUREMENT P ROCEDURE 206
9.2 R ESULTS AND D ISCUSSION 207
9.3 C ONCLUSION 210

XVII
10 FINAL CONCLUSION AND OUTLOOK 212
10.1 C ONTINUOUS EMULSION FRACTIONATION 213
10.2 T EMPERATURE MODULATED OPTICAL REFRACTOMET RY 215
10.3 O UTLOOK 217
REFERENCES 220

1 I NTRODUCTION

2
In food industry, the processing of fat containing products requires good
knowledge of the composition of the fats and their crystallization behavior. Fats are
multicomponent syste ms which are mainly composed of triacylglycerides (TAGs).
Structuring fats are use d e.g. to stabilize water or air in products like margarine or ice
cream. In addition, the proper crystallization influences shelf life, mouthfeel, and
spreadability of the desired products. Decades ago, the application of so-called
hydrogenated fats as structuring fats was popular. Th ese were obtained by the
saturation of oils with hydrogen (hydrogenation) lead ing to fats with different degrees
of saturation and, hence, different melting profil es which increased the appl ication
range dramatically.
However, the use of hydrogenated fats in food industry diminished drastically, it
almost vanished. The r eason is that during hydrogenation of oils an d fats if the pr ocess
is not com pleted, trans -fatty acids are formed. In the past, by choos ing the right de gree
of hydrogenation, such partially hydrogenated oils were use ful to adjust the desired
product propertie s. Even though if hydrogenation is complet ed no trans -fatty acids are
formed the public acceptance of the process is low as it is difficult to teach the
differences between partial and full hydrogenation to the consumer. Therefore, the
industry searched for alternatives to the hydrogenation process. Another modification
process is interesterification, where the fatty acids at the glycerol backbone of the TAGs
are chemically or enzymatically exchanged. Nevertheless , this exchange occurs
randomly and, thus, the prediction of the product properties is difficult.
In this thesis, the selected modification technique was a fractionation process. In
contrast to hydrogenation and intereste rification, which use chemicals to modify the
properties of oils and fats, fractionation is a solely physical process. The invention of
the technology of fractionation to separate high from low melting fat is ascribed to
Hippolyte Mège Mouriès (1817 – 1880) in the late 1860s. It uses the di fferences in the
melting points of different fractions, for example of palm oil, to separ ate the low
melting part (olein) from the high melting part (st earin). This separation can be
performed several times to yield even more specific fats for ce rtain applications.
Nowadays, three different fractionation methods are kno wn. On ly two of them ar e
usually applied, solvent fractionation for specialty fats and dry fractionation for larger
amounts of fat to be fractionated (like palm oil). Both are batch processes. The first

3
one use s solvents like hexane or acetone to decrease the viscos ity of the slurry
containing crys tallized and liquid fat. However, an expensive solvent recover y is
indispensable to obtain a fat which i s food gra de. Ano ther di sadvantage of this process
is the hazardous w orking environmen t.
In dry fractionation, the feed is crystallized so that large crystals are formed which
do not entrap liquid oil forming a slurry with the surrounding liquid phase. The slurry
is separated by membrane press filtration. Due to the lack of the washing step, the
separation efficiency is lower for the dry than for the solvent fractionation. However,
sequential fractionation steps can yield diverse fractions sh owing desired propertie s for
different applications, respectively. The third method, which is not frequently used
today, is the deterge nt fractionation process. It uses surfactants to em ulsify the yielded
fat fraction which is the n separated by ce ntrifugation.
The new process of emulsion fractionation, which was started to be develop ed in
this thesis, combines the mechanism of forming stable droplets as for the detergent
fractionation process with a conti nuous separation ste p but actually without the use of
a detergent. The aim of the emulsion fractionation process is the direct frac tionation of
the valuable fraction of high melting TAGs like PPP. No rmally, to generate this
fraction, several fractionation steps need to be performed in the dry fractionation
process.
The process is as follows. A cold water stream is inj ected into a warm liquid oil
stream, composed of a high and a low melting fr action. At the cold surf ace of the water
droplet the high melting fraction of the fat crystallizes and stabilizes the droplets,
forming a Pickering emulsion. This emulsion is fed into a decanter centrifuge where
continuous separation between the liquid phase and t he water drop lets stabilized by
crystallized fat occurs. The process on ly uses water as an entrainer whi ch can be
recycled and is re latively cheap. Moreover, the continu ous proces s has advantages over
a batch process because it decreases the inter-batch variabil ity of the resulting products
and saves energy costs. Additionally, the particle size distribution of the product is mor e
uniform.
To decrease the complexity of the process, both steps, the crystallization and the
separation, were studied indepe ndently using palm oil an d a mixture of rapes eed oil
with a defined amount of high melting fat (hardstock) as model systems . The ultimate

4
objective is the use of seed oils like sunflower or rapeseed as the raw material for the
fractionation process. However, d ue to the low amount of saturated TAGs in seed oils,
first the pr ocess needs to be fully understood before a highly effective fractionation
method can be designe d.
The parameters like crystallization tem perature need to be deter mined precisely to
design the process explained above. A reliable and robust alternative to well-estab lished
but expensive laboratory devices would be de sirable for bo th researc h and industry .
Therefore, this thesis includes the feasibility study of a new method to determine phase
transitions whic h are crucial for the food industry. The crystallization of fats is decisive
for the new fractionation process but also for the product quality and, thus, the resulting
shelf life. For example, the melting sensation of chocolate is based on the proper
crystallization of coc oa butter during the product ion.
Moreover, the investigation of the melting behavior is di rectly lin ked to th e
crystallization process because it determines the consumer acceptance. For exa mple,
this comes into focus if the consumer uses the margarine right from the fridge whi ch
should be spreadable at this temperature. In the last decades, the investigation of
crystallization and melting behavior of fats is done by differential sc anning cal orimetry .
This method monitors the heat flow during heating and cooling of the sample, which
changes if a phase tra nsition occurs. Other methods to cha racterize product proper ties
are pulsed nuclear magnetic resonance and powder x -ray diffraction. How ever, all o f
the mentioned methods are m ainly applicable f or laboratory work since the devices are
expensive, the handling is quite complex, and the procedures are often time-consuming.
Hence, a reliable but robust, cheap and fast method to determine the phase
transitions of fats would be desirable. This is why in this thesis the determination of
the refractive index is taken into account as another possibility to c haracterize fats and
oils. The refractive index differs for the solid and the liquid state of a fat. This
phenomenon was used to stu dy the phase behavior of lipid component s and fats using
temperature modulated optical refractomet ry (TMOR) .
In addition to traditional refractometry, this device can modulate the temperature
around a set mean temperature with a predefined amplitude and period. This
modulation and the resulting phase shift of the answer of the refractive index is used
to compute the thermal expansion coefficient  which then indicat es the temperature

5
of ph ase transition. Additionally, the thesis illustrates th at TMOR can be used to
determine the SFC of f ats and oils or even pol ymorphic transitions.
In this thesis, the possibility to perform a continuous emulsion fractionation
proces s was evaluated. Therefore, the process was studied by dividing it into the two
single processes, crystallization and separation, as well as by studying the entire process.
The aim of the study was to show that it is possible to separat e a model fat system
by continuous emulsion fractionation technology. Thus, th e foll owing questions need
to be answered to desig n a reliable continu ous emulsion fract ionation process.

1. Which centrifugal forces need to be applied to separate the liquid phase from
the water droplets stabi lized by fat c rystals?
2. How is the temper ature develop ment over the process (melting of the crystals)?
3. Which paramete rs take influence on the separation efficiency, e.g. flocculation,
viscosity, throughput, temperatu re?
4. What are the parameters influencing the crys tallization in general and on the
water droplet surf ace?

In addition to the process, also questions concer ning the analysis need to be
addressed .

1. Is temperatu re modulated o ptical refract ometry (TMOR) an ap plicable me thod
to determine the crystallization and melting behavior of alkyl components and
thus to charact erize systems before e mulsion fractionation?
2. What is the proper sample handling to use TMOR as a method to determine the
phase behavior of fats and oils?
3. Is it possible to det ermine the phas e behavior of fats and oils with TMOR?
4. Can the application of TMOR be expanded to the determination of SFC and
polymorphic transitions ?

The characterization of di fferent properties of fats and oils like melting behavior
or solid fat content by TMOR could be used to determine the best conditions for the
outlined emulsion frac tionation process.

6
First, to answer all the stated questions, the required fundamentals of this thesis
are elucidated in chapter 2 to generate an overview of the basic knowledge. Th e chapter
covers the composition of triacylglycerides, the prin ciples of their crystallization
behavior, explains the ph enomenon of polymorphism and gives background
information needed to perform the emulsion fractionation process like the formation
of a Pickering emulsion. Subsequently, the experimental and analytical methods used
in this thesis are explained (see chapte r 3 ).
The results and discussion section is divided into two sections concerned with
ei ther the emulsion fractionation or the TMOR method. The resul ts of the separation
step during the emulsion fractionation depending on the emulsion composition are
given. The experiments cond ucted in a batch-wise operating benchtop centrifuge and
in a continu ous decanter centrifuge are di scussed in this part of the results and
discussion sec tion (see chapter 4 ).
The investigations of the applicability of TMOR to study pure substances lik e
n -hexadecane, palmitic acid and tripal mitate are shown in chapter 5. The adaption of
the sample handling and proper measurement procedure for fats and oils is
subsequently given in chapte r 6 . This procedures were applied to determine the
crystallization and melting behavior of edible fats (e.g. partial ly and fully hydrogenated
palm oil) by TMOR (see chapter 7 ). This section is completed by the applicability of
TMOR to determine the SFC of edibl e fats (e.g. coconut oil, palm oil) which is
evaluated in chapter 8. In addition, the possibility to determine polymorphic transitions
using TMOR is give n in chapter 9.
In the last part of the thesis, a conclusion of all conducted experiments is given
(see chapter 10 ). In addition, advices for future experiments are specified and further
areas of researc h are elucidated.

2 F UNDAMENTA LS

8
This chapter is dedicated to the important topics related to fats and oils and are
required as the background information for this thesis. First, the co mposition of fats
and oils is explained including the principle structure of triacylglycerides as well as a
brief explanation of minor components and their respective influence on physical and
chemical properties. The increasingly used palm oil is key to this thesis and, thus, one
chapter about its origin and composition is presented. In addition, the phase behavio r
of fatty acids, triacylglycerides (TAGs ), fats, and oils is described shortly because this
knowledge is re levant to understand the crystallization behavior of fats and oils
explained in 2.2 .
The subchapter on fat crystallizati on explains nucleation and growth. Furthermore,
the phenomenon of polymorphism is elucidated and its importance during fat
crystallization and especially fat fractionation is clarified. Particular attention is
subsequently paid to fra ctionation as a common fat modificat ion technique.
After a short intro duction into hydrogenation and interesterification, the focus is
on the various fractionation te chniques available. The solvent and detergent
fractionation are explained followed by the description of the dry fractionation process.
Finally, the new idea of emulsion fractionation is presented and substantiated with
literature about Pickering emulsions and the crystallization of fat at the water/oil
interface of emulsions a s well as the se paration in a decanter c entrifuge.
2.1 Fats & Oils
What we refer to as f at is always a multicomponent system and therefore it s
behavior depends on the composition of the raw material (Flöter, 2012) . Therefore, the
structure of the main components of fats and oils is given in the following chapte r.
2.1.1 Composition of Fats & Oils
The basic molecules of fats and oil s are triacy lglycerides (TAGs, Figure 2.1 c) ,
accompanied by minor amounts of mono- and diacylglycerides as well as other minor
components such as phospholipids, phytosterols and tocols (Gunstone, 2013). TAGs
are made of fatty acids, which are either based on n -alkanes (Figure 2.1 a) or n -alkenes
referring to saturated and unsaturated fatty acids, respectively. While n -alkan es and
n -alkenes end with a methyl gro up, fatty acids end with a carbo xyl group (see Figure

9
2.1b) In the no menclat ure e.g. C16 :0 for palmitic acid (Figure 2.1 b) , 16 refers to the
number of C-atoms of the alkane or alkene-cha in an d 0 refers to the fact that there are
no do uble bonds . So for example C18:1 is a fatty acid with 18 C-atoms in the alkene
chain and a do uble bond (oleic acid ) or C18:2 with 18 C-atoms and two double bonds
in the alkene-chain (linoleic acid) . TAGs are composed of a glycerol backbone esterified
with three fatty acids (carbon chains) (Sato, 2001; Himawan, Starov and Stapley, 2006;
Douaire et al. , 2014) .

Figure 2.1 : Structure of a n -alkane ( n -hexadecane (a)), a fatty acid (palmitic acid (b)) and a
triacylglyceride (tripalmitate (c)).

TAGs are th e main components with a mass percentage up to 98 % in oils. The ir
structure is given schematically in Figure 2.2. R I , R II and R III are fatt y acid residues
which determine the physical properties of the TAGs. To describe the position of the
fatty acids at the glycerol molecule the so called stereospecific numbering ( sn ) is used.
The biological synthesis of TAGs is enzyme-promoted and the pathway typically results
in vegetable fats with an unsaturated fatty acid at the sn -2- pos ition (R II ) of the glycerol
backbone whereas the sn -1,3-positions (R I and R III ) are preferentially occupied by
saturated fatty acids (Gunstone, 2013) .

10

Figure 2.2 : Composition of a triacylgly ceride molecule with the fatty acid residues R I , R II and R III
(modified, Sato 2001).

The fatty acids can be subdivided into the three categories depending on their
degree of saturation, shown in Figure 2 .3. Saturated fatty acids have no double bonds
whereas monounsaturat ed fatt y acid s (MUFAs) have one double b ond. If two or more
double bonds occur the fatty acids are called po lyunsaturate d (PUFAs). The group of
PUFAs can be further sub divided into  -3 and  -6 fatty acids depending on the
C-atom at which the first double bond is present counting from the methyl end group
(  -end). The double bonds are in vegetable oils naturally aligned in cis- configurati on,
whereas the trans -config uration is not common.
In addition, the fatty acids can differ in the number of carbon atoms (the chain
length) or have additional functional residues like hydroxyl or epoxy groups (Gunstone,
2013). Furthermore, the arrangem ent of fatty acids residues on the glycerol backbone
takes influence on the physical properties like the melting point of the TAGs
(Gunstone, 2013; Douaire et al. , 2014). Hence, the composition of fats with respect to
fatty acids determine s their physical, nutritional and chemical propert ies and is
therefore crucial for t he application of lipids in f oods (Gunstone, 2013 ) .
The composition of TAGs with re spect to fatty acids is different depending on the
source. For example, fat originating from coconut or palm kernel, so called lauric oils,
is highly saturated. In contrast, soybean and rapeseed oil have a higher amount of
linolenic acid (C18:3) . In general, these oils consist of 80 % of mono- and
polyunsaturate d fatty acids (Noureddini, Teoh and Clements, 1992; Przybylski, 2011) .
All in all, palmitic, oleic and linoleic acid are the most common fatt y acids in vegetable
oils. Cocoa butter is an exception and has an unusual TAG composition with mainly
so -called HUH TAGs (H = saturated fatty acid with 16 or 18 carbon atoms ,
U = unsaturated fatty acid). In contrast to ve getable oils, animal fats contain mainly
saturated fatty acids (Gunstone, 2013) .

11

Figure 2.3 : Fatty a cid structures: sa turated (s tearic acid), monounsaturated (oleic acid) and
polyunsaturated (linoleic acid).

A minor component of general interest are trans fatty acids (see also 2.1.2) . Trans
fatty acids are a result of incomplet e hydrogenation of vegetable oils (U auy et al. , 2009) .
The trans configurat ion in at least one do uble bond is necessary to call a fatt y acid " trans
fatty acid" . The reason why trans fatty acids came into focus is that they were fou nd to
be related to an increased risk for cardiovasc ular diseases caused by the increase of the
serum cholesterol level (Mozaffarian, Rimm, et al. , 2004; Mozaffarian and Clarke, 2009) .
There is also evi dence to assume that breast cancer as we ll as syste mic inflammation is
related to a high intake in trans fatty acids (Mozaffarian, Pischon, et al. , 2004; Dhaka et
al. , 2011). High uptake can also shorten pregnancy and could have an influence on
growth and development of the central nervous system especially of infants and kids.
According to some studies, there is also a clear correlation between trans fatty acids
intake and colon cancer, di abetes, and obesity (Uauy et a l. , 200 9; Dhaka et al. , 2011) .
The main trans fatty acid originating from industrial hydrogenation is elaidic acid
( trans -18:1) .

12
The complete exclusion of fats and oils in nutrition is, however, not an appropriate
option to avoid these risks. An intake of less than 20 % of the daily calorie intake
coming from oil s and fats can actually result in a bad absorbance of vitamins, in
particular vitamin E, and a lack of essential fatty acids which cannot be synthesized by
the body itself (Dhaka et al. , 2011) . This could disturb processes in the body lik e
immune response or sk in appearance.
In order to reduce trans fatty a cids in fats, the hydrogenation process needs to be
modified in pressure, temperatu re or catalyst . The hydrogenation process is used to
modify unsaturate d fatty acids by the addition of hydrogen atoms and yie lds fatt y acids
at the TAGs with different degrees of saturation and, hence, di fferent application
possibilities of the TAGs (see also 2.3.1). Other promising strategies reducing trans fatty
acids include inter alia interesterification (see also 2.3.2) or the exchange of par tially
hydrogenated oils by oils with a differe nt TAG composition (Uauy et al. , 2009; Dhaka
et al. , 2011). A very promising possibility to gain structuring lipids is the fractionation
of palm oil (see chapter 2.3.3). Thus, the subsequent chapter focuses on palm oil, its
cultivation, producti on and its exceptional com position.
2.1.2 Palm Oil
Palm oil already gained muc h attention in the beg inning of the 1990s for its widely
use in confectionary, shortening or margarine producti on (D’Souza, DeMan and
DeMan, 1990). At the beginning of this century, palm oil got even more attention as
replacement of hydrogenated fats due to the desired elimination of trans fatty acids in
food produ ction. This elimination became a challenge for food processing because the
physical properties of the products have to be maintained, while simultaneously an
increase of the nutritional value was demanded (Gunstone, 2013) . These challenges
could be overcome using fractionated palm o ils (see 2.3.3) .
Malaysia an d Ind onesia are the leading countries in palm oil plantation. The oil
palm ( Elaeis guineensis ) was brought t o Malaysia in the mid 19 th century and, since then,
its plantation area has grown rapidly (Basiron, 2005). Today, the tenera hybrid
(crossbreeding of dura and pisifera ), which yields around fo ur tons oil per hectare, is the
most plan ted oil palm (MPOB, 2009; Lin, 2011) . In trials, other oil palm varietie s
yielded over eight tons per hectare, which makes the oil palm the most efficient among

13
all vegetable oil prod ucing plants (Basiron, 2005). However, he tenera hybrid is mainly
used due to its high yield per bunch and the fatty acid composition of the harvested oil.
The various fatty acid compos ition at the TAGs offers the possibility to yield many
different fractions w ith distinct prope rties.
The oil pa lm has so called fre sh fruit bunches, which are compose d of many small
palm fruits having the s ize of a walnut. The unripe palm fr uits have a black color, while
the red or orange colored ones are ready to be harvested. The palm fruit contains a
mesocarp and a palm kernel, which is surrounded by an endocarp (see Figure 2.4). The
palm oil is obtained from the mesocarp and is mainly composed of palmitic and oleic
acid (MPOB, 2009) .

Figure 2.4 : Fruit bunch of the oil p alm with palm fruits co mposed of the kernel (white), the s hell
(black) and the mesocar p (yellow) ( https://blog.wwf.de/palmoel-stud ie/ , accessed 05. December
2017)

In the mill, the fresh fruit bunches are loosened up to separate the oil palm fruits
from each other. The oil yield per bunch can vary between 18 and 24 %. After the fruits
have been discharged a sterilization step is done using ste am before threshing the palm
fruits. The sterilization is required to avoid further hydrolysis which could lead to the
transformation of TAGs into partial glycerides and free fatty acids . Further
consequences of the sterilization are the preconditionin g of the mesocarp for th e
subsequent processing and to ease the separation of the mesocar p from the palm
kernel. Crude palm oil is extracted after di gestion which increases the release of oil. The
process to obtain palm kernel oil is similar and described elsewhere (Basiron, 2005) .

14
During further processing in the mill free fatty acids (FFAs) are removed to prevent
the oil from lipid oxidation. In addition, color and flavor are removed to make its
application range as wide as po ssible and provide a longer storage time without product
changes. In the degumming step, the phospholipids in the crude palm oil are
precipitated by phosphoric acid and subsequently re moved with bleaching clay along
with other undesired compone nts like trace metals or remai ning water. Afterwards, the
slurry of oil and bleaching earth is set under vacuum at 95 -110 °C for at least half an
hour to ensure a proper process a nd then filtered to gain a light orange palm oil. If
elevated levels of free fatty acids are present a neutralization step is executed prior to
bleaching. Otherwise, the free fatty acids are removed in the subsequent step. In the
deodorization step, the oil is heated under vacuum up to about 250 °C to remove
undesired volatile compounds like ketones and aldehydes as well as free fatty acids
(Basiron, 2005). The flow chart of the above described process to obtain the so called
light-colored RBD pa lm oil (refined, bleac hed, deodorized) is dep icted in Figure 2.5

Figure 2.5 : Flow chart of the RBD p alm oil extraction process.

oil palm • fruit bunches are harves ted
fruit
bunch
• bunch separ ation
• st eriliz ation
• diges tion
• extr action/ pr essing
crude
palm oil
• degumming
• bleaching
• desodoriz ation
RBD
palm oil
• r efined, bleached, deodoriz ed palm oil

15
The fatty acid and TAG composition of palm oil is quite unique because it is
roughly composed of equal amounts of saturated and unsaturated fatty acids.
Braipson-Danthine and Gibon (2007) found a typical TAG di stribution in different
palm oils of about 43 - 49 % HUH (monounsaturated TAGs), 38- 44 % HUU
(diunsaturated TAGs), 5-9 % HHH (trisaturat ed TAG s) , and 6-8 % UU U
(triunsaturate d TAGs). The main fatty acids present in palm oil are palmitic (P), oleic
(O), stearic (S) and linoleic acid (L). They are combined to the main TAGs POP
(30.0 %), POO (25.0 %), POL (1 0.3 %), POS (5.9 %), PPP (5.3 %), and OOO (4.6 %).
Typical percentages of the occurrence of these TAGs in palm oil are given in brac kets.
Thus, palm oil became a very popular raw material for the fractionation process since
it yields differ ent fractions with distinctive properties .
A typical fractionation tree of palm oil is shown in Figure 2.6. Palm stearin, the
high melting fraction, and palm olein, the low melting fraction, can be further
subdivided into hard and mid stearin or soft palm mid fraction and super olein,
respectively. Thes e fractions have distinctive chem ical compositions and are thus used
to achieve the desired physical state of diverse final products. The iodine value (IV)
shown in Figure 2.6 gives the degre e of saturation (the higher the IV the lower the
degree of saturation) (Kellens et al. , 2007). However, on e needs to be careful because
various combinations of TAGs yield similar i odine values.

Figure 2.6 : Schematic fractionation tree of palm oil (adapted and modified from Timms 1997 and
Kellens et al. 2007 ).

palm oil
IV 51- 53
palm st earin
IV 32- 36
hard st earin
IV <15
mid s tearin
IV 40- 45
palm olein
IV 56- 59
soft palm mid
fr action
IV 42- 48
super olein
IV 64- 66

16
Table 2.1 : Main TAG composition of palm oil, palm stearin and palm olein (M= myristic acid ( C14:0) ,
P= palmitic a cid (C16:0), St= stearic acid (C18:0), O= oleic acid (C18:1), S= saturated fatty acid, U=
unsaturated fatty acid) (modified from Kellens et al. 2007).
TAG

palm oil (%)

palm stearin (%)

palm olein (%)

PLL/MOL

2.7

1.5

0.6

OOL

1.9

1.1

2.1

POL /S t LL

10.7

5.9

12.0

PLP/MOP

10.4

7.5

10.9

POO

22.7

12.9

24.5

POP/PLSt

30.3

27.5

30.2

PPP

6.1

26.5

1.7

StOO

2.5

1.5

3.1

POSt

5.5

4.8

6.0

PPSt

1.2

5.3

0.2

UUU

6.0

3.3

6.7

SUU-USU

38.6

21.8

42.9

SSU -SUS

47.5

40.7

48.3

SSS

7.9

34.1

2.1

The variations in the TAG composition of palm oil , stearin and olein are listed in
part in Table 2.1. Palm stearin is rich in saturated fatty acids whereas palm olein consists
of TAGs with a higher amount of unsatura ted fatty acids.
Beside TAGs, which are the major components of edible fats and mainly determine
the physical behavior of fats during proces sing and production, minor components lik e
mono- and diacylglycerides (MAGs and DAGs) as well as FFAs or phospholipids are
also present (Smith et al. , 2011) . Even though many more components are present in
edible fats only the three minor components MAGs and DAGs as well as
phospholipids are shortly described in the following . Most of these other small minor
components are removed during refining. However, in palm oil, a high amount of
diglycerides remains because their vapor p ressure is too low to be removed during the
deodorization step (Smith et al. , 2011; Gunstone, 2013). The amount of partial
glycerides in the end product varies depending on the feedstock and the fractionation
method used (Smith et al. , 2011) .
The difference between MAGs and DAGs is the number of fatty acid moieties
esterified to the glycerol backbone. MAGs have a glycerol backbone with only one fatty
acid residue (see Figure 2.7 a) . The other two sites are occupied by a hydroxyl group,

17
giving the molecule a polar head and thus increasing its sur face-active properties . The
MAGs are either formed during biosynthesis or by hydrolysis of di- and triglycerides
(Gunstone, 2013) . In a DAG molecule, two site s instead of one of the glycerol
backbone are esterified with a fatty acid moiety (see Figure 2.7 b) . Also, DAGs show
surface-active properties and are often used as e mulsifiers (in the same way like MAGs)
in the cosmetics and food industry. Jacobsberg and Ho (1976) found no correlation
between the free fatty acid and the diglyceride content but concluded that the DAGs
resulted from an incomplete biosynthesis of TAGs and not from TAG hydrolysis
(Jacobsberg and Ho, 1976) . In Figu re 2.7c a phospholipid is depicted schematically . It
is similar to DAGs but with a phos phate group esterified to the third glycerol hydroxy l
group. Lecithin, which is ordinarily a mixt ure of differe nt phospholipids, is use d in the
food industry as emulsifier (Smith et al. , 2011) .

Figure 2.7 : Three schematically displa yed minor components based on glycerol with varying
moieties: monoacylglyceride (a), diacylgly ceride (b), and phosp holipid (c).

All present minor com ponents as well as the various TAG s in edible fats influence
the crys tallization of palm oil a nd thus impact the fractionation process and by th at the
end products (Jacobsberg and Ho, 1976) . So far, the influence of all the components
on crystallization are no t yet completely unde rstood. For example, Smith and
coworkers (2011) found promoting effects on crystallization for some ph ospholipids

18
while other phospholipids inhibit crystallization. Consequently, the occurrence of the
aforementioned minor components is essential for the characteristics of the final
product.
2.1.3 Phase Behavior
Before discussing the ph ase behavior of mixtures of various TAGs, a brief
introduction into phase behavior in particular on solid-liquid equilibria should be given.
In Figure 2.8, three possible solid-liquid phase equilibria in a binary system a re
schematically depicted. The phase behavior in graph a) shows complete miscibility in
the solid and the liquid phase and a two phase solid-liquid region. At higher
temperatures there is a singl e liquid phase which consists of A and B in all compositions
(Flöter, 2014a). If complete immiscibility in the solid phase occurs (Figure 2.8b),
component A and B exist as pure solids. The eutectic point describes the point at which
the single liquid phase c oexists with the solid A and the solid B. (Flöt er, 2014a).
A so-called hylotrope can be found in systems with complete miscibility in the solid
and the liquid phase. However, due to interactions between the molecules of
component A and B the liquid -solid t wo-phase region exhibits either a temperature
maximum or minimum (see Figure 2.8c). The hylotrope is the point at which the
coexisting liquid and solid phase have the same compositi on at eithe r the maximum or
minimum temperature. As a consequence, the liquid -solid two-phase region is divided
into two sections. In the one section the liquid phase coexists with a solid phase s 
which is rich in component B. In the other section, the liquid phase coexists with a
solid phase s  which is r ich in component A (Flöter, 2014a).
The phase behavior of binary systems explained above are quite simple because
there exists eithe r a mixture of components A and B or there is complete immisc ibility.
Further complexity arises if one mixed crystal rich in B coexists with a mixed crystal
rich in A. Three ex amples of this kind of phase behavior are depicted in Figure 2 .9. An
immiscibility at low temperatures of the two components leads to a solid-solid
two-phase region with an upper critical solution temper ature (top of the s  + s  region).
At higher temperatures than the upper critical solution temperature there is a single
solid phase with complete miscibility of components A and B. At even higher

19
temperatures there is a liquid-solid two-phase region. Above the two-phase region there
is a single liqu id phase (see Figure 2.9a).
In Figure 2.9b, a system with complete miscibility in the liquid phase, an eutecticum
and two coexisting solid phases, one rich in A (s  ) and one rich in B (s  ) is shown. At
lo w temperatu res there is a sol id -solid two-phase region. A solid phase s  whic h is rich
in compone nt A but also contains component B coexists with a solid phase s  which is
rich in component B with some component A included. The composition of the solid
phases s  and s  changes slightly with the temperature up to the eutectic temperature.
For temperature s higher than the eute ctic temperature the changes in the compos ition
of s  and s  are more pr onounced when s  bec omes richer in B and s  becomes richer
in A . At the eutectic temperature one observes coexistence of the liquid phase l and the
two solid phases s  and s  . At higher temperatures before on ly one single completely
miscible liquid phase is observed. One finds two different liquid-solid two-phase
regions, the s  -l and the s  - l region (Flöter 2014).

Figure 2.8 : Solid-liquid phase diagrams; a) nearly ide al mixing, solid and liquid phase are c ompletely
miscible; b) eutectic behavior, A (s A ) and B (s B ) are completely immiscible in t he solid phase; c) com plete
miscibility of the liquid and the solid phase with a high temperature hylotrope, where t he coexisting
solid and liquid phase have the same c omposition (modified, Flöter 2014a).

A system exhibiting peritect ic behavior is displayed in Figure 2.9c. Similar to a
system with an eu tectic point, at the peritectic temper ature ther e is a phase equilibrium
of a solid phase rich in A (s  ) and a solid phase rich in B (s  ) as well as a single liquid
phase (l). However, different than for the eutectic temperature one observes a single

20
liquid-solid two-phase region (here: s  in coexistence with l). At temperature s lower
than the peritect ic te mperature a liquid-solid and a solid-s olid tw o-phase region as well
as two single solid phases occur. This behavior is found for systems consisting of
components with large difference in the pure component melting points (Flöter 2014).

Figure 2.9 : Solid-liquid phase behavi or, solid phase with partial immiscibility; a) complete miscibilit y
in the liquid phase and p artial immis cibility in the solid phase at low temperatures; b) eutectic system
with mixed crystals s  and s  ; c) peritectic system (modif ied, Flöter 2014a).

So far, the explanations were given for systems consisting of two pure components.
However, fats and oils are multicomponent systems of n- alkanes, fatty acids, and TAGs.
Consequently, the phase behavior of fats and oil s is more complex than all the depicted
phase diagrams shown in Figure 2 .8 and Figure 2.9. However, the ph ase behavior of
these binary systems form the base for the understanding of the complex ph ase
behavior of fats and oils. In general, it can be stated that the complexity increases for
mixtures consisting of n -alkanes to fatty acids to TAGs.
The melting point of n -alkanes is basically determined by their chain length and
increases with increasing chain length. In addition, it has been shown that the
complexity of the phase behavior of binary systems containing n -alkanes of which the
aliphatic chains differ by two carbon atoms also increases with increasing chain length
(Mondieig et al. , 2004) .
The phase behavior of binary sy stems of fatty acids is in it self even more complex
but at the same time the changes in the behavior is dramatically systematic. A summary
of different studies is given by Floeter et al. (2018) showing that binary systems of two
saturated fatty acids differing by two, four or six carbons atoms show a simi lar complex

21
phase beha vior thoug h at differe nt temper atures. In addition, the temperature range in
which the liquid-solid two- phase regions occu rs decreases with increasing chai n length
of the fatty acids. If a saturated fatty acid is mixed with a monounsaturated fatty acid,
the phase behavior is less complex than for a system of two saturated fatty acids; though
it still shows eutectic behavior. The asymmetry of the phase diagram increase s wit h
increasing chain length of the saturated fatty acid shifting the eutectic composition
towards the pure monounsaturated fatty acid. Thereby, the do uble bond of the
monounsaturated fatty acid hampe rs the packing of the sa turated and unsat urated fatty
acid adjacent to each other within one crystal structure and thus the ph ase behavior
with respect to the occurrence of the solid phase . Binary mixtures of two
monounsaturated fatty acids or a monounsaturated and a saturated fatty acid show a
systematic change of the phase behavior with the chain len gth as show n in Figure 2. 10 .

Figure 2. 10 : Phase behavior of oleic acid (black solid) mixed with stearic (blue, dashed double dotted),
lauric (green, dotted), petr oselinic (orang e, dashed), gondoic (red, dashed dot ted) and asclepic (grey
solid) acid (adapted from Floeter et al. 20 18).

Figure 2. 10 gives the phase behavior of oleic acid mixed with stearic, lauric,
petroselinic, gondoic or asclepic acid. All mixture s reveal eutectic behavior where at the
composition of the eutectic point shifts to higher concentrations of oleic acid and the
eutectic temperature decreases in the order of stearic (C18:0) → lauric (C12:0) →
petroselinic (C18:1,  6  12) → gondoic (C20:1,  11  9) → asclepic acid (C18:1,

22
 11  7). It can be summed up that the phase behavior of fatty acids is influenced by
the degree of saturation a nd the chain length of the fatty acid as well as the positi on of
the double bond in the unsaturated chain (Floeter, Haeupler and Sato, 2018). The
complexity of the phase behavior increases even more if more than one double bond
is present.
The combination of fatty acids to triglycerides makes the phase behavior of systems
containing TAGs even more complicated. Studies are often limited to one or two TAGs
in order to decrease the intricacy. Natural fats are, however, co mposed of a mixture of
TAGs (Calliauw et al. , 2010) . Beside the influence of mixing TAGs, the polymorphic
behavior of each TAG needs to be additionally considered. TAG miscibility in the solid
phase ultimately depends on the polymorphic form (Takeuchi, Ueno and Sato, 2003;
Himawan, Starov and Stapley, 2006; Floeter, Haeupler and Sato, 2018).
The packing of the TAGs depends on both, the fatty acid moieties as well as their
respective position on the glycerol backbone. Sometimes TAGs pack more easil y
together if the fatty acid order at the TAG is not the same (e.g. POP/OPO) (Himawan,
Starov and Stapley, 2006) . In the ph ase behavior one distinguishes between complete
miscibility in the solid phase, occurrence of an eutecticum, and the formation of
so -called molecular compounds which are actually mixed crystals with a fixed ratio of
components A and B (Floeter, Haeu pler and Sato, 2018) . For example, eutectic
behavior was found for all polymorphs for POP-PPO mixtures due to the steric
hindrance due to the double bond of the unsaturated fatty acid residue (Sato, 2001) .
Depending on the conditions, TAGs occur in di ffere nt polymorphic forms.
Consequently, at some conditions a mixture of two specific TAGs are completely
miscible, at other conditions they form an eutecticum (Kellens et al. , 1991; Takeuchi,
Ueno and Sato, 2003).
One important aspect for the work at hand is the study of the solid -liquid phase
behavior of system containin g fats and/or oils. As pointed o ut above, fats a nd oils are
a mixture of a number of components. With each component the phase behavior
becomes more complicated (Floeter, Haeupler and Sato, 2018) . For pure components
such as e. g. n -alkanes, methods su ch as DSC or other calorimetry allow a quite accurate
determination of the solid -liquid phase transition (e.g. melting point) though
parameters as heating or cooling rates affect the accuracy of the determination. For

23
multicomponent systems, the determination of the solid-liquid p hase behavior is more
complex as already the systematic phase diagrams of binary systems ( Figure 2.8 and
Figure 2.9) show (Zh ou and Hartel, 2006).
The use of fats and oils as substitutes for animal fats or trans -fats requires the
knowledge, in particular, of the phase transition from the liquid to the solid phase. As
fats and oil s are multicomponent systems there is no t one single transition point but a
number of po ints describing the transition from the liquid phase to the liquid-solid
two-phase region depending on temperatu re, pressure and composition of the liquid
phase. In gener al, these phase transitions can be described by the equality of the
chemical potentials of a component i (which is on e of the constituents of the system)
in the coexisting phases (= phases in equilibrium at given temperature and pressure),
e.g. at the eutectic temperature of a system displaying the phase behavior as given in
Figure 2.9b. This is give n by the following e quation
𝛍 𝐢,𝐋 = 𝛍 𝐢, 𝐬 𝜶 = 𝛍 𝐢, 𝐬 𝜷

Eq. 2.1

where  i, L is the chemical potential in the liquid phase,  i ,  the chemical po tential
of component A, and  i,  the che mical p otential of component B in the coex isting
phase (Flöter, 2012).
In general, the chemical potential of a component i in phase K (as K is liquid or
solid) can be desc ribed by
𝛍 𝐢,𝐊 = 𝛍 𝐢,𝐊
𝟎 + 𝐑𝐓 𝐥𝐧 (𝐱 𝐢 ∙ 𝛄 𝐢 ) 𝐊

Eq. 2.2

where  i ,K is the chemical potential in the phase K,  i,K 0 the chemical potential of
the pure component i, R the gas constant, T the temperature, x i the mole fraction of
component i in phase K and  i the activity coefficient of component i (Himawan, Starov
and Stapley, 2006).
Using this equation for the description of the solid -liquid phase equilibrium and
simplifying the eq uation leads to the so-calle d Hildebrand eq uation:
𝐥𝐧 𝐱 𝐢 ≈ ∆𝐇 𝐦,𝐢 ∆𝐓
𝐑𝐓 𝐦,𝐢 𝐓 ≈ ∆𝐇 𝐦,𝐢
𝐑 ( 𝟏
𝐓 − 𝟏
𝐓 𝐦,𝐢
)

Eq. 2.3

with the mole fraction x i of component i,  H m,i the heat of fusion of component
i, T m,i the melting temperature of component i, R the gas constant and T the
experimental temperature. This equation is quite commonly used to estimate the

24
solubility of component I in a liquid phase (or in other words to d es cribe the liquidus
area). For fats and oils it can be only used as a first approximation as fats are
multicomponent mixt ures occu rring in more than one solid phase
2.2 Fa t Crystall ization
The crystallization of fats is one of the most important and complex processes in
the food industry. It is both, important for the desired final product and for
intermediate processes like fractionation. In food technology it is one of the crucial
steps fo r products containing fats as a structure giving component. Examples for its
commercial application are margarine, ice cream or chocolate. Dependent on the
application, fat crystals give me lting stability at ambient temperature (margarine),
provide the desired mouthfeel (chocolate) and incorporate and stab ilize air bubbles (ice
cream). The so -called mouthfeel is re lated to the size of the crystals, which are
de tectable b y humans at around 2 0- 25  m (Flöter, 2014a).
The crystallization behavior is primarily given by the TAGs in the mixt ure .
Furthermore, mino r components like di- and monoacylglycerides, waxes or
phospholipids influence the crystallization process (Sato, 2001) . The resulting crystals
contribute to shelf life, functionality and consumer perc eption (Hartel, 2013).
The crystallization process can be considered as the transformation from a mainly
disordered liquid into an ordered solid state (Flöter, 2014a) . Regarding the
crystallization process, especially nucleation and growth rate decisively contribute to
the size and amount of formed crys tals, which is in turn important for the final product
properties. In order to obtain the proper product characteristic s or the desired
fractionation, nucleation, growth, and polymorphic transformations need to be
optimized. In the previous chapter the ph ase behavior in general and in detail of
systems containing fats was discussed. The di splayed phase diagrams give the
equilibrium cond itions. However, during the processing of fats equilibrium is not
achieved. The equilibrium conditions give the circumstances which can optimally be
reached and thus give the lower value of the gradient re presenting the driving f orce for
a process. Additionally it has to be taken into account that t he equilibrium is often

25
achieved very slow in the given systems and t his process is probably not completed
within the shelf life of s ystems like chocolate or marga rine (Flöter, 2012).
Crystallization consists mainly of two steps: nucleation and growth. Nucleation is
the accumulation of molecules into a cluster until it reaches the so-called critical nucleus
size to favor the incorporation instead of the dissolution. During crystal growth,
material from the liquid phase is attached onto the already formed crystal (Flöter,
2014a) . In crystallizatio n not only the ph ase equilibrium needs to be known and studied
but also kinetic aspects and driving forces. Among the se, supersaturation and
recrystallization need to be considered because of their influence on the initiation of
crystallization and the resulting shelf life of the products, respectively (Hartel, 2013) .
To decrease the complexity, the explanations given are mainly limited to pure TAG
systems. This needs to b e considered if the crystallization behavior of fats and oils as
complex mixture s is considered.
2.2.1 Nucleation
The initiation of nuclei can be important for the further progression of a process.
Hence, it is necessary to briefly discuss this first step durin g crystallization. In his
review, Hartel (2013) defines the nucleation as “the formation of crystals from liquid
state”. In the work at hand this means that nucleation can also be described as an
accumulation of molec ules and is the crucial step to control if solid phases occu r. This
accumulation needs to be present long enough to start crystal growth for which the
nuclei serve as growth s ites (Flöter, 2014a). During this step , the TAGs start to align in
an ordered way to form a nucleus whi ch later serves as a growth site. Supersaturation
as a driving force is a required prerequisite for nucleation (Hartel, 2013). It can be
explained as the state a t which the concentration of the solutes in a solvent is higher
than the solubility at given conditions . Thus, it is the difference in composition between
the actual state and the e quilibrium state (Flöter, 2014a).
Though commonly the supersaturation is used to determine the driving force for
crystallization it is more accurate to use the difference in the chemical potential. The
chemical potential describes the partial molar Gibbs energy of a component i in a phase
K. Here, one would use the differences between the chemical potential of component
i in the supersaturat ed solution and the chemical potential of component i in the

26
saturated sol ution (the equilibrium state). When using the chemical potential difference
instead of solely the concentration difference (supersaturation) also the interactions
between al molecu les in solution a re taken into account.
As already mentioned above, the chemical potential is the partial molar Gibbs
energy. In classical thermodynamics the Gibbs energy is used to determine the
energetically favorable state, in other words which phase is stable at a given temperature
and pressure. In general, the phase which is described by the smallest Gibbs energy is
the phase which is stable (see Figure 2. 11 ). In Figure 2. 11 the Gibbs energy of a pure
component is give n as funct ion of temperat ure. G s gi ves th e Gibbs energy of the solid
phase, G L the one of the liquid phase. At the melting temperature T m where the solid
and liquid phase coexist, e.g. are in equilibrium, the Gibbs energy of the liquid phase is
equal to the Gibbs energy of the solid phase. At temperatures higher than the melting
temperature, the Gibbs energy of the liquid phase is smaller than the one of the solid
phase. Thus, the liquid phase is the stable on e in this temperature region. At
temperatures lower than the melti ng te mperature T m , the Gibbs energy of the solid
phase is smaller and, he nce, the solid phase is stable.
If kinetics are slow, the equilibrium state, e.g. the phase which is stable at a given
temperature and pressure , is not achieved immediately. Then a me tastable state occurs.
This happens if a solution contains a solute in a concentration which is higher than in
its saturated state. The difference in the Gibb s energy of the metastable state and the
equilibrium state is the real driv ing force . In the cas e of a pure component, if the liq uid
phase is subcooled, e.g. the components exist in the liquid state even though
temperature and pressure are such that the solid state is the energetically most
favorable, the liquid phase is the metastable state. The Gibbs energy differenc e  G
gives the driving force required to overcome the activation energy to change into its
stable state (se e Figure 2. 11 , T 1 ) (Fl öter, 2014a).
The formation of molecule clusters (nucleation) makes the formation of an
interface necessary which requires energy input . The tra nsfer of molecules from the
liquid into the crystal releases energy. The di fference in the energy of these processes
mainly determines the required driving force to form crys tal s (Flöter, 2014a).

27

Figu re 2. 11 : Schematic illustration of the relationship between temperature and Gibbs free energy for
a pure component at constant pressure ;  G is the driving force fo r crystallization at T 1 , T m is the
melting p oint of the pure component , G s the Gi bbs energy of the soli d phase (short dashed line), G L
the Gi bbs energy of the liquid phase (long dashed line); the red line indicates t he m ost stable state at
the corresponding temperature (modified, Flöter 2014).

A nucleus nee ds to overcome a critical size r* before it is energetically favorab le to
grow further (see Figure 2. 12 ). Therefore, the mole cules need to accumulate into
clusters larger than this critical size (r*). Otherwise it is energetically more favorable for
the molecules in the cluster to dissolve again . Because the detection of the critical
nucleus is often impossible, the term “induct ion time” is introduced which describes
the time until the detection of crystals . Logically, it depend s on the detection method
applied (Jacobsberg and Ho, 1976; Flöter, 2014a).
During nucleation three ways of nuclei formation are possible . First, there exists
homogeneous nucleation in which the molecu les collide in a homogeneous liquid
phase. Second, heterogeneous nucleation at impurities lik e dust or at irregularities of
the crystallizing equipment can occu r. The third possibility is nucleation initiated by
already existing crystals. T his can be either cal led seeding (nucleation on purpose) or
secondary nuc leation (unintentional nuc leation) (Hartel, 2013; Flöter, 2014a).
Although ho mogeneo us nucleation is used to describe the phenomeno n of
nucleation, it plays a minor role in the industry. Heterogeneous nucleation occurs
preferentially since it is energetically more favorable than homogeneous nucleation .

28
During heterogeneous nucleation, the energy required to form a surface is le ss, in
particular if the interfacial tension between the crystal and the foreign surface is low.
Commonly, heterogeneous nucleation rather occurs at low levels of supersaturation. A t
higher supersatura tion, homogeneous nucleation appears dominant due to the higher
driving force which allows to overc ome the activation energy and thus does not require
existing surfaces for crystallization (Flöter, 2014a).

Figure 2 . 12 : Dependency of Gibbs free energy G o n t he size of f ormed nuclei r; r * is the critical radius
of nucleus, r 0 is the radius where  G=0; liquid state (I), accumulation of molecule s below the critical
size r* (II), nucleus with sufficient size r > r* (III), crystal with r > r 0 (IV) (modified, Flöter 2014 ).

The advantages of seeding (controlled heterogeneous secondary nucleation) are the
control of the crystal size distribution, the requirement of a lower level of
supersaturation also due to improved secondary nucleation and the templating effect
of the seeds for the desired structure of the crystal (Calliau w et al. , 2010 , Flöter, 2014a) .
Subsequent to nucleation, crystal g rowth is the second step in the crystallization process
and is explained in detail in t he following sub-chapter.
2.2.2 Crystal growth
A prerequisite for crystal growth is a liquid system in a supersaturated state wit h
present nuclei of critical size. The m olecules need to di ffuse from the bulk to the crystal
surface and att ach onto it . I n Figure 2. 13 the concentration of the solution forming the

29
crystal is illustrated. Th e diffusion of molecules towards the crystal surface happens due
to differences in the chemical potential. Due to enthalpy difference s of the liquid
solution and the crystal, att achment of molecules on the crystal results in the release of
heat (so-called latent heat). If this heat is not transported away from the crystal surface,
a local temperature increase which changes the equilibrium conditions and thus
decreases th e driving force, is deserved. Other limitations of crystal growth are, e.g. a
limited diffusion of new molecules to the growth site or a reduced availability of growth
sites and slow conf ormational processes at them (Hartel, 2013; Flöter, 2014a).
Even though nucleation is a prerequisite for growth, after growth has started
nucleation and growth occur simultaneously. Crystal growth already happens at a low
level of supersaturation if growth sites are present. If these are rare, apparent bulk
crystallization is limite d. In general, with incre asing supersaturation the nucleation rate,
and therefore the number of growth sites, increases. The consequence is a higher crystal
growth rate . With a high nucleation rate the cr ystals grow in a small di stributi o n of
sizes. Thus, the resulting crystal size can be controlled by choosing a specific
temperature or temperature range during crystallization. For example, increasing the
temperature after generating nuclei reduces the nucleation rate. In this case, persis ting
crystal growth can solely lead to larger crystal sizes. I n contrast, small crystal sizes ,
which are favored in the food industry because of their abili ty to stabilize and structure
more efficient compared to large crystals is, achieved by simultaneously producing a
large number of nuclei during cr ystal growth (Flöter, 2014a).

Figure 2. 13 : Attachment of material from the liquid onto the alread y solidified and ordered material
(here for TAGs); crystallized TAGs (I), adsorption of TAGs onto the grow th sit e (II), di ffusion of
TAGs (III), composition of the bulk (IV) (modified, Flö ter 2014).

30
When studying fat crystallization besides supersatura tion, nucleation and growth
also recrystallization ne eds to be considered . It is drive n by the difference between the
manufactured metastable and the equilibrium state and can cause product defects like
coarsening of ice c rystals or the formation of f at bloom in chocolate (Harte l, 2013) .
2.2.3 Polymorphism
Many long chain compo unds possess the abili ty to form different crystal str uctures
due to a variety of possible arrangements of the molecules in the unit cell. This
phenomenon is called po lymorphism and is of technical and scientific interest (Sato
and Garti, 1988; D’Souza, DeMan and DeMan, 199 0; Gu nstone, 2013; Hartel, 2013;
Flöter, 2014a). As a consequence of the di fferent molecular packing, different
polymorphic forms of the same material have disti nct prop erties, most promin ently
different melting points (Gunstone, 2013) . The kno wledge of the polymorphic
behavior of fats and oils is crucial for many food produc ts l ike choco late or margarine .
Depending on the product the best properties can actually be achieved with specific
polymorphic forms which are metastable (D’Souza, DeMan and DeMan, 1990; Metin
and Hartel, 2005; Himawan, Starov and Stapley, 2006; Sato and Ueno, 2011; Marangoni
et al. , 2012) .
Some authors relate the po lymorphism in TAGs to their stereo chemical
configuration (Jacobsberg and Ho, 1976) . The occurrence of different solid phases
(polymorphism) has been studied for a large number of pure components. For fats,
which are multicomponent systems, less is known. This is due to the fact that the
complexity of the polymorphic behavior increases with the number of distinct TAG s
in the mixture. In particular, d ifferences in chain length and unsaturation of the
esterified fatty acids have a strong influence ( D’Souza, DeMan an d DeMan, 1990) .
In polymorphism, monotropic and enantiotropic behavior need to be
distinguished. The different forms of polymorphism can be explained using the Gibbs
energy as function of the temperature (see Figure 2. 14 ). In general, G L describes the
Gibbs energy of the liquid phase, G S,I of the solid phase I and G S,II of the solid phase
II. At a given temperature the stable phase is the ph ase which is chara cterized by the
smallest value of Gibbs energy. The points at which the curves cross give the phase
equilibria of the respective phase.

31
In Figure 2. 14 a, the so -called monotropic polymorphism is shown. The
intersections of the curves describing G L , G S,I and G S,II occur, one intersection of G L
and G S,I and one of G L and G S,II . In the given te mperature range, the curve for G S,I lays
under the curve for G S,II . This indicates that the solid form S,II is not the stable phase
in the given temperature range. If this phase is ob served, then it is in the metastable
state. This behavior is called monotropic polymorphism because only on e of the solid
phases is stable.
In Figure 2. 14 b, t he so-called enantiotropic polymorphism is depicte d. Here more
than two intersections of the Gibbs energy curves are found, intersection of G L and
G S,I at T m,SI , intersection of G L at G S, II at T m,SII , and G S,I and G S, II at T I → II . For
temperatures T > T m,SI the liquid phase is stable, for Tm,SI < T < T I → II the solid phase
SI is stable and for te mperatures T < T I → II the solid phase SII is stable. The respective
other phases are metastable and would not be observed if equilibrium is established
immediately.

Figure 2. 14 : Gibbs free energy of solid and liquid phases of a pure component over temperature,
 G=driving f orce at T I . a) monotropic polymorphism, T m,S,I = melting point s olid SI, T m,S,II = melting
point solid SII; b) enanti otropic polymorphism, T m,S,I = melting p oint solid SI , T m,S,II = melting point
solid SII, T II → I = transition tempera ture of solid I and II (modified, Flöter 2014).

32
As already mentioned above, due to kinetics the equilibrium state is not established
immediately so that the metastable phases can be observed sometimes for very long
time. The so-called Ostwald´s rule of stages says that a system rather favors the
conversion into an energetically similar state instead of converting it into the
energetically most favorable one (Ostwald, 1897). This means that the metastable phase
might occur instead of the energetically favored stable phase. The kinetics of
polymorphic transitions such as the transition from SII to SI at T I → II depend on the
crystal composition, th e structure and temperatu re, shear or pressure (Flöter, 2014a).
Above polymorphism is explained using an ex ample of a c omponent which forms
two different solid states. Depending on the nature of the component more than two
solid forms can occu r.
For glycerides, Larsson (1966) defined three polymorphic forms differing in their
sub cell structure. Wille and Lutton (1966) found six polymorphic forms for cocoa
butter to be differentiated. I n Fig ure 2 . 15 the most common polymorphic forms of
TAGs are shown and explaine d in the following .

Figure 2. 15 : Subcell structures of the polymorphic form s  ,  ’ and  (modified, Sato and Ueno 2005 ;
Lopes et al. 2015).

The  polymorph shows a hexagonal sub cell structure and is the energetically least
favorable one. It has the lowest melting point and thus it is also the least stable form
due to its lose molecular packing (Gunstone, 2013). The fatty acid residues are aligned
in a double chain length structure (s ee Figure 2. 16 b). In the double chain packing, the
TAG molecules can either align in the turning fork or the chain configuration as shown
in Figure 2. 16 a (Fl öter, 2014a) .

33

Figure 2. 16 : Different configurations of TAGs: a) chair (left) and tuning fork (right) configuration,
b) double chain length struc ture, c) triple chain length structure (modified, Sato and Ueno 2005).

The  ’ and  polymorphs (see Figure 2. 15 , right) are the more stabl e forms and
provide a more efficient packing due to the tilted arrangement of the alkyl ch ains
(Gunstone, 2013). The fatty acids can either be arranged in a do uble chain ( Figure
2. 16 b) or a triple chain (Figure 2. 16 c) length structure. For example, the triple chain
lengt h occu rs if the sn -2-pos ition of the TAGs is occupied by an unsatu rated fatty ac id
(Flöter, 2014a) . The sub cell of the  ’ polymor ph is orthorhombic .
In particular, TAGs with an unsaturated fatty acid at the sn -2-position and two long
chain saturated fatt y acids at the sn- 1- and sn -3-positions (HUH) sh ow good stru cturing
properties (e.g.  V form of cocoa butter), slow polymorphic transiti on and have been
found to form stable  -crystals causing chocolate bloom or POP graininess (D’Souza,
DeMan and DeMan, 1990; Koyano, Hachiya and Sato, 1990; Sato, 2001; Bot and Flöter,
2013; Kang et al. , 2013) .
The  polymorph shows a triclinic parallel p acking and along with the triple chain
length structure (see Figure 2. 16 c) the densest packing of the molecules is favored
(Koyano, Hachiya and Sato, 1992; Sato, Ueno and Yano, 1999) . Symmetrical TAGs
have a tendency to form stable  crystals, whereas unsymmetrical compounds favor the
 ’ form due to the possible molecular packing (Gunstone, 2013). Each polymorphic
state corre sp onds to a different pa cking of the hydrocarbon chains and depen ds on the
chain length and the angle of tilt due to double bonds if present in the fatty acid moieties
(Jacobsberg and Ho, 19 76) .

34
The generation of distinct polymorphs is a matter of pri mary nucleation kinetics
and the application of adequate driving force, e.g. supersaturation. In Figure 2. 17 a the
relation between nucleation/ cooling rates and preferred occurrence of polymorphic
form is given. In Figure 2. 17 b the curves for the respec tive Gibbs energy are given. The
 form can be induced by fast cooling. Once its respective supersaturation is reached,
it nucle ates faster than the  ’ form or the  f orm (G unstone, 2013; Flöter, 2014a). The
nucleation and growth of  lead to a reduc ed driving force (supe rsaturation) for  ’.
Nevertheless ,  ’ nucleation takes place. Once  ’ nuclei gro w further the driving
force for the  nucleation vanishes due to the depletion of molecules available for
crystallization due to the ir incorporat ion into  ’ crystal s . As a consequence ,  ’ crystals
continue to grow further while  crystals dissolve completely (Flöter, 2014a). In
principle, th e same mechanism between the  ’ polymor ph and the stable  polymorph
during gro wth is observ ed. However, due to kinetics the metastable  ’ cryst als ex ist for
extended periods of time.

Figure 2. 17 : a) Nucleation rate of different polymor phic forms in relationship with the cooling r ate,
nucleation of  (dotted line) , nucleation of  ´(dashed line), nucleation of  (solid lin e); T m (  ) = melting
point of polymorph  , T m (  ´) = melting point of polymorph  ´, T m (  ) = melting point of polymorph
 (modified, Flöter 2014); b) Gi bbs free energy ov er temperature for three different polymorphs with
the liquidus line GL;  -form: highest G and low est melting point (T m,  ),  ´-form: medium G and
medium melt ing point (T m,  ´ ),  -form: lowest G and highest melting point (T m,  ) (modified, Sato 2001).

35
In general, each polymorph can be crystallized directly from the melt . The
transition from one polymorph int o another is only possible from the less to the more
stable one (  →  ´ →  ). The understanding of the parameters influencing the phase
transition is complicated but some rules of thumb exist. For example, with increasing
chain length of the TAG residues the transition rates with respect to the transitions
from one polymorph to the other decreases (Jacobsberg a nd Ho, 1976) .
The TAGs prese nt in a fat determine the po lymorphic and crystallization behavior .
For fats lik e cocoa butter and palm oil, POP and SOS are the common and dominant
TAGs. D’Souza and coworkers ( 1990) stated, that asymmetric TAGs, which means
the same fatty acid residue at the sn -1,3- and sn - 2-position but a different one at the
sn -1,3-position, yield mostly a  ´ crystal structure. In contrast, for symmetric TAGs
where the sn -2-position fatty acid moiety differs from the residues at the sn - 1,3-position,
the  polymorph is the favorable state.
The desired product characteristic s determine which polymorphic form needs to
be formed. For example, in margarines and spreads the  ´ polymorph gives a smooth
texture , whereas the  -form may lead to disadvantageous structural defects such as
graininess (D’Souza, DeMan and DeMan, 1990) . A possibility to achieve the proper
polymorphic form is the addition of seed crystals which occur in the desired
polymorphic form. The addition of seed crystals during the process leads to a
crystallization at higher temperatures (Sirota and Herhold, 2000) . If seeds are added in
the metastable zone where the driving force is already high enough to start nucleation
excessive homogeneous nucleation is prevented . Often, this is done during chocolate
manufacturing to directly crystallize in the  V-form so that a tempering step to avoid
chocolate bloom is n ot required (Metin and Ha rtel, 2005) .
The process of seeding typically involves the same type of material as the one to
be crystallized. Alternatively, crystals of a similar material can be used. This effect is
called templating and could also be considered as heterogeneous nucleation . Koyano
and coworkers ( 1990) claimed that successful seeding with foreign crystals requires
similarities i n chain length and polymorphic properties . Her e the nucleation site sh ows
a similar surface structure to the molecules to be cr ystalliz ed (e.g . emulsifiers with same
fatty acid composition in fat crystallizatio n at an oil/water inter faces) (Flöter, 2014 a).

36
2.3 Fat Modific ation
Since the re quirements for the widespread applications of edible fats and oils it i s
crucial that their physical properties can be adjusted. In the following section, the three
modification techniques hydrogenation, interesterification and fractionation are
explained.
2.3.1 Hydrogenation
The old est of the three modification processes explained in this chapter is
hydrogenation. It was invented to improve the stability against oxidation of fat s
containing polyunsaturated fatty acids ultimately leading to the saturation of the fatty
acid . Hydrogenation is an exothermic reaction during which hydrogen molecules are
added to the fatty acids. The catalyst (predominantly nickel) and the concentration of
hydrogen molecules are the main factors which determine the reaction kinetics
(Dijkstra, 2014b).
The process conditions need to be balanced in concern for the prop er working
temperature since on the on e hand higher temperature s pro mote the formation of t rans
fatty acids but are favorable for the selec tivity of the hydrogenation process on the
other hand (Dijkstra, 2014b) . Because of this competition between isomerization and
hydrogenation during hydrogenation trans fatty acids may be formed. Hydrogenation ,
even if completely executed, is immediately related to trans fatty acids in the public
discussion and, thus, the application of this technique is very limited (Kellens and
Calliauw, 2013). Therefore, isomerization of double bonds is favored if the catalytic
effect is di minished. The formation of trans fatty acids is undesired since they are related
to an increa sed risk fo r e.g. cardiovascular diseases (Dhaka et al. , 20 11; Kellens and
Calliauw, 2013) .
The so-called iodine value (IV) is often used in chemistry to determine the degree
of unsaturation of fatty acids. It relates to the fact that the double bonds of unsaturate d
fatty acids react with iodine. The high er the iodine value, the more do uble bonds are
pr esent. The relationship between the iod ine value and trans fatty acid content in fish,
soybean and palm oil is shown in Figure 2. 18 . As expected the iodine value found for
fish oil is larger than for soy bean oil than for palm oil. The trans fatty acid content if

37
plotted as function of the iodine value expresses a maximum. This maximum is
commonly found at the typical mean iodine value of the spec ific oil. For all show n oils
it shows also that for low iodine values (< 20) the trans fatty acid content is less than
15 %. The oils are so-called partially hydrogenated until the iodine value reaches a l ow
value (IV < 5). These fats are called fully hydrogenated and can be use d for the
structuring of fat-conta ining food products si nce they are low in trans fatt y acids.

Figure 2. 18 : Relationship between iodin e value a nd trans fa tty acid content of fish oil (solid line),
soybean oil (dashed line) and palm oil (dotted line) (mo dified, Kellens and Calliauw 2013) .

In summary, the hydrogenation process yields a hig her degree of saturation of the
fatty acids and changes an oil into a fat. In addition, the melting point increases with
increasing degree of saturation (Kellens and Calliauw, 2013) . The progress of
hydrogenation can be measured by the decrease of the i odine value a nd t he incre ase of
the melting point (Dijkstra, 2014b). An advantage of hydrogenation is, hence, that the
sourcing of the oils is less importan t because diffe rent oils could be proces sed into fats
of desired properties.
2.3.2 Interesterificatio n
Another method to modify physical properties, e.g. melting of fats, such that they
fit the product requirements is the interesterification. Therefore, the fatty acids attached
to the glycer ol bac kbone of the fat are exchanged. The technology of interesterification
was invented in the 19 th ce ntury .

38
So far, two variations of the process namely the chemical and enzymatic
interesterification are applied . Both have the advantage to be applicable for a wide range
of possible raw mate rials (Dijkstra, 2014a).
Interesterificat ion was firstly used in the 1920s during the search for cheaper butter
and became more famous in the 1950s, when researchers started to use sodi um
methoxide as a catalyst enhancing the chemical intereste rification . (Kellens and
Calliauw, 2013). The main parameters influencing the process are the oil quality, th e
type, and the concentration of the catalyst. In addition, the op timized amount of
catalyst yields less by-products and thus results in less oil loss (Kellens and Calliauw,
2013). Interesterification is always followed by a deodorization step to remove minor
co mponents such as form ed FFAs which are prone to oxi dation (Dijkstra, 2014a).
During the process, which is performed batch-wise in the industry, the fatty acid
residues of the TAGs are statistically rearranged over the TAGs present. Figure 2. 19
shows a possible interesterification between a trisaturated TAG (SSS) and a
triunsaturated TAG (OOO). I n this example, the interesterification yields a TAG with
two saturated fatty acids at the sn -1,3-position and an unsaturated fatty acid residu e at
the sn -2-position (SOS). These HUH TAGs are important in the production of cocoa
butter equivalents (CBEs) due to their structure providing propert ies. It is important to
mention here that the depicted TAG resulting from the interesterification is just one of
many possible fatty acid residue co mbinations. For chemical interesterification, sodium
methanolate is a common used chemical catalyst (Dijkstra, 2014a). Probably, even
though not completely verified, a carbonyl addition is the basic principle during this
reaction (Kellens and Calliauw, 2013). However, the TAGs resulting from chemical
interesterification cannot be controlled in a targeted manner. Thus, apart from the TAG
SOS (12.5 %) shown in Figure 2. 19 , also the TAGs SSO (25 %), OSO (12.5 %), OOS
(25 %), SSS (12.5 %), and OOO (12.5 %) po ssibly result from the interesterification
process. The prob abilities for each TAG composition to occur are given in the brackets
assuming a randomly p erformed process and a statistical distribution.
A fast reaction normally takes about 30 min. It can be stopped by addition of water,
leading to the formation of soaps and free fatty acids. Addition of e.g. citric acid reduces
the soap formation. The formation of both, free fatty acids (FFAs) and fatty acid methyl
esters (FAMEs) is u ndesired because of the lower yield of the desir ed TAGs.

39

Figure 2. 19 : Interesterifica tion example; a tris aturated TAG (top le ft, 3x st earic acid) interesterified
with a triunsaturated TAG (bottom left, 3x oleic acid) can yi eld a HUH TAG (right, 2x stearic
( sn -1,3-position), 1x oleic aci d ( sn - 2-positio n)).

The interesterification process can alternatively be performed enzymatically as
mentioned above. This process can be conducted at milder conditions and provides a
stereotypic acyl exchange (Kellens and Calliauw, 2013). It uses lipases to catalyze the
abovementioned rearrangement of the fatty acids at the glycer ol backbone. There are
sn -1,3-position selective and non-selective lipases available. The stereo specificity is very
helpful to produce cocoa butter equivalents (CBEs) fro m palm mid fraction by
exchanging some of the palmitic acid residues at the sn -1,3-positions with stearic acid
moieties (Cowan, 2014). Starting from the sa me feedstock, the TAG composition
resulting from a ste reo selective lipase and from a chemical approach can differ greatly.
2.3.3 Fractionation Technol ogies
The main reasons to apply fractionation is the enrichment of valuable TAGs, the
removal of undesired minor components or the production of different valuable
fractions each with different application ranges and thus presenting alternatives to
hydrogenated fats (Timms, 199 7) . In fat fractionation, fractional crystallization is
combined with a separation step to receive di fferent fractions with distinct properties.
The sol ubility of each TAG in the different fraction determines the separation. The
separation step depends on the melting points of the various fractions. Therefore ,
fractionation is currently the only technology which yields fats with specific properties
solely based on a physic al process (Timms, 1997; Dhaka et al. , 2011) .

40
The undesired wide melting range of palm oil is due to the variety of reasonably
similar TAGs in the mixture. Because of this it is po ssible to separate palm oil into
different fractions with distinctive properties (Stöver, Eggers and Stein, 1983) .
Therefore, palm oil became the most fractionated fat in the world (Talbot, Smith and
Cain, 2006). It is produced in large amounts (around 64 Mio. tons between 2016 and
2017) and is highly produc tive (4 t/ha/yr., other veg etable oils  0.5 t/ha/yr.) (MPOB,
2009; Yan, 2017).
During palm oil fractionation, mainly a good quality olein is aimed due to its
application as Asian table or salad oil (Stöver, Eggers and Stein, 1983;
Braipson-Danthine and Gibon, 2007; Kellens et al. , 200 7). How ever, applying various
subsequent fractionation ste ps leads to a vari ety of different fractions for di vergent
applications (Kellens et al. , 2007) . Once hydroge nation was abandoned, palm oil and its
fractions became increasingly important for the food industry. The following
explanations are hence focused on palm oil fract ionation.
In a simplified way, palm oil can be subdivided into four groups of TAGs with a
different melting point each: PPP (65 °C), PPO/POP (34 °C), POO/OPO (18 °C) and
OOO (5 °C). These different TAG groups can be separated using fractional
crystallization. The mixture of TAGs is crystallized at a certain temperature before
separating the two fractions. If no liquid oil remains in the s olid fraction, the separation
efficiency (SE) of the process is 100 %. To achieve a separation efficiency of 90 % or
higher, solvent fractionation needs to be used whereas dry fractionation leads to a SE
of max. 60- 70 % (Harris, 2014) .

Figure 2. 20 : Schematically depicted fractionation of a slu rry into stearin (solid fraction with entrained
olein) and olein (liquid fraction).
feed
(slur r y of stearin and olein)
stearin
solid
olein in stearin
olein

41
In Figure 2. 20 fractionation is schematically shown. The yield of the fractionation
is given by
𝐲𝐢𝐞𝐥𝐝 𝐬𝐭𝐞𝐚𝐫𝐢𝐧 = 𝐦 𝐬𝐭𝐞𝐚𝐫𝐢𝐧
𝐦 𝐟𝐞𝐞𝐝

Eq. 2.4

In Eq. 2.4, m stearin is the mass of the stearin fraction after separation and m feed the
mass of the slurry at the start of the process .
The separation efficiency (SE) is a measure ho w successfully the high melting
components (solids) were s eparated from the feed. It is expressed by the ratio of th e
mass of solid (m solid ) an d the mass of ste arin (Flöter, 2014b) .
𝐒𝐄 = 𝐦 𝐬𝐨𝐥𝐢𝐝
𝐦 𝐬𝐭𝐞𝐚𝐫𝐢𝐧

Eq. 2.5

With the solid fat co ntent of the feed
𝐒𝐅𝐂 𝐬𝐥𝐮𝐫𝐫𝐲 = 𝐦 𝐬𝐨𝐥𝐢𝐝
𝐦 𝐟𝐞𝐞𝐝

Eq. 2.6

the yield can be expre ssed by
𝐲𝐢𝐞𝐥𝐝 𝐬𝐭𝐞𝐚𝐫𝐢𝐧 = 𝐒𝐅𝐂 𝐬𝐥𝐮𝐫𝐫𝐲
𝐒𝐄

Eq. 2.7

Both parameters, separation efficiency and yield, are used to determine the quality
and the efficiency of a fractionation process. If the valuable fraction is stearin, the
entrainment of olein should be as low as possible and a maximum separation efficiency
is targeted. To increase the separation e fficiency , large cubic crystals are desirable to
facilitate the removal of olein out of stearin . Possibilities are the solvent or detergent
fractionation which incre ase the separation efficiency even furthe r (Flöter, 2014b).
In general, three different fractionation technologies (dry, solv ent and detergent)
are applied for oils. Special fractionation processes lik e supercritical carbon dioxide
extraction or molecular distillation are too expensive and not applicable for bulk oil and
are therefore not discus sed within t his work (Kellens and Calliauw, 201 3) .
All fractionation processes include four steps in common: melting, nucleation,
crystal growth, and separation. The oil is melted completely before nucleation is
initiated by controlled cooling. Nucleation and growth are done by specific temperature
regimes. Once the crystals have grown to the desired size, the crystallized material is
separated from the liqui d (Timms, 2006) .

42
In the dry fractionation technology, the fat is crystallized from the slurry without
any additives such as solvents or surfactants and subsequently filtere d. To increase the
yield the filter cake is additi onally sque ezed i n e.g. a membrane filter press (Kellens and
Calliauw, 2013) .
In solvent fractionation solvents such as hexane are used to dissolve the fat and
thus increase the separation efficiency (Harris, 2014). The presence of the solvent not
only generates a significantly increased volume of feedstock, it also reduces the
crystallization tempera ture significantly. This makes the process only usable for high
value fractions.
The third method is the detergent fractionation also known as Lanza or Lipofrac
method. Here , the crystals are emulsified in the aqueous phase by addition of a
detergent to the slurry prior to the se paration in a centr ifugal field.
The process parameters and the complexity of the process d epend on the desired
valuable fraction. This means, for instance, that different fractionation techniques are
required whether a low melting fraction or HUH TAGs are the desired fract ion.
(Hamm, 1986). In addition, the separation of trisaturate d TAGs (HHH) from
triunsaturated TAGs ( UUU ) is less complicated because they are not soluble in each
other. More complicated is the separation of H UH and HHU TAGs from each other
because they have both similar melting points and the solubility in the solid phase is
about the same. The entrainment of liquid oil (m ainly UUU and HUU) in the resulting
filter cake made of the solid fraction (mainly HHH or HUH) and determine h e
separation e fficiency. There are two possible mechanisms responsible for entrainment ,
the inter- and intra-particle entrainment of the liquid phase. The first on e ref ers to the
entrainment between crystal aggregates, whereas the second option relates to the
entrapment of oil in s ide the crys tal aggregates (Hamm, 1986) .
Dry fractionation is the cheapest fractionation technique whereas solvent and
detergent fractionation show higher separation efficiencies. However, both are afflicted
with high costs, diminishing consumer acceptance, and hazardous working conditions.
Nevertheless, all three mentioned fractionation me thods ar e still of importance and are
therefore explained in de tail in the following c hapter .

43
2.3.3.1 Dry Fractio nation
Palm oil is often fractionated using the discontinuous dry frac tionation process
which is composed of two main steps, crystallizat ion and separation. T he first ste p can
be described as a fract ional crys tallization based on the distinctive melting points of the
different fract ions. This means that at a certain temperature, one fraction is already
crystallized while the other is still in the liquid state. This invention is attributed to
Hippolyte Mége-Mouriés who tried to separate different fractions from tallow to
produce cheaper butter. In addition, traders and sailors, who imported palm oil,
observed that natural fractional crystallization happened on the ship. In this way, the
slight shaking helped the denser crystals to settle yielding a more cold resistant oil
behind on t op of the crystallized mate rial in the barrels (Kellens an d Calliauw, 2013) .
Supercooling is necessary to initiate the crystallization, which can also lead to the
attachment of undesired lower melting material onto the crystal surface yielding a
poorer se paration eff iciency (Stöver, Eggers and Stein, 198 3). Ng and Oh (1994) found
that the high melting fraction of palm oil needs to undergo a supercooling of 22 °C
before crystallizing. In contrast, a lower supercooling was necessary for the low melting
fraction, probably because the high melting fraction crystals acted as seed crystals. In
addition, during crystallization transformation from one polymorphic form into
another can occur which causes re lease of the late nt heat. The latter is linked to a local
temperature increa se which can st op the crys tallization for a short time which then
results in an increase in supersat uration (Stöver, Eggers and Stein, 1983).
In the following section, the process shown in Figure 2. 21 is explained in detail.
The feed slurry is melted in the feed tank until no crystals remain. Subsequently, the
melt is cooled down in the crystallizers to the temperature at which the high melting
fraction of the mixture crystallizes. This step is, so far, a batch process. The cooling
rate is crucial for the final separation efficiency because it impacts the size of the crystals
and the amount of liquid oil in corporated into them. The higher the cooling rate, the
smaller the formed crystals which are on the on e hand accompanied with a lower
amount of incorporated oil but on the other hand have a relatively large surface at
which liquid oil can attach. In contrast, a slower cooling lea ds to less nuclei resulting in
larger crystals (Pe tersson, Anjou a nd Sandström, 1985) .

44

Figure 2. 21 : Schematic se tup of a dry fractionation process with a continuous (top) and a batch
(bottom) separation (modifi ed, Calliauw 2017).

The crystal size and shape are important for both, the final product application as
well as the fractionation performance . While in prod uct applications, s mall crystals are
desired due to their flexible structuring properties, for a good fractionation
performance, large crystals with a low tendency to bind liquid oil are desirable resulting
in a less porous filter cake (Stöver, Eggers and Stein, 1983 ; Bot and Flöter, 2013). The
challenge is, hence, to find an adequ ate balance between the size of the crystals and the
amount of oil remaining in the crystals. The generation of larger c rystals is done by
increasing the temperature after generating enough nuclei to exclusively promote crystal
growth instead of t he formation of further nuclei (Flöter, 2014a) .
In other words, t he supersaturation is changed stepwise. Thereby, it needs to be
large enough to create a dri ving force for the crystallization of the desired crystals, bu t
should not be too large so that undesired c rystal formation an d growth can be
prevented (Kellens and Calliauw, 2013). In addition, the viscosity of the slurry should
stay moderate during the increase of the amount of c rystallized material to avoid
slowing down the proc ess too strongly (Calliauw et al. , 2010).

45
The formed slurry consists of the crystallize d high melt ing fraction and the still
liquid low melting frac tion. It is kept in a buffer tank until it is separated in the second
step, the separation (Calliau w, 2014). The separation step was developed and enha nced
from vacuum belt to membrane press filters or centrifuges (Timms, 2006; Kellens and
Calliauw, 2013) . In the batch proces s, after the first filtration a membrane filter press is
used to squee ze the filter cake so that the stearin (high m elting fraction) releases as
much of the liquid olein (low melting frac tion) as possible . For the continuous process
either vacuum filters or decanter centrifuges are used for the separation of stearin and
olein. However, the continuous process is rarely used in the industry.
In palm oil dry fractionation, two ways of generating a palm mid fraction (PMF)
can be applied. Either the olein is generated in the first step and separated from the
stearin which is further fractio nated or the stearin is removed in the first step leavin g
the olein fraction for further fractionation. The advantage of the first approach is that
the high melting TAGs of the stearin help to nucleate the fractions in both steps and
the PMF is ob tained mo re easily (Talbot, Smith and Cain, 2006) . In addition, during
the fractionation of stearin, also a hard stearin fraction is generated with a sharp melting
range. While for dry fractionation two steps are required to obtain hard stearin for
solvent fractionation only one step is nec essary.
One disadvantage of the dry fractionation is its usually batch-wise operation.
Moreover, dry fractionation shows a lower separation efficiency and precision with
respect to the se paration of the fract ions compared to other methods. The precision is
rather low due to liquid sticking onto crystals or liquid being trapped in agglomerates
so that it is hard to separat e the olein from the ste arin (Stöver, Eggers and Stein, 1983).
Further, the kinetics of the crystallization is rather slow. The crystallization time
mainly determin ing the processing time ranges from 5 hours to 3 days (Kellens and
Calliauw, 2 013). Such a long processing time is one of the other main disa dvantages of
the dry fractionation process.
2.3.3.2 Solvent Fra ctionation
In the proces s of solvent fractionat ion, solve nts like ace tone or hexane are used to
increase the separation efficiency. The solvent addition to the feedstock leads to a
variation of the solubility of the TAGs and a very low-viscous filtrate. Moreover,

46
solvent fractionation often generates a higher separation efficiency compared to dr y
fractionation because the stearin filter cake can be washed with solvent purging the
solid from the trapped olein (Stöve r, Eggers and Stein, 1983; Kellens and Call iauw,
2013).
Like dry fractionation, the solve nt fractionation process is comp osed of the
aforementioned two steps crystallization and separation. Now, the crystallization
happens in a solvent like acetone or hexane . These components are used as solvents
due to the high solubility of minor and major components and their low heat of
evaporation leading t o less energy required for the solvent re covery (Harris, 2014) .
Due to the addition of solv ents, the crystallization temperat ure decreases which
leads to a viscosity increase (Vanhoutte et al. , 2003) . However, the solvent present in
the liquid phase (e.g. acetone with 0.3 mPa) reduces the viscosity of oils which ranges
at relevant fractionation temperatu re from 30 -100 mPa when no solvents are present.
The subsequent increased molecular diffusion leads to faster crystallization (30 min in
solvent fractionation) and possibly even to a continuous crystallizatio n (Harris, 2014) .
The lower viscosity helps during filtration because it limits the entrainment of liquid oil
in the filter cake and this leads to an increase of the separation efficiency (Timms, 1997;
Kellens et al. , 2007; Harris, 2014) . To sum up, solvent fractionation generally yields a
cleaner product resulting in a sharp melting stearin, a higher stearin yield as well as a
higher process efficiency due to a one step process, and fast crystallization (Harris,
2014) .
As mentioned, one of the most important process steps in fractionation technology
is the nucleation of fat crystals. Therefore, the metastable zone, needs to be considered.
This degree of supersaturation without substantial nucleation is practically the
difference betwee n the temperatures at the cloud point, where some nuclei are already
visible, and the clear point (Talbot, Smith and Cain, 2006) . Another important factor
to consider are the shape and size of the crystals correlating with the washing of the
liquid fraction from the solid crystals. The macroscopic appearance of the crystals can
be modi fied by adjustment of the cr ystallization conditions (Vanhoutte et al. , 2003). For
palm oil fractionation, hexane and acetone show the same selectivity. However, better
olein qualities are achieved, if hexane is used as a solvent. In contrast, acetone should
be used if the palm mid fraction is rich in POP (T imms, 2006).

47
The remaining lower melting TAGs can either be entrapped in the crystals or
remain in t he bulk oi l (Timms, 2006). The removal of the liquid from t he solid cr ystals
can be critical in solve nt frac tionation. Crys tals generally tend to entrain more liquid oil
during solvent fractionation compared to dry fractionation. Better separation is
achieved by washing the stearin with fresh solvent after filtration . This leads to the
removal of the remaining liquid oil, which is a mixture of oil a nd s olvent, yielding 90 %
SE. The solvent needs to be fully recovered. This particularly means the decrease to a
level of 100-200 ppm in the low m elting product. Moreover, the slurry must be further
cooled down. All these aspects lead to a significant increase of the pro duction c osts in
comparison to the dry fractionation. Consequently, the higher separation efficiency an d
yield of solvent fractionation can compensate the higher costs on ly for high value
products (Hamm, 1986). Furthermore, solv ent fractionation plants po se a substantial
higher hazard potential for operators and the environment. Hence, the use of solvent
fractionation is only economical if a high separation efficiency is necessary such as for
specialty fats like c ocoa butter equivalents or replacer s (Stöver, Egg ers and Ste in, 1983;
Timms, 1997; Kellens et al. , 2007; B ot and Flöter, 2013; Harris, 2014).
2.3.3.3 Detergent Fractionation
The detergent fractionation, which is also named Lipofrac or Lonza method, is
composed of a crystallization step identical to the dry fractionation procedure, but
additionally followed by the usage of a detergent solution an d a separation in a
centrifuge (Stöver, Eggers and Stein, 1983; Timms, 2005) . It can thus be assigned to
the dry fractionation technologies (Hamm, 1986). The process is based on emulsion
technology because a dete rgent solution com posed of wat er and surfa ctants or we tting
agents is used (D effense, Tirt iaux and Charleroi, 1985) .
After crystallizing the f eedstock, the added det ergent suspends the already formed
crystals in the aqueous solution and hence entrain them from the lipi d phase. The
density difference between the a queous detergent solution with the entrained fat
crystals and the lower melting liquid oil fraction makes the separation easier using
centrifugal force (Timms, 2005). Normally, a wetting agent lik e sodium lauryl sulfate
with an electrolyte (e.g. magnesium sulfate) is used during this process (Kellens et al. ,

48
2007). The high costs of the used detergent solution and its recovery are reasons why
the detergent process is at present practically n ot applied in the fractionation i ndustry.
2.3.3.4 Compariso n of Fractionation Tec hnologies
In Table 2.2, a summary of different parameters concerning the dry, solv ent and
detergent fractionation is given. It shows that dry fractionation has the lowest
separation efficiency but the lowest costs. In addition, it is not as hazardous as solvent
fractionation and produces less waste than the other two fractionation technologies.
Both fractionation techniques, in which crystallization is performed from the molten
lipid phase (dry and detergent fractionati on), show higher entrainment compared to
solvent fractionation, which includes a washing step with solvent (Hamm, 1986) .
However, the hazard and costs make this process less pop ular. In contra st, the cheaper
and environmentally more friendly process of dry fractionation has a separation
efficiency inferior to solvent and detergent fractionation. This limits the functionality
of the fat phases produced by dry fractionation. Thus, there is a demand for a new
fractionation technique, which yields high separation efficiencies with out the ex cessive
use of time, supplementary material or energy. The proposed ne w approach for the
fractionation of edible f ats is key to this thesis and is subsequ ently discussed in det ail.

49
Table 2.2 : Compa rison of c osts, yield, efficiency, advantages and disadv antages of the three
fractionation methods dry, solvent a nd detergent.

Dry

Solvent

Detergent

Costs

lowest 9

highest 9

medium 9

Duration

5 h – 3 d 1
10 h 3

30 min cryst. 4

3-4 h 7

Stearin yield
(%)

17 2
40 6
20 - 21 10

10 - 11 2
37 - 40 10

20 6
17 - 23 10

IV Stearin

32 2
46.7 6

8 2

24.9 6

Entrainment of
oil (% of
stearin)

30 2
47 - 50 10

5 2

35 - 52 10

Liquid to solid
ratio (L/S)

0.5-1.0 (press
filtration) 9
> 1.0 (vacuum
filtration) 9

< 0.5 9

0.5-1.0 9

Advantages

- cheap 9

- high selectivity 5
- short
crystallization
time 8
- easier filtration
due to lower
viscosity 8
- easier heat
transfer 8
- less entrained oil 8

- cheaper than
solvent 5

Disadvantages

- long residence
time 1
- low separation
efficiency 10

- high energy
costs 5
- high material
costs 5
- safety issues 1
- consumer
acceptance 1

- chemical costs 5
- recovery of
polluted water 5
- consumer
acceptance 1

1 (Kellens and Calliauw, 2013) 2 (Timms, 2006) 3 (Stöv er, Eggers and Stein, 1983) 4 (Harris, 2014)
5 (Ricci-Rossi and Deffense, 1984) 6 (Deffense, Tirtiaux and Charler oi, 1985) 7 (Cornelius, 1977) 8 (T imms,
2005) 9 (Hamm, 1986) 10 (Timms, 1997)

50
2.4 Emulsion Fra ctionation
A continuous frac tionation process woul d be an energe tically favorable and t hus a
desirable process. A possibility would be emulsion fract ionation which is, like all other
fractionation technique s, based on the two dist inct sub -processe s of crystallization and
separation. The combination of the abov e into a continuous process decreases the
inter-batch variability, improve energy efficiency and yield a more uniform crystal size
distribution (Kellens and Calliauw, 2013) . In such a process one starts with an emulsion.
On the interface between droplets and continuous liquid phase crystallization of the
respective fraction of fat occurs. Separation of these droplets with crystals from the
liquid phase would lead to the desired fraction. The combination of these processes
into one continuous process requires the knowledge about the respective underlying
pr ocess steps. Therefore, the theoretical background of the crystallization at the oil and
water interface along with the separation in a decanter centrifuge as well as the
systematic setup idea of the emulsion fractionation technology are given in the
following sub-chapters .
2.4.1 Emulsions and Pickeri ng Stabilization
An emulsion is a thermodynamic unstable t wo-phase system composed of a
continuous phase and a di spersed liquid phase. Two different types of emulsions are
relevant for the food industry. The first one is water-continuous with oil droplets as the
dispersed phase (O/W emulsion). The second one is oil-continuous with dispersed
water droplets (W/O emulsion). These emulsions are stabilized either by the addition
of an em ulsifier (Figur e 2. 22 a and b), by Pickering, e. g. the stabilization of the dr oplets
due to parti cles on the droplet surface (Figure 2. 22 c) or a combinat ion of both ( Figure
2. 22 d).
Emulsifiers are usually amphiphilic molecules characterized by a lipophilic and a
hydrophilic part. These molecules arrange themselves in a way that the lip ophilic tail
reaches into the oil phase whi le the hydrophilic head is in contact with the water phase.
These molecules decrease the surface tension between the oil and the aqueous phase,
thus, generate the emulsion with droplets surrounded by surfactants . A potential
prolongation of the stability of an emulsion depends on the type of emulsifier and in

51
particular on the steric hindrance of the droplets avoiding coalesc ence . Emulsifiers can
be classified by their so -called HLB-value (hydrophil ic -lipophilic -balance). The
HLB-value describes the affinity of the emulsifier to stabilize O/W (8-15) or W/O
(3 -8) emulsions. Emulsifiers with HLB-values lower 3 show anti-foaming p ropertie s.
Emulsifiers with HLB-values higher than 15 are used to enhance solubilizing
(Schuchmann and Köhler, 2012) .

Figure 2. 22 : Different types o f emulsion: a) oil - in -water emulsion stabilized by emulsifie rs, b)
water- in -oil emulsion stabilized by emulsifiers, c) water - in -oil emulsion stabilized by fat cr ystals/
particles, d) water- in -oil emuls ion stabilized by emulsifiers and fat crystals/ particles.

There are different mechanisms for emulsion stabilization. For example, charged
moieties of emulsifiers lead to a repulsion of the droplets wherea s large moieties o f
emulsifiers stabilize an emulsion due to ste ric hindrance. This special case of emulsion
stabilization by particles is called Pickering stabilization. Pickering was the first one who
observed that solid particles can serve as stabilizing agent for emulsions (Pickering,
1907). Hence, emulsions stabilized in a steric manner by fat crystals after their
adsorption onto the interface are called Pickering emulsions and are important in f ood
products like mar garine, mayonnaise or ice cream (Pawlik et al. , 201 6) .
In the case of the aforementioned food products, fat cry stals are the stabilizing
agents as s hown in Figur e 2. 22 c. In cosmetic and pharmaceu tical products, other
particles can also be used to stabilize emulsions. The particles at the interface in a
Pickering emulsion lead to less coalescence and a decreased mass transfer of
components from inside the droplets to the surrounding liquid phase due to their
function as barrier or shell at the inter face (Pawlik et al. , 2016) .

52
An oil-continuous matrix in which the emulsion droplets are stabilized by fat
crystals is i mportant for many food products . Depending on the nature of the fat
crystals in combination with the continuous phase the emulsion is stabilized or
dissolved (Hodge and Rousseau, 2005). In a system with a water continuous phase, they
mostly lead to partial coalescence of the droplets. In contrast, at the interface fat crystal s
in oil continuous systems m ay serve as Pickering particles, thus having a stabilizing
effect. Due to adsorption of fat crystals onto the interface sedimentation, flocculati on,
and coalescence are avoided (Hodge and Ro usseau, 200 5) . Platelet shaped and small
crystals as well as crystals formed by in situ crystallization are known to increase th e
stability of W/O emulsions (Frasch-Melnik, Norton and Spyropoulos, 2010; Douaire
et al. , 2014).
The stabilizing effect of fat crystals can be enhanc ed by the addition of surfactants .
These increase the polarity of the naturally not amphiphilic fat crystals (Frasch-Melnik,
No rton and Spyropoulos, 2010). Moreover, crystallization directly at the interface can
be promoted by so -called templates, which lower the activation energy (see Figure
2. 22 d). These templates impact the crystal arrangement, whi ch in turn influences the
emulsion stability itself (Douaire et al. , 2014) . Improved stabilization was observed if
the hydrophobic part of the ap plied emulsifier has a similar length a nd structu re as the
TAGs in the oil (Awad, Hamada and Sato, 2001) . Furthermore, the addition of
emulsifiers increases the crystallization temperature (Douaire et al. , 2014) . This was also
fond for higher cry stal concentrations (Frasch-Melnik e t al., 2010) .
The addition of mono- and diglycerides with a higher melting point lead to a
crystalline interface ("shell") in W/O emulsions (Douaire et al. , 2014) . Additionally,
added saturated monoglycerides for example could serve both as surfactants and as
seed for TAGs to crystallize. Small crystals are preferred for the production of Pickering
emulsions because they give a high surface coverage of the droplets and by this a bette r
stabilization. Low crystal concentrations lead to less stable emulsions. The desired small
and numerous crystals are formed at high cooling rates, hence at larger driving force
for the in situ crystallization. In addition, the increased amount of available growth sites
at high cooling rates leads to a faster depletion of high melting components for crys tal
growth (Flöte r, 2014a) .

53
In addition to the faster nucleation and the higher amount of produced crystals,
added emulsifiers influence polymorphism due to templating. Due to present crystals
the creation of the in situ formed crystals is influenced. This control of the polymorphic
form can be crucial for the stability of emulsions , e.g. needle-shaped crystals ( β ) tend
to destabilize emulsions, whereas platelets improve the stabilization. Some authors
found that specific addit ives lead to specific pol ymorphic forms (D ouaire et al. , 2014) .
However, one needs to be careful because crystallization in emulsions differs from
bulk systems (see chapter 2.2). As mentioned above, in situ crystallization depends on
the emulsion type, the nucleation process, the lipid phase composition and the
molecular packaging. The crystallization of oil in an O/W emulsion requires more
supercooling (thus supersaturation) because the amount of impuritie s in the droplet s is
statistically low (Douaire et al. , 2014). Nevertheless, for this thesis, O/W-emulsions play
a minor role. The focus is on Picke ring W/O -emulsions, where the crystallization
mainly takes place on the water droplet su rface and not in the bulk. From this
sub -chapter, it can be concluded that the existence of an oil/water interface has the
potential to severely influence the TAG crystallization and that additives allow to
manipulate the nucleation process. In summary it can be said that the functionality of
the Pickering particles can be influenced by the continuous ph ase but also by the choice
of emulsifier (Pawlik et al. , 2016).
2.4.2 Decanter Centrifuge
The separation of two materials with different densities can be performed in a
centrifuge. If a continuous process is desired, a ho rizontal decanter centrifuge can be
used. The se are often used to clarify liquid , classify particles or to remove moisture
from solids (Gleiss and Nirschl, 2015). Decanters can be applied in a wide range of
concentration of solids (up to 60 %) and particle sizes (Beiser et al. , 2004) . In the oil
and fat industry, decanter centrifuges are so far used , for example, in the olive oil
industry to separate th e oil from the seeds after squeezing (Altieri, Di Renzo and
Genovese, 2013) . Here, the pro cess executed is a sedimentati on process under
increased driving forc es.

54
A decanter mainly consists of a bowl and a screw. A setup is schematically shown
in Figure 2. 23 . The bowl has a conical (right side, Figure 2. 23 ) and a cylindrical part
(left side, Figure 2 . 23 ). At the end of the cylindrical part in the middle of the decanter,
the feed is inserted. There exist various parameter s which influence the se paration in a
decanter. Concerning the material, the particle size of the dispersed ph ase as well as the
viscosity of the continuous phase influence s the process and also the amount of fee d.
Stokes law can be used to calculate the sedimentation velocity v of a particle p in a
continuous phase c :
𝐯 = 𝐠 ∙ (𝛒 𝐩 − 𝛒 𝐜 )
𝟏𝟖 𝛈 𝐜
∙ 𝐝 𝐩
𝟐

Eq. 2.8

This velocity is influenced by the density of the particles

p , the density

c and the
viscosity

c of the conti nuous phase, and the diame ter of the particles d p .
In addition, the g-force plays a decisive role and can be varied in the decanter by
changing the rotat ion speed of the bowl. In the used equipment ( Lemitec MD 80), the
g-force during the separation can be varied between 2 00 -3,000 g. Further parameters
are the differential speed between the screw and the bowl (1 -200 rpm) as well as the
length and the angle of the drying section (both constant in the used device). The
retention time of the liquid also influences the separation of the material and can be
adjusted by the vol ume flow into the decanter and the filli ng volume of the device. The
latter can be changed by the addition of different weirs, which increase the filling level.

Figure 2. 23 : Lab scale decanter (Lemitec MD 80) with an inlet (left), an exit f or the liquid phase (left,
bottom), an exit for the solids (right, bottom) and a weir to adjust the filling level (s mall grey dash).

55
The conical part of the decanter is necessary to dry the separated soli ds . The drying
zone prior to the ejection of the material is visualized i n Figure 2. 24 . The length of
both, the drying and the clarifying zone can be theoretically adjust ed by the height of
the weir, ill ustrated with the yellow arrows in Figure 2. 24 . If the weir inner diameter is
decreased and thus the height of the weir increases, the amo unt of liquid in the decanter
and hence the filling level increases. This leads to a short er drying zone at the conical
end of the decanter in which the solids are transported by the inner screw. This
movement is generated because the bowl and the screw rotate at different speeds
(1 -200 rpm in the case of a Lemitec MD 80).

Figure 2. 24 : Working principle of a decanter with a clarifying zone (left, red lin es and arrows) and a
drying zone (right, yellow line and arr ows).

In this thesis, a lab scale decanter built by the company Lemitec was used for the
emulsion fractionation . In contrast to an industrial decanter (the largest conveying
250, 000 l/h (Beiser et al. , 2004)), the lab scale decanter (see in Figure 2. 25 ) has a
comparatively low t hroughput of approximately 40 l/h ena bling small-scale
experiments.

56

Figure 2. 25 : Lab scale decanter (Lemitec M D 80)
2.4.3 Concept of Emulsion Fract ionation
The conceptual setup of the continuous emulsion fractionation process is depicted
in Figure 2. 26 . The basic idea is to continuously generate a Pickering emulsion by
sending cold water droplets into a hot oil stream. This process initiates the
crystallization of the high melting fat fraction at the wate r droplet surface (step 1). The
resulting slurry, a Pickering emulsion, water droplets (blue dots after s tep 1) stabilized
by fat crystals (orange dashes) disperse d in liquid oil (yellow bac kground).

Figure 2. 26 : Theoretical setup of the c ontinuous emul sion fractionation process; 1) crystallization of
fat at water droplet surface, 2) separation of high and low melting fraction in decanter, 3) separation of
high melting fraction and water in decanter, 4) recycling of water, 5a) yielded low melting fractio n
(olein), 5b) yielded high melting fra ction (stearin)

57
In step 2, th e separation in the decanter centrifuge takes place. Due to density
differences, the denser water droplets carrying a high melting fat c rystal shell sediment
out of the oil phase . The density of the oil is approximate ly 0.92 g/cm 3 (Coupland and
McClements, 1997 ), the density of the water droplets is 1. 00 g/cm 3 (Coupland and
McClements, 1997) and the density of fat crystals is stated to lay between 1.02 g/cm 3
(Sato et al. , 2001) and >1.04 g/cm 3 (Bailey and Singleton, 194 5). During this process
step, olein whi ch is the low melting fraction exits the decanter as the liquid fraction
(step 5 a). After the separation o f the olein from the slurry, the water droplet s with
attached fat crystals are heated up until the fat melts. Subseque ntly, the resulting
undefined emulsion is separated into an aqueous and a lipid phase by means of a
centrifuge (step 3). In this step the second product, the high melting frac tion ste arin, is
obtained (step 5b). The outlined process allows to reuse the water (step 4) and optimize
energy consumption by recovering thermal e nergy from the streams generate d.
The ambition of the outlined emulsion fractionation process is to oper ate
continuously at low conversion costs compared to solvent or detergent fractionation .
It is further aiming at selective crystallization by the choice of the appropriate
crystallization conditions, exclusive use of water as entrainer, and high separation
efficiencies.

3 E XPERIMENTA L M ETHODS

59
This chapter is dedi cated to the main experimental methods use d in this thesis.
Both the b asic measurement principle an d the used procedur es are outlined for
differential scanning ca lorimetry, temperature modulated op tical refractometry, gas
chromatography, nuclear magnet ic resonance, polarized light microscopy, and powder
X-ray diffraction. In addition, their relevance and application in the field of fat
technology are highlig hted.
3.1 Differential Scanni ng Calorimetry
Phase transitions like melting and crystallization as well as glass transitions can be
determined by differential scanning calorimetry (DSC). Thus, it is a wid ely applied
technique in the food industry (Menard and Sichina, 2000). This subchapter gives an
overview of its basic pri nciple and its m ain applications in the area of fat technology.
3.1.1 Principle of determ ination
The DSC method is based upon the differences in the thermal conductivity of tw o
samples. The setup is shown in Figure 3.1, where a reference and a sample pan are
placed in a temperat ure controlled cabinet. During heating or cooling, the difference in
the vol tage between the two pans is recorded by the instrument and converted into
temperature di fferences . The sensors are located at positions for the reference and
sample crucible.

Figure 3.1 : Schematic setup of a heat -flux DSC with a reference pan and a sample p an for the
determination of the voltage di fference  V.

60
The temperature difference is converted into a thermogram where the heat flow is
depicted as a function of temperature. This conversion is shown in Figure 3.2. where
the temperature of the reference pan, indicated by the grey lin e, increases linearly over
time. In co ntrast, the temperature recorded for the sample pan (bla ck line) shows a
plateau starting at time t 1 . At that time, a phase transition commences in the sample
which means that the thermal energy is either absorbed (melting) or emitted
(crystallization) by the sample but not used to heat or cool the pan. Thus , the
temperature stays constant until t he phase transition is completed and t he temperatu re
increases again linearly .

Figure 3.2 : Experimental data of a heat flux DSC; upper graph: thermocouple voltage (proportiona l
to temperatur e) plotted against tim e, sample signal (black line) and reference signal (grey line); l ower
graph: converted voltage signal into temperature difference of the refe rence and the sample (modified,
Netzsch 2017).

The detected temperature difference is converted into peaks which are plotted
against the temperature, the so called thermograms. Two possible resulting schematic
thermograms are depicted in Figure 3.3. A phase diagram of a system constituted of a
high and a low melting component is given. The red, dashed line shows the liquid to
solid transition of a mixture with an overall composition of x a . The area between the
solidus and the liquidus line describes the mixture of a solid and a liquid phase . There ,
the low melting comp onent of the mixture in its pure state would already be liquid

61
while the high melting one would still be solid. The width of this two-phase region
(with respect to temperature) decreases approaching the composition of the pure
components. For a pure component, the resulting melting peak (red solid curve) is
almost discrete, meaning that the transition from solid to liquid occurs at one distinct
temperature. For a mixture, a broader me lting peak is ob tained because at each
temperature two phases (solid and liquid) of different compositions exist. Only after
the liquid phase has reached the overall composition x a the phase transition has been
completed.

Figure 3.3 : Phase diagram of a two -component mixture with schematic DSC thermograms of a
mixture (red, dashed line) and of a pur e component (red, solid line).

Hence, the composition of the mixture influences the shape of the peak.
Conversely, the shape of the peak can be used to characterize a sample. Moreover,
parameters like the peak temperature (transition temperature) as well as the onset and
offset temperatu re of the phase transition are helpful parameters for the
characterization of a mi xture. The onset temper ature of a phase tra nsition in a mixture
can be considered as the crossing of the solidus line into the two-phase region.
Subsequently, the crossin g of the liquidus line can be related to the offset temperature
of the transition. In addition to the different characteristic temperatures, the heat of
fusion (  H) can be determined for not too complex systems by the area under the peak.
Since the heat of fusion depends on the mass of t he sample, the mass needs to be

62
determined as exactly as possible. The determination of all four parameters is shown in
the schematic t hermogram for a cry stallization and a melting process in Figure 3.4 . An
important term for this data processing is the baseline. The zero baseline is based on
the equal heating of the empty reference pan and the sample pan without a phase
change. A proper baseline is required to calculate the enthalpy of fusion. In additi on,
the intersection of the extension o f the baseline and the tangents of the peak (grey
dashed lines in Figure 3.4) are necessary to de termine the onset and offset of phase
transitions.

Figure 3.4 : DSC thermogram o f crystallization and melting; T on = onset temp erature, T peak = peak
temperature T off = offset temperature, c = crystallization, m = melting.

Commonly, a positive peak in a DSC thermogram refers to a crystallization process
which is an exothermic process (release of heat). A melting process is an endothermic
process showing a nega tive peak in the DSC t hermogram because of the negative heat
flow due to the uptake of heat.
Besides the composition, also the scan rate influences the width of the peak. For
example, a too high cooling rate during the crystallization of a pure component would
lead to a temperature lag in the sample behind the temperature in the syste m. Thus, the
peak becomes broader and the transition temperature is found at a higher crystallization

63
temperature than the actual equilibrium temperature. To decrease the influence of the
thermal lag a small scan rate would be desirable. This also leads to a higher resolution.
However, while enhancing the sens itivity, a small scan rate may also incr ease the noise
(Chiu and Prenner, 2011) . Hence, a good balance between resolution and sensitivity
needs to be foun d for DSC measurements .
3.1.2 Application in the fi eld of fat technology
DSC is applied widely i n the field of fat technology to determine phase transitions.
Fats and o ils are com posed of a mixture of various TAGs. This can make the
interpretation of the DSC thermog rams more complicated but if the method is applie d
with care the data can be used to characterize different fats and oils . The received
experimental data is influenc ed by both, the chemical compositi on of the fat as well as
the applied parame ters (scan rate, sample mass).
Considering the composition of fats, it can be said that fats with high amounts of
saturated fatty acids need more energy to be melted wh ich results i n a higher specific
melting enthalpy. However, one must be careful determining the enthalpy from DSC
thermograms because the baseline may differ before and after the phase transition
(peak) (T an and Che Man, 2002b). In addition, as mentioned above, the heat of fusion
is an extensive property whi ch makes it necessary to determine the weight of the sample
properly.
Hence, to gain proper data, the sample mass needs to be determined precisely i n
advance since it has an impact on the resulting thermograms and the determined
transition temperatures. Saeed and coworkers (2016) showed that the specific melting
enthalpy increases slightly with increasing sample mass. The determination of the
melting temperatu re was affected at lower sample masses (< 5 mg) while no effec t was
seen at larger sample masses (> 5 mg). Therefore before comparing data, it is important
to always weigh in the s amples in the sa me weight range.
In addition, Saeed and coworkers ( 2016) presented that the sc an rate influences the
peak temperature and the specific melting enthalpy for mainly pure components. The
same was found by Tan and Che Man (2002), who showed the effect of the scanning
rate on the thermal profile of different fats. They examined four scanning rates (1 , 5,
10, 20 °C/min) from 80 °C to - 80 °C and back to 80 °C. High scanning rates resulted

64
in a smooth melting curve with less peaks than lower rates. This may lead to a loss of
information. Furthermore, high scanning rates resulted in an increase of the determined
melting point and the computed area under the peak. Also Vanden Poel and Mathot
(2006) found that at high cooling rates the crystallization temperature is shifted towards
lower temperature s while high heating rates lead to a shift of the determined melting
temperature to higher temperatures. In addition, the offset temperature can be affected
by the scanning rate shifting to higher values with increasing scanning rate (Tan and
Che Man, 2002b). Consequently, it might be interesting prior to analysis to investigate
comparable samples with diffe rent scan rates to identify the best sc an rate for the given
investigation.
Besides the peak, onset and offset temperatures, also the number of peaks can
change with the scanning rate. Often more than one endotherm ic peak occurs during
melting which can be expl ained by the variet y of composition in TAGs (SSS, SUS,
SUU, UUU) or the appe arance of polymorphism (melting-recrystallization). Overall it
can be said that the complexity increases with the number of different TAGs
comprising the mixt ure (Tan and Che Man, 2002b).
DSC should be use d with caution t o differentiate polymorphic forms. More peaks
could be visible at slow scanning rat es because the TAGs have more time to rearrange.
This leads to the conclusion, that the scan rate takes a big influence on the
determination of the cr ystallization behavior of TAGs. The higher the scanning rate
the lower the determined exothermic peak temperature. Furthermore, the slow
scanning rate gives the TAGs m ore time to re -organize whi ch results in more
interactions and thus le ads to co-crystallizati on (Tan and Che Man, 2002b) .
3.1.3 Procedures
To determine the crystallization and melting behavior a device from Netzsch (DSC
204 F1 Phoenix, NETZSCH-Geraetebau GmbH, Selb, Germany) was used. 5 - 10 mg
of sample was weighed into aluminum pans. Then, the samples in the DSC pans were
molten by increasing th e temperature to 80 °C and holding it there for 10 min.

65
Afterwards the samples were cooled at defined rates (2 °C/min, 5 °C/min or
10 °C/min) to the desired stabilization temperature, which varied between - 50 °C and
5 °C. After a defined crystallization time (between 10 min at - 50 °C and 30 min at 5 °C,
respectively) the samples were heated to 80 °C again at predefine d rates (2 °C/min,
5 °C/min or 10 °C /min) to determine the melting profile.
In addition to the determination of the phase transitions, DSC data received at a
scan rate of 5 °C/min were also used to calculate the solid fat index (SFI). Therefore,
the area under the pea k was subdivided and partially integrated. The r elation betwee n
the partial area and the complete area under the peak was d efined as the amount of
solids at the specific temperature (Bentz and Breidenbach, 1969; Nassu and Guaraldo
Gonçalves, 1995). All experiments were at least performed in duplicate. The analysis of
all thermograms was performed with the NETZSCH Prote us – Thermal Analysis
software version 6.1.0 provided by the ma nufacturer.
3.2 Temperature Modu lated Optical Refracto metry
The measurem ent of phase transitions is cruc ial in fat technology to determine e.g.
organoleptic properties. The standard method is the well-established DSC which is
described above. Alternatively, the temperature modulated optical refractometry
(TMOR) could be applied. It uses the difference in the refractive index n between a
solid and a liquid phase to compute the phase transition temperature. Its basics like
refractometry and temperature modulation are explained in the following chapter al ong
with the principle of TMO R and its previous applicat ions apart from fat cr ystallization.
3.2.1 Refractometry
The refractive index n of a substance or component is its property to diffract light
and is expressed as the relationship between the propagation speed of light in the
medium c m in comparison to vacuum c 0 (se e Eq. 3.1). The refractive index is also called
optical density and, hence, a denser material leads to a high er n .
𝐧 = 𝐜 𝟎
𝐜 𝐦

Eq. 3.1

66
The difference in the refractive ind ices of two materials is a result of their distinct
refraction of ligh t. In Figure 3.5, an incident ray is goin g through medium 1 with the
refractive index n 1 . At the interface between the two media, the beam is partly reflected
(with the same angle as it is impinge d upon the i nterface

1 ) and partly refracted. The
angle of the refracted ray

2 depends on the refractive index n 2 of medium 2. The
so -called Snell ´ s law g ives this relation betwee n the refrac tive indices and the angl es of
the beam (Carlton, 2 011) :
𝐧 𝟏 ∙ 𝐬𝐢𝐧 𝛉 𝟏 = 𝐧 𝟐 ∙ 𝐬𝐢𝐧 𝛉 𝟐

Eq. 3.2

Figure 3.5 : Refractive index n 1 of medium 1 and n 2 of medium 2 (modified, Carlton 2011 ).

The determination of the refrac tive index is temperatu re dependent and quite easy
if a pure component is investigated. A mixture of two components makes the
determination of the refractive index more complex. However, it migh t be possible to
calculate the ratio of tw o miscible components with different r efractive indices from
the resulting r efractive index. The change of t he refractive index o ver the c omposition
range is based on the mixing rule. Hence, th e amount of each comp onent can be
calculated as indicat ed in Figure 3.6 .

67
For the investigations of fat, both isothermal and dyn amic measurements are
helpful. In addition, a robust and convenient device would be desired if the method
should be applied in practice e.g. in a factory. Therefore, the application of a
refractometer with additional temperature m odulation was investigated to determine
the crystallization and melting behavior of fats and alky l components.

Figure 3.6 : Mixing rule of a binary sys tem com posed of two miscible components with distinct
refractive indices.
3.2.2 Fundamentals of the Temperature Modulate d Optical Refractomet ry
The total reflection is required to determine the re fractive ind ex of a solid or a
turbid liquid sample. To generate a total reflection of the beam, the incident ray needs
to enter the sample with an angle larger than the critical angle of total reflection

c (se e
Figure 3.7 ).
In addition to basic refractometry, the temperature modulated optical
refractometry (TMOR) provides more information due to an additional applied
temperature undulation. The temperature of the prism is precisely co ntrolled by a
Peltier element. Prior to the measurement, a period (in seconds) and an amplitude
(in °C) of the undulation are set. T he equations given in this sub-chapter to enhance
the understanding of T MOR are taken from M üller et al. (2013).

68

Figure 3.7 : Total refl ection of a beam: at a critical angle of the incident ray (  c ) the beam is reflected
at the in terface between medium 1 and 2 (red arrows ), rays wi th a n entrance angl e larger than  c are
completely reflected (  1 , black arrows).

The basis for TMOR is the correlation between the refractive index n and the mass
density of a substance

. Both are related to the specific refractivity r , which is the molar
refractivity of a component divided by its density. T his is expressed by the
Lorentz-Lorenz relation:
𝐧 𝟐 − 𝟏
𝐧 𝟐 + 𝟐 = 𝐫 ∙ 𝛒

Eq. 3.3

The relationship between the density and the refractive index allows that the
refractive index can be used to determine the volume expansion coefficient

. It is
calculated from the r efractive index n and its ch ange over temperature T ( 𝛹 = 𝑑𝑛
𝑑𝑇 ):
𝛂 = −𝟔𝐧
(𝐧 𝟐 + 𝟐)(𝐧 𝟐 − 𝟏) ∙ 𝚿

Eq. 3.4

To obtain data on phase transitions with only the determination of the refractive
index and its dynamic temperatu re derivative, the linear response theor y (LRT) is use d .
It relates the temperature perturbations to the variations of the refractive index of a
sample . The determination of the thermo-optical properties of a sample 𝛹 𝜔
∗ makes a
sinusoidal perturbation of the temperature with a suitable amplitude 𝐴 𝑇 ,𝜔 necessary.
The amplitude needs to be small so that LRT can be applied because a linear response
is crucial for the modulat ion (Müller e t al. 2013).

69
Additionally, the phase shift at a given ang ular fre quency 𝛷 𝜔 and the static
equilibrium refractive index 𝑛 0 need to be considered for the calc ulation of the
frequency dependent re fractive index
| 𝐧 𝛚
∗ (𝐭) | = 𝐧 𝟎 + | 𝚿 𝛚
∗ | ∙ 𝐀 𝐓,𝛚 ∙ 𝐬𝐢𝐧 ( 𝛚𝐭 − 𝚽 𝛚 )

Eq. 3.5

The applied sinusoidal temperature modulation results in a delayed but also
sinusoidal answer of th e refractive index ( see Figure 3.8 ).

Figure 3.8 : Modulation of TMOR: undulated temper ature (black solid curve) and the answer of the
refractive index (green solid line) delayed by the phase shift  (modified, Müller et al. 2013).

TMOR delivers reliable values for 𝛹 and 𝑛 0 (average refractive index in one
modulation period ). These are u sed to calculate the co mplex volume expansion
coefficient 𝛼 𝜔
∗ with Eq. 3.6, which is based on
| 𝛂 𝛚
∗ | = −𝟔𝐧 𝟎
(𝐧 𝟎
𝟐 + 𝟐)(𝐧 𝟎
𝟐 − 𝟏) ∙ | 𝚿 𝛚
∗ |

Eq. 3.6

The phase shift 𝛷 caused by the delayed answer of the refractive index is used to
compute the re al and imaginary part of the volume ex pansion coefficient


𝛂 ′ ≡ 𝐑𝐞 ( 𝛂 𝛚
∗ ) = | 𝛂 𝛚
∗ | 𝐜𝐨𝐬 ( 𝚽 𝛚 )

Eq. 3.7

𝛂 ′′ ≡ 𝐈𝐦 ( 𝛂 𝛚
∗ ) = | 𝛂 𝛚
∗ | 𝐬𝐢𝐧 ( 𝚽 𝛚 )

Eq. 3.8

70
The computed complex volume expansion coefficient is ne eded for the calculation
of the real Re (

) and imaginary Im (

) part. The real and imaginary volume expansion
coefficient allows to identify transition temperatures such as glass transition or phase
transitions such as crys tallization or me lting. These phase transitions can either be glass
transitions or crys tallization and melting processes . The me lting behavior of a TAG
over temperatu re is schematically r epresented in Figure 3.9. The me an refractive index
decreases with increasing t emperature , thus it is higher in the solid state than in th e
liquid state. The phase transition temperature is indicated by peaks of the imaginary
thermal expansion coefficient ( Im (

) ). A peak of the real volume ex pansion coefficient
( Re (

) ) indicates a real phase transition such as crystallization or melting whereas a step
shows a glass transition. In the figure below, the mean refractive index shows a drop
and both Re (

) and Im (

) show peaks at the phase transiti on temperatures.

Figure 3.9 : Schematic melting process of a TAG determined by TMOR; mean refractive index ( n mean )
and real ( Re (  )) and imaginary (Im (  )) part of the volume expansion coefficient  over temperature.

The thermal volume expansion coefficient can also be determined over time, which
makes se nse if one temperature is chosen and t he undulation is perfor med around this
temperature. This quasi-isothermal measurement allows the determination of e.g. the
induction time of a glass transition or a crystallization . A ramp at a certain scan rate is
required for a dynamically determined t ransition temperature. With the dynamic

71
measurement the melting and crystallization behavior of fats and oils in the allowed
temperature range of TMOR could be investigated. Since the investigation of lipid
systems by refractometry is not new, the next subchapter shortly summarizes this
application area.
3.2.3 Refractometry in Fat Technology
Even more complex than the application of refractometry for single components
or TAGs is the investig ation of fats and oils. There is not only a two-component but a
multi-component system prese nt. The application of refractometry for fats and oils
needs to be performed with care. Not only the liquid phase influences the
determinati on of the refractive index for mixtures as occurring during fat crystallization
but also the solid one. The group of Kaufmann and Thieme (1954) investigated the
crystallization of fats, e.g. cocoa butter and coconut oil, with refractometry. The
investigation of the only solid phase was performed by crystallizing the sample on the
prism directly from the solution or by melting it on the prism . They f ound that even
though the sensitivity of the determination refractive index is less for solid than for
liquid phases, it is still possible. The refraction ind ex increases with increasing fatty acid
chain length and increasing amount of double bonds (Kaufmann and Thieme, 1954) .
In addition, they id entified a minimum stabilization time of about 15 min before a stable
value of the re fractive index was ac hieved (Kaufmann, T hieme and Wöhlert, 1955 ) .
They also found that the curve of the refractive indices as a fu nction of temperature
differed for distinct polymorphic forms of cocoa butter. This leads to the assumption
that also the solid ph ase of a fat mixture takes influence on the refractive index .
Furthermore, it seems that less stable solid phases have a higher refractive index
compared to more stable ones. This could be due to the fact that less stable crystal
modifications have a lo wer melting point. Hence at the same temperature, they contain
a higher liquid fraction compared to a more stable polymorph. Consequently, the
refractive index already decreases at a lower temperature. The transformation from one
polymorphic form into another is a zero order react ion whi ch means the rate of
transition is ind ependent of the previous present amount of polymorph ( Kaufmann
and Thieme, 1954 ; Kaufmann, Thieme and Wöhlert, 1955) .

72
Kaufmann and Thieme (1954) als o stated that the refractive index of mixtures lies
in between the refractive ind ex of the liquid and the one of the solid phase. Thus, this
refractive ind ex can be calculate d with the mixing rules. In addition they indicate that
the refractometer determines a geo metric mean of the refractive indices of the liquid
and the solid fraction (Kaufmann and Thieme, 1954). This relationship is depicted in
Figure 3. 10 . The measured refrac tive index can be related to a certain solid fat content
(SFC). Moreover, it can be plotted against temperature resulting in a melting curve
which is deter mined by the me asurement of the apparent re fractive index.

Figure 3. 10 : Refractive index n pl otted schematically against SFC (left) and temper ature (right).

The crystals of TAGs, fatty acids, fatty acid alcohols, and hydrocarbon chains show
anisotropic behavior, which results in different refractive indices for introduced ligh t
with different angles. In contrast, the melted sample shows an isotropic refractive
curve, which means that the introduce d light is reflected in the same way in all directions
(Kaufmann and Thieme , 1954) .
In the liquid phase of TAG mixtures they found that the refractive index decreases
by 0.00038 per 1 °C . Moreover, the sharpness of the melting interval can be used to
identify the prese nt fat (Kaufmann and Thieme , 1954) .

73
3.2.4 Procedures
The refrac tometer used for the TMOR measurements was an Abbemat 350
displayed in Figure 3. 11 (Anton Paar Op toTec GmbH, Seelze-Letter, Germany). It uses
the angle of the total reflection at 589 nm to precisely determine the refractive index.
The principle of the r efractometer is explained shortly. The device uses reflec ting light
instead of transmitting ligh t. The LED beam hits the sample which is placed on the
prism at different angles. At the interface between prism and sample, a part of the b eam
is reflected and another part is refracted by the sample as explained above (see Figure
3.7). The reflected light is used to determine the critical angle of tota l reflection and is
used to calculate the refractive index of the sample in relation to air (taken from the
Abbemat 350/550 m anual).

Figure 3. 11 : Refractometer Abbemat 350 used for temperature modulated optical refractometry.

The applicability of TMOR is the best for transparent soft matter. H owever, this
thesis shows, that also opaque materials like fats and lipid components after
crystallization can be analyzed. One needs to be car eful beca use the approach can only
be used if the refractive index changes lin early in one period of modulation. For the
interpretation of the data, it needs to be considered if ph ase separation (“fractionation”)
of the sample t ook place during the measurem ent. This could lead to re sults whic h are
not representative for the complete sample. Therefore , the sample is only analyze d in a
volume directly at the prism with the dimensions 1 mm x 1 mm x 1 µm.

74
Three different procedures were pe rformed to determine phase transitions
temperatures, solid fat content, and polymorphic behavior.
For the determination of phase transition temperatures all samples were placed
directly on the prism and subsequently heated to 80 °C under the predetermined
conditions. Subsequently, after a stab ilization time of 10 min the samples were cooled
down to 5 °C. All samples were at least performed in duplicate. As the method was
never used before for the experimental dete rmination of phase transition temperatu res
of fats or TAG mixtures, the method was used with three di fferent emphases. First,
distinct scan rates (2 °C/min, 5 °C/m in, and 10 °C/min) were chec ked on their
applicability. Second, different amplitudes (0.25 °C, 0.5 °C, and 0.75 °C) were und er
investigation. Finally, distinct periods (30 s, 60 s, and 120 s) were applied.
For the determination of the solid fat content (SFC) the AOC S temperature profile
for non-stabilizing fats was applied (AOCS, 2009a). The fat was heate d up to 80 °C and
kept at this temperature for 10 min. After cooling to 60 °C and a stabilization time of
10 min, the sample was cooled down to 0 °C. This temperature was held for 60 min
before the sample was heate d to the desired temperature, re spectively 10 °C, 15 °C,
20 °C, …, 60 °C. The stabilization time was 30 min during which the undulation was
applied with a period of 30 s and an amplitude of 0.5 °C. The temperature profile is
depicted in Figure 3 . 12 (black solid line). The mean refractive index was used to
calculate the SFC. Before each measurement, the heating to 80 °C with the following
stabilization steps at 60 °C and 0 °C was performed with each sample. To shorten the
required time, three different approaches were taken. First, a stepwise heating was
applied after 30 min of stabilization at each temperature (see Figure 3. 12 , grey solid
line). Second, the time for stabilization was decreased from 30 min to 5 min (see Figure
3. 12 , black dashed line). Third, the stepwise conce pt was done starting at 60 °C for
30 min before the sample was cooled stepwise in 5 °C steps to respectively 55 °C,
50 °C, 45 °C, …, 0 °C (see Figur e 3. 12 , grey dashed cu rve).

75

Figure 3. 12 : Temperature profiles of the SFC determination with TM OR, AOCS method with 30 min
stabilization time (black solid line), forward proceeding measurement with 5 min (black dashed line)
and 30 min stabilization time (grey solid line), backward proceeding m easurement (grey dashed line).

Two me thods were alm ost eq ually applicable. The AOCS proce dure always lead to
good results. In addition, the forward -approach with a stabilization time could repl ace
this procedure for reason s of measurement time reduction. Both, the forward-approach
with a stabilization time of 5 min and the backward-approach did not lead to
satisfactory results. The reasons could be not enough time to yield a stable sample at
the measurement temperature in the first case or to lack a driving force for
crystallization in the second case.
For the determination of polymorphic transitions the sa mples were molten on t he
prism and kept for 10 min at 80 °C. Subsequently, the sample to generate a less stable
polymorph was cooled down to 4 °C at a scan rate of 10 °C/min and kept there fo r
10 min. Next, the sample was heated to 80 °C at 2 °C/min, with the modulation
parameters of a period of 30 s an d an ampl itude of 0.5 °C. The real and imag inary part
of the volume expansion coefficient  were then compu ted. To yield a more stable
polymorphic form, the sample was cooled to 45 °C after the stabilization time of
10 min at 80 °C. Afterwards, the sample was kept at this temperature for 60 min b efore
it was heated at the same conditions as describe d above. The two temperature profiles
are given in Figure 3. 13 .

76

Figure 3. 13 : Temperature profiles used f or TMOR to yield a less stable polymorph (grey das hed line)
or a stable polymorph (black solid line).

3.3 Pulsed Nuc lear Magnetic Resonance
One of the important technique s for fat technologists is the pulsed nuclear
magnetic resonance (pNMR). Its principle is described only briefly below before its
application in the field of fat te chnology is outlined. Further more, the followed
procedure is explained.
3.3.1 Principle of pNMR
In general, NMR is used to characterize material based on its protons and neutrons.
In particular, only the principle of NMR appli ed in fat technology is outlined due to the
fact that a deeper background would be beyon d the scope of this thesi s.
A sample exposed to a strong magnetic field will experience the orientation of its
protons where each hydrogen nucleus acts as a little magnet (Van Putte and Van Den
Enden, 1974; van den Enden et al. , 1978) . The magn etization of the sampl e is
proportional to the number of protons prese nt (Van Put te and Van Den Enden, 1974).
After the sample is plac ed in a static ele ctromagnetic field the nuclei align themsel ves.
The appl ication of pN MR in the area of fat technology is based on the assumption,
that the hydrogen nuclei of fat relax differently in the liquid and the solid state leading

77
to a different spin-spin relaxation time t 2 (van Boekel, 1981). This t 2 is determined by
the receiver coil of the pNMR. Sin ce there exists a difference in t 2 for solids (10 µs) and
liquids (10- 20 ms) , a so lid - to -liquid ratio can be determined by pNMR (Van Putte and
Van Den Enden, 1974).
The application of a strong radio frequency pulse perpendicular to the existing
magnetic field (90°) leads to a change of the orientatio n of the protons (Petersson,
Anjou an d Sandström, 1985). If a pulse of a different frequency is applied, the
magnetization is disturbed and the reorganization of the molecules in the magnetic field
differs for different nuc lei (Van Putte and Van Den Enden, 1 974) .
Subsequently, the removal of the pulse results in the relaxation of the sample,
which retu rns to its equilibrium stat e. The relaxation t ime f rom this disorientation i nto
the former orientation is re corded with the NMR (s pin-spin relaxation time). This time
differs betw een protons in so lid or liquid phases. The relaxation time i n a solid is f aster
because the protons are packed closer in a dense network and can exchange their energy
faster (van den En den et al. , 19 78; Petersson, Anjou and Sandström, 1985). In addition,
the number of present protons influences the signal at the receiver co il. If the energy
fades, the system relaxes and returns to its initial state (Petersson, Anjou and
Sandström, 1985).
Peyronel and Marangoni (2014a) describe the background of pNMR in more detail .
However, for this thesis only th e application in fat technology is relevant, which is
outlined in the following sub -chapter.
3.3.2 Application of pN MR in fat technology
The solid fat content (SFC) is an important property if one is concerned with the
sensorial behavior of fat containing products, e.g. the quantification of the structuring
potential (Augusto et al. , 2012). Thus, the knowledge of the solid fat index ( SFI) or
content (SFC) is crucial for formulations and process control in fat technology
(Chapman and Sunlight, 1959; Van Putte and Van Den E nden, 1974; Mills and van de
Voort, 1981a; Pe tersson, Anjou an d Sandström, 1985) .
Nowadays, the determination of the SFC is mostly performed using pNMR.
Before, other methods suc h as dilatometry, density meters, wide-line NMR or DSC
were used (Walker and Bosin, 1971 ; Mills and van de Voort, 198 1b; Petersson, Anjou

78
and Sandström, 1985). Dilatometry, which is based on the change of the specific
volume at different temperatures is only ap plicable up to 50 % solids. It is an empirical
but reproducible method (Mills and van de Voort, 1981a). It is based on the different
densities of the solid and liquid phase of fats (Van Putte and Van Den Enden, 1974) .
Until the 1980s it was t he most accepted met hod to determine the solid-liquid content
in fats and oils (Mills and va n de Voort, 1981 b) .
Today, the most often applied method to determine the SFC is pNMR (see Figure
3. 14 ). It can be per formed in an indirec t and a direct manne r. The direct method uses
%𝐬𝐨𝐥𝐢𝐝𝐬 = 𝐟 ∙ 𝐬 ′
𝐟 ∙ 𝐬 ′ + 𝐥 ∙ 𝟏𝟎𝟎 = 𝐬
𝐬 + 𝐥 ∙ 𝟏𝟎𝟎

Eq. 3.9

to calculate the amount of solids. Here, s' is the soli d signal after ca. 10 µs, f the
correction factor for the dead time of the rece iver coil, s the “ true" solid signal and l the
liquid signal aft er ca. 70 µs.
The indirect met hod is the more accur ate one and uses the eq uation
%𝐬𝐨𝐥𝐢𝐝𝐬 𝐚𝐭 𝐭°𝐂 = 𝟏𝟎𝟎 − 𝐥 𝐓
𝐥 𝟔𝟎
∙ 𝟏𝟎𝟎 ∙ 𝐥 𝐬, 𝟔𝟎
𝐥 𝐬,𝐓

Eq. 3. 10

Here, l T represents the liquid signal of the sample at T °C, l 60 is the liquid signal at
60 °C, l s, T and l s, 60 are the signals of a refer ence oil (e.g. soybean oil) at T °C and 6 0 °C,
respectively and 𝑙 𝑠, 60
𝑙 𝑠,𝑇 as a correction factor (Pe tersson, Anjou and Sa ndström, 1985).

Figure 3. 14 : Principle of pulsed nuclear magnetic resonance, decay of the signal after a 90° pulse ((van
Boekel, 1981).

79

The direct method determines the signal immediately after the 90° pulse. However,
a correction factor f is necessary which takes the solid fat protons into account to
compensate the signal due to the dead time of the NMR receiver (van Putte and van
den Enden, 1973; Mills and van de Voort, 1981a) . The f- factor depends on the dead
time itself as well as the composition of the solid (polymorphic form, type of fat, crystal
size) and should therefore be chosen carefully (van Putte and van den Enden, 1973;
van den En den et al. , 1978; Petersson, Anjou a nd Sandström, 1985) . A mean factor can
only be use d if the applied temperature re gime is the same and the samples have a
similar composition (van Boekel, 1981) . A mean f -factor of 1.37 is used instead of
determining it by the indirect me thod. This is a mean value for oils and fats. The more
accurate way would be to dete rmine the f -factor separately for each fat under
investigation (Van Putte and Van Den Enden, 1974). The fact that the f -factor decreases
with increasing solid fat content should be kept in mind (van Putte and van de n Enden,
1973) .
The ind irect pNMR method uses the signal of the liquid phase to determine the
SFC (van Boekel, 1981) . It is character ized by the sign al of the liquid part after 70 µ s
(number of protons in the liquid) in re lation to the signal of the completely melted
sample (total number of protons). To ensure good results, th e spin-lattice relaxation
time t 1 is crucial. Hence, the number and time between pulses need to be chosen
carefully . For example, the t 1 for  crystals is shorter compared to  crystals (Van Putte
and Van Den Enden, 1974) . Therefore, also the po lymorphic form and thus for
tempering fats the correc t sample handling prior to the measurement is cruc ial because
fats high in POP, SOS, POS (2 -oleodisaturat ed TAGs) tak e a longer time to stabiliz e
(Petersson, Anjou and Sandström, 1985) . One needs to mention, that this method
neglects that the number of protons in the liquid and solid ph ase can slightly differ (van
Putte and van den En den, 1973) .
The SFC curve generated by the pNMR, whereby the percentage of solids is plotted
ove r temperature, is also called N-line. This curve is used as an important quality
parameter for every oil and fat. Typical SFC curves for cocoa but ter and palm oil are
depicted in Figure 3. 15 .

80

Figure 3. 15 : SFC lines for cocoa butter (black solid line) and palm oi l (black dashed line).

In addition to the mentioned principles , empiric al models can be used to describe
N-lines. Based on NMR and DSC dat a from different literature sources, Augu sto et al.
(2012) used three different models to describe the sigmoidal appearance of a SFC curve.
The aim is to model changes in the SFC behavior in a process before trying it. The
Gompertz model describes the SFC curve the best compare d to the other two models
(power decay and logistic) especially at high and low SFC values. The power and the
decay model tend to overestimate the SFC values at a low solids content and to
underestimate the SFC at a high amount of solids. In add ition, polynomial functions
do not describe the S-shape of t he SFC curve and can only be used in a cer tain part of
the curve to describe it (Augusto et al. , 2012). The use of a model can be helpful to
describe the SFC of different fats and fat mixtures witho ut the need to determine
experimental data over the whole tem perature range.
Since the pNMR in fat technology is often used as a benchtop equipment, NMR
glass tubes are used as sample holder (AOCS, 2009b). Errors can occur if the
crystallization is different over the who le NMR tub e, both in height or width of the
tube. In add ition, the determination of the solid fat content with pNMR via the
orientation of the hydrogen nuclei is only an a pproximation of the SFC which c an lead
to errors. Also, the usage of the direct or indirect method can result in differences in
the SFC value. Thus, the same method needs to be use d if values sh ould be compa red.

81
3.3.3 SFC Determination P rocedure
As me ntioned before, the tempering of fats is c rucial for th e proper det ermination
of the SFC. Tempering i s the systematic heating and cooling of a fat sample to achieve
a desired crystal network (Moens et al. , 2015). This is important for fats which show a
complex polymorphic behavior like cocoa butter (Petersson, Anjou and Sandström,
1985). The same temperature and tempering history leads to better comparability
between the two me thods of NMR and dilatometry (Madison and Hill, 1978). For fat s
like cocoa butter, which have an oleic acid at the sn -2-position, te mpering is even more
important to receive comparable SFC results due to their characteristic crystallization
properties (Petersson, Anjou and Sandström, 1985). Van Putte and van den Enden
(1973) di scovere d that a tempering step enhances the accuracy of the NMR method
and leads to a lower content of solids compared to the in the meantime applied
dilatometry. The tempering leads to a smaller number of solids below the tempering
temperature (Fu lton, Lutton and Wille, 1953; Walker and B osin, 1971) .
The tempering procedure of the direct AOCS method Cd 16b -93 for
non -stabilizing fats and oils like margarine was slightly mo dified and used in this the sis
(AOCS, 2009b). It is c omposed of the following four ste ps:
1. Melt the sample at 80 °C for 10 min
2. Stabilize the sam ple at 60 °C for 10 min
3. Keep the sample at 0 °C for 60 min
4. Heat the sample to the desired measurement tempe rature and keep it there fo r
30 -35 min be fore the measure ment
3.4 Gas Chromat ography
The analysis of the fatty acid composition is performed using gas chromatography
(GC). The basic princi ple is outlined before the required preparation proce dure for the
determination of fatty acids is explained. Subsequently, the applied procedure is
elucidated.

82
3.4.1 Principle of Gas Chrom atography
The GC is used to detect vol atile components in the mixture. In this study, the
different components in a mixture are separated within the GC column due to their
distinct molecular weight. The separation efficiency is influenced by the choice of the
packing material of the column and the applied flow rates and temperatures. Basically,
the equipment is composed of a column in an oven which can be heated at a defined
temperature rate and can be kept constant at a desired temperature. The device is
completed by a detector. For the detection, the temp erature at which the volatile
components are evaporated needs to be established. At the end of the column a
detector analyzes the volatile substances. A carrier gas, nitrogen in the example shown
in Figur e 3. 16 , carries the volatile compounds through the column after the sample
injection. The smaller components move faster through the column in comparison to
larger and, thus, heavier compounds leading to an earlier detection of the smaller
components. The chosen column affects the retention of the components depending
on its packing material and their interact ion with the sampl e. In the giv en case, a fl ame
ionization detector (FID) is used which generates a flame by the combination of air and
hydrogen. The volatile substance is ionized in the flame and the released electrons are
detected. The detected electrons are converted into a peak which is plotted over time
leading to a chromatogram. The retention times can be used after calibration to assign
a peak to a component and to c ompare their conce ntration in the sa mple.

Figure 3. 16 : Principle of a gas chromatograph with nitrogen (N 2 ) as carrier gas, an injector on the top
left of the column in the oven and air and hydrogen (H 2 ) to gener ate the flame for the FID (flame
ionization detector) at the top of the oven recording a chr omatograph of the fatty acid methyl esters.

83
The determination of fatty acids is crucial in the fat technology because different
fatty acids esterified on to the glyc erol backbone lead to different physical and chemical
properties. The analysis of fatty acids using gas chromatography require volatile
substances. Unfortunately, neither TAGs nor fatt y acids are vol atile and need to be
modified. Thus, the fatty acids are first removed from the glycerol backbone.
Subsequently, the fatty acids are methylated to generate the volatile fatty acid methyl
esters (FAM Es). For both steps the addition of a methylation agent (e.g.
trimethylsulfonium hydroxide) is required (see Figure 3. 17 ). The FAMEs are then
injected int o the GC for analysis.

Figure 3. 17 : Chemical reaction of trimethylsulfonium hydroxide (TMSH) with a fatty acid (FA) with a
carboxyl chain residue R leading to a fatty acid methyl ester (FAME), dimethyl sul fide (DMS) and wa ter.

Errors can occur if the methylation of the TAGs has not been conducted
completely or if residues of former measurements are still on the column. The latter
was tried to be avoided by rinsin g the column after every measurement with met hyl
tert -butyl ether ( MTBE) .
3.4.2 Procedure
The sample of which the composition should be analyzed is dissolved in MTBE.
About 10- 20 mg were weighed in and 4 m l of MTBE were added. After the sample was
dissolved, 100 µl of the sample solution were transferred into a GC vial. 100 µl of
internal standar d solution (C17:0 in MTBE) were added together with 10 µl of a
trimethylsulfonium hydroxide (TMSH) solution (Machery -Nagel GmbH & Co. KG,
Düren, Germany). The latter was used to get volatile FAMEs. The internal standard
ensures a reliable quantification. The sample was left at room temperature for 2 h to
ensure proper m ethylation.

84
For the analysis of the FAMEs a gas chromatograph (GC-2010 Plus) equipped with
an auto injector (AOC-20i) from Shimadzu was used (Shimadzu Corporation, Tokyo,
Japan). The car rier gas was nitrogen (6.8 ml/min), hydrogen and air we re used to ignite
the flame in a ratio of 1/10 ( 40 ml/ 400 ml ). The GC was equipp ed with a sili ca capillary
column from Fisher Scientific (Trace TM TR -FAME GC Column, Shimadzu
Corporation, Tokyo, Japan) with a di ameter of 0.25 mm and a length of 120 m. The
stationary phase in the column has a film thickness of 0.25 µm. After injection, th e GC
oven was kept at 100 °C for 5 min. Subsequently, the oven was heated to 250 °C at a
rate of 4 °C/min. The end temperature of 250 °C was kept for 45 min. Each GC run
lasted for 85 min and was performed in duplicate . The d ata processing was done with
the software provided by the manufacture r (Lab Solutions, Version 5.85, Shimadzu
Corporation, Tokyo, Ja pan).
3.5 Polarized L ight Microscopy
One of the main microscopic methods used for the investigation of fat
crystallization is polarized light microscopy (PLM). Its principle and procedure are
outlined below.
3.5.1 Principle of PLM
Light microscopy uses transmitting light to display a specimen using different
objectives to achieve different magnifications. A special case is the use of polarized light
to display only birefringent specimen. Anisotropic materials reflecting a beam of light
in two perpendicular directions due to differe nces in the refractive index depending on
the polarization of light, are called birefringent (Wright, Narine and Marangoni, 2000) .
This birefringence is utilized in the polarized light microscopy (PLM). In Figure 3. 18
the principle of the PLM is displayed. The light from the bottom of the microscope is
oriented by a polarizing filter so that it can only pass in on e direction. On the top of
the microscope an analyzer is placed. Both the polarizer a nd the analyzer are arranged
perpendicular to each other (90°) thus no light is coming through. If a birefringent
specimen is placed between the co ndenser (bundling the beam) and the objective
(magnification) a bright object appears in the micrograph where as isotropic materials
stay dark.

85

Figure 3. 18 : Schematic setup of a polari zed light microscope with two fil ters (grey rectangles), a
polarizer ( at the light source) and an a nalyzer after the specimen framing two lenses (light blue ovals),
the condenser and the objective as well as the specimen.

Errors in the analysis of polarized light m icrographs can occur if the polarizer and
analyzer are not set properly aligned i n a 90° a ngle to ge nerate the corr ect optical path.
Additionally, the preparation of the sample could always influence the macroscopic
appearance. The coverage by the glass slide could have an impact e.g. on the
crystallization behavior if the sample is not completely molten before insertin g the
microscopy slide in the Linkam stage. The used glass slides need to be clean e.g. to
avoid any nucleation sit es for crystallization.
3.5.2 PLM for the investiga tion of fat crystals
Since fat crystals show birefringence, their macroscopic appearance can be
investigated by polarize d light microscopy. The polarized light micrographs were
captured at 5x, 10x or 20x magnification using a Zeiss AxioScope (Carl Zeiss Jena
GmbH, Jena, Germany) equipped with a camera (AxioCam ICm1, Carl Zeiss Jena
GmbH, Jena, Germany). The image analysis was done with the ZEN Software provided
by the manufacturer (Carl Zeiss Jena GmbH, Jena, Germany). If an exact temperature
or a temperature profile was required a temperature-controlled stage from Linkam
Scientific Instrume nts (Surrey, UK) was used. Depending on the desired experiments,
heating a nd cooling sc an rates of 2 °C/min, 5 °C/min or 10 °C/min were applied. The
respective te mperature profiles are given i n the corre sponding chapters.

86
3.6 Powder X-Ray Dif fraction
The correct determination of the polymorphic form of the solid phase is crucial
for many food products like chocolate or margarine. Hence, powder X -ray diffraction
(XRD) is an impo rtant method in this area. Subsequently, its basic principle is explained
and adopted to the fiel d of fat technology.
3.6.1 Principle of XRD
This method uses an X-ray beam to inv estigat e the internal structure of a material
by scattering the impinging beam. X -rays have a short wavelength and thus a high
energy level (~120 eV to ~120 keV). Therefore, they can penetrate the matter under
investigation (Pe yronel and Marangoni, 2014b).
A req uirement to generate peaks in X-ray diffraction patterns is that the sent X- ra y
and the scattered beam are in-phase. Hence, Braggs law, given in equation Eq. 3. 11 , can
be used to determine a typical XRD pattern where the intensity is plotted against the
diffracted angle ( usually 2  ).
𝛌 = 𝟐 ∙ 𝐝 𝐡𝐤𝐥 ∙ 𝐬𝐢𝐧(𝛉)

Eq. 3. 11

The spacing in between two lamellar planes is indicated by d hkl , where the indices
hkl are referred to a fam ily of atomic plan es based on the Miller indices which are given
to explain the orientati on of the atomic pla nes (Peyronel and Marangoni, 2014b).
A ty pical set up of a diffract ometer is shown in Figure 3. 19 . The X-ray tube and the
detector always move at the same time to keep the ratio of  /2  . In this study, they
move up to 25° 2  to allow X-rays from all desired angles to hit the sample (2  = angle
of diffracted beam). Normally, the specimen is hold on a temperature controlled sample
holder.
A systematic error can occur if the sample mold is continuously over or under filled
because the device takes the proper flat filling level of the cavity as the zero position
for the procedure. In add ition, during data processing errors can occur if the baseline
and the peak fits are not set properly or e qually.

87

Figure 3. 19 : Setup of a X-r ay diffractometer (modified, Peyronel & Marangoni 2014 ).

3.6.2 Application of XRD in F at Technology
XRD is one of the main methods to study fat polymorphism. There are two main
sections which are mainly investigated in fat technology: the long spacing (1-15° 2  )
and the short spacing (16- 25° 2  ), where 2  represents the angle of the diffracted beam.
The long spacing is also kno wn as th e small angle X -ray scattering (SAXS) region and
the short spacing as the wide angle X-ray scattering (WAXS) region. The extension of
the SAXS region angle depends on the chain length of the fatty acids because it
corresponds to the p lanes formed by the methyl end groups. In contrast, the short
spacing is independent of the chain length of the fatty acids and corresponds to the
cross sectional packing (D’Souza, De Man an d DeMan, 1990; Pe yronel and Marangoni,
2014b).
The cross sectional packing correlates with the po lymorphic form. The main forms
in fat technology are the so -called  ,  ´, and  polymorphs. The  form corresponds
to a hexagonal cross sectional packing while the  ´ aligns in an orthorhombic manner.
The  po lymorph is characterized by a triclinic structure (Sato and Ueno, 2005) . Th e
determination of the polymorphic form is crucial due to their different structure and
thus distinctive melting points. More detailed informatio n is given in the fundamentals
section (chapter 2.2.3). The specific procedures applied in this thesis are explained in
the following sub-chapt er.

88
3.6.3 Procedure
For the dete rmination, a Multiflex powder X-ray diffrac tometer ( RigakuMSC Inc.,
Toronto, Canada) was used. Approximately 150 µl were placed on the sample holder
and analyzed at the corresponding temperature. The copper X-ray tube ( λ =1.54Å,
Cu/K α 1) was operated at 40 kV and 44 mA together with a 0.5° diversion slit and
scatter slit. In addition, a 0.3 mm receiving slit was used. The analysis was performed
moving the X-ray tube and the detector from 1.0-3.0° 2  and from 17.0-25.0° 2  to
cover the relevant ranges in the wide and small angle region at a scan speed of 1°/min
with a step size of 0.02°. The XRD patterns were analyzed using the Jade 9.0.1 XR D
software (Rigaku MSC Inc., Toronto, Canada). Two different procedures were
performed to identify di fferent polymorphic forms of fully hydrogenated palm oil
namely  ,  ´, and  . To generate the less stable polymorphic form, the sample was
placed onto the slide in the liquid stat e and transferred directly into the fridge at 4 °C
where it was kept for 10 min. The sample holder in the X-ray di ffractomete r was cooled
to 4 °C by a water bath to keep the same temperature during the examination. The
more stable poly morph was genera ted by kee ping the samp le for 60 min at 4 5 °C. The
sample holder was se t at 45 °C during the me asurement.

4 O N THE FEASI BILITY OF CONTINUOUS EM ULSION
FRACTIONATION

Michaela Häupler* , Miro Kirimlidou, Wiebke Wilms -Schulze Kump,
Alessandra Parisi, Leonie Wagner, Eckhard Flöter

TU Berlin, Departm ent of Food Process Engineering , Seestrasse 13, 13353
Berlin, Germany

Submitted to the European Journal of Lipid Science an d Technology (2018).
Copyright Wile y-VCH Verlag GmbH & Co. KGa A. Reproduced with permission.

The following chapter is a s ubmitted manuscript.

90
Abstract
In this study, the feasibility of a co ntinuous emulsion fractionation process was
evaluated. The conceptual design of the process entails that a cold -water stream is
emulsified into a hot oil stream such that a Pickering emulsion em erges. The water
droplets serve as entrainer whi ch is removed conjointly with the attached fat crystals
continuously in a decan ter centr ifuge.
Results on the kinetics of crystallization give guidance f or the process design in the
future. The sedimentation experiments in the benchtop centrifuge showed on the one
hand that the prepared emulsions were often insufficiently stabilized to withstand the
centrifugal forces (3,000 g). On the other hand, flocculation occurred as a problem
since the highly porous sediments cause proh ibitively low separation efficiencies. The
results for the decanter centrifuge lead to the conclusion that an accumulation of
hardstock at the solid discharge took place because a significantly increased level s of
water droplets in the solid discharge were accompanied by decreased levels of hardstock
in the lipid phase.
The findings show the difficulty to identify the correct process window for this
new process and indicate further research need s. Even though general feasibility was
illustrated by the data obtained currently achievable separation efficiencies are
unacceptably low.

Key words : Emulsion fractionation, Pickering emulsion, centrifugation, emulsion stabilization,
palm oil fractionation

Practical applications
A new continuous emulsion fractionation process is developed as an alternative for
the typical palm oil fractionation proce ss. the designed process aims at a more specific
fractionation of tripalmitate, higher separation efficiencies as well as a more energy
efficient operation.

91

Figure 4.1 : Graphical abstract of the p rocess window for the continuous emuls ion fractionation
process.
4.1 Introduction
The production of functional fat phases necessitat es the application of oil
modification techniques. The process to eliminate partially hydrogena ted fats starte d in
the second half of the nineties of the previous century due to conce rns about the health
ri sk associated with the consumption of trans -fatty acids. Since then the consumer
awareness with respect to chemical modification of food materials has significantly
grown. Even though chemical interesterificat ion has widely been substituted by enzyme
catalyzed reactions, processes which leave the molecular structure of raw materials
unchanged appear to be most desirable considering consumer acceptance.
Fractionation which is widely applied to palm oil is such a process. Duri ng fractionation
a fat composition is divided into fract ions, high melting stearin and low melting olein,
by solely physical processes. Typically fractional crystallization in combination with a
separation, often in a membrane filter press, is performed to separate the solid high
melting triacylglyce ride (TAG) fraction from the low melting one (Deffense, Tirtiaux
and Charleroi, 1985; Kellens et al. , 2007; Bot and Flöter, 2013; Kellens and Calliauw,
2013). Palm oil can be separated into different fractions with varying purposes like olein
(frying or salad oil), stearin (m argarine, shortening), or palm mid fraction (cocoa bu tter
equivalent, coating) on repeate d applicati on of the process (Timms, 1997; Kel lens et al. ,
2007).

92
However, fat fractionation processes are performed batch wise or semi -
continuously. Hence, a continuous process would be a desirable alternati ve (Kellens
and Calliauw, 2013). Shorter process times omitting the slow crystallization process an d
improved separation efficiencies would be meaningful targets. The separation
efficiency is in essence the content of solid material in th e high melting fraction. In
other words, it expresses how muc h liquid low me lting fraction is included in the filter
cake. In dry fractionation achievable separation efficiencies are usu ally limited to levels
below 60 %. This value can be im proved by dilution of the system with organic
solvents, wet fractionation. A raft of disadvantages, however, accompanies the wet
fractionation process. Higher energy consump tion, larger volumes to process, and
increased safety precautions render wet fractionation only suitable for high value
fractions, such as in the production of cocoa butter equivalents (CBE). The suggested
emulsion fr actionation process possibly offers a solution si nce a direct frac tionation of
high melting TAGs could be possible in a continuous way. The basic idea is that cold
water droplets are inj ected into a hot oil strea m, which contains high and low melting
TAG fractions to be separated. If process conditions are set accordin gly the high
melting TAGs crystallize around the water dro plets forming a Pickering emulsion
(Pickering, 1907; Flöter, 2009) i n analogy to th e margarine manufacturing process. The
process design entangles that this possibly transient emulsi on is subseque ntly separated
from the liquid low melting fraction in a decanter centrifuge. The separation in a
centrifugal field is promoted by the fact that th e water droplets function as entrainer.
The process ste ps are illustrated in Figure 4.2 .

Figure 4.2 Principle of emulsion fractionation pr ocess, crystallization of high m elting TAGs at water
droplet surface (1), separ ation of water dro plets and liquid oil (2), heating and separation of high melting
fraction and water (3), recirculation of water (4), resulting products olein (5a) and s tearin (5b).

93
Obviously, the process has some resemblance to the Lonza process. But in the case
considered here the two crucial elements of the process, crystallization and separation
have also to be considered with respect to their kin etics. Taking a closer look at the
different processes occurring either simultaneously or sequential it becomes apparent
that the identification of a viable process window is far fr om easy. Figure 4 .3
schematically depicts the process window looked for. The crystallization at the water
droplet surface has to progress to the level that the target TAG’s are crystallized u p to
equilibrium level to deliver a high relative yield. Secondly, the solid material needs to
be able to stabilize the interface. Th ese processes, crystallization and surface coverage
depend on among others thermal energy balances , heat and mass transfer and the
cumulative droplet surface area. The separation in a decanter centrifuge allows for
several process parame ters to be se t. These are primarily the actual centrifugal force by
means of rotational speed of the bowl, the differential speed between bowl and screw,
and the residence time. Insid e the decanter the emulsion should neither break, loss of
entrainment, no r flocculate because this will negatively affect the density differences
and separation efficien cy. Density difference being an obvious m ust (Gleiss and
Nirschl, 2015) for successful process implementation. It is envisage d that deformation
of fat covered droplets at the inner surface of th e bowl is beneficial for the separation
efficiency because the porosity of the droplet packing would be re duced.

Figure 4.3 : Schematic position of the process window for emu lsion fractionation, progress of
process es over time le ading to crystallization (black solid line), emulsion break up – subject to
centrifugal force (grey solid l ine), and flocculation (grey dashed line).

94
Additional means to manipulate the boundaries of the process win dow are, a priori
not desired though, the addition of other components . For example, could surfac tants
serve as templates to speed up crystallization and lead to the desired TAGs
accumulating at the water/oil interface (Awad and Sato, 2001, 2002; Awad, Hamada
and Sato, 2001). To increase density differ ences between droplets and oil phase
materials such as s alts or sugars could be dissolved in the aqu eous phase.
To this en d the study presented here documents the initial ex periments to evaluate
the feasibilit y of the continuous e mulsion fractionation proce ss.
4.2 Materials a nd Methods
A commercially available stored Pickering emulsion was used as a model syste m to
investigate the process. This emulsion was expecte d to be the most di fficult to separate.
Pronounced flocculation including a space -filling network is indicated by the fact that
20 % of dispersed phase combin ed with less than 2 % crystalline fat material result in
a homogeneous, non- sedimenti ng thin paste. Th is 20 % water and 80 % oil/fat
mixture contained a dditionally a small amount of emulsifier (lecithins).
A so-called freshly prepared Pickering emulsion was prepared containing 20 %
distilled water and 80 % oil/fat. The oil mixture was composed of 95 % rapeseed oil,
0.5 % distilled monoglycerides, and 4.5 % fully hydrogenated palm oil. Both oils were
provided by ADM (Hamburg, Germany). The oil mixture was heated to 80 °C and kept
at this temperature while stirring for at least 30 min to remove all crystal memory.
Subsequently, the oil mixture was po ured into a cylinder and distilled water (80 °C) was
added. The emulsion was prepared using a disperser (T25 Ultra-Turrax ® , IKA ® Werke
GmbH & Co. KG, Staufen, Germany) for 1 min at 8.000 rpm and for 1 min at
24.000 rpm. To initiate crystallization, the emulsion was put in a n ice bath and sheared
with the disperse r for 5 min at 8.000 rpm.
The so-called dynamic Pickering emulsion was no t prepared batch wise but
continuously by mixing cold water (1°C) and a mixture of rapeseed oil and fully
hydrogenated palm oil (60 °C) in a T-piece in a ratio of 20 % water and 80 % oil
mixture. The oil mixture s contained 90 to 95 % rapeseed oil, 9.0 to 4.5 % fully
hydrogenated palm oil, and between 1.0 and 0.5 % distilled monoglycerides (Dimodan ®

95
HR -Kosher, DuPont Nutrition Biosciences ApS, Braband, Denmark) to serve as a
template for the crystallization of fully hydrogenated palm oil at the water drop let
surface. This st atic mixing set up resulte d into the formation of a Picke ring emulsi on.
The samples were poured into centrifuge tubes directly after prep aration and
centrifuged at 4,500 rpm (  3,000 g) in a la b scale ce ntrifuge (Sigma 2-15, Sigma
Laborzentrifugen GmbH, Osterode, Germany) for 60 min, taking a photograph every
10 min. For the dynamic emulsions only one photograph after 10 min was taken since
the emulsion was n ot stable.
The Pickering emulsions were also separated con tinuous ly in a lab scale decanter
centrifuge (MD 80, Lemitec, Berlin, Germany) at di fferent accelerations and differential
speeds.
The crystallization and melting behavior of materials and fractions was investigated
by differential scanning calorimetry ( D SC ) using a device from Netzsch (DSC 204 F1
Phoenix, NETZSCH-Gerae tebau GmbH, Selb, Germany). Samples were heated to
80°C and the temperature was kept for 10 min to erase the crystal memory.
Subsequently, the samples were cooled down to - 50 °C at 10 °C/min. The sample s
were kept at - 50 °C for 10 min prior to heating to 80 °C at 10 °C/min. About 10 mg
of each sample was weighed into aluminum pans. An empty aluminum pan served as
reference. The peak analysis was do ne with the software provided by the DSC
manufacturer ( NETZSCH Prote us – Thermal Analysis s oftware vers ion 6.1.0).
The microscopy was performed using a polarized light microscope from Zeiss
(AxioScope A.1, Carl Zeiss Jena GmbH, Jena, Germany) equipped with a digital camera
(AxioCam ICm1, Carl Zeiss Jena GmbH, Jena, Germany). Either a 10x or a 20x
magnification was used and the micrographs were recorded at room temperature. T he
ZEN Software provided by the manufacturer was applied to do the image analysis (Carl
Zeiss Jena GmbH, Je na, Germany).
The samples were analyzed by gas chromat ograph (GC) to determine the fatty acid
composition of the obtained fractions. In detail, 10 - 20 mg were di ssolved in 4 ml of
MTBE. 100 µl of sample solution and 100 µl of internal standard solution (C17:0 in
MTBE) were added together with 10 µl of a trimethylsulfonium hydroxide (TMSH)
solution (Machery -Nagel GmbH & Co. KG, Düren, Germany). The sa mple was left at
room temperatu re for 2 h to ensure proper me thylation. The FAME a nalysis was done

96
with a gas chromatograph (GC - 201 0 Plus, Shimadzu Corporation, Tokyo, Japan). A
silica capillary column (Trace TM TR -FAME GC Col umn, Shimadzu Corporation,
Tokyo, Japan, 120 m x 0.25 mm x 0.25 µm) was used with nitrogen as the carrier gas
(6.8 ml/min) for the analysis. The sample was heated from 100 °C to 250 °C at a rate
of 4 °C/min. Data analysis was done with the software provided by the manufacturer
(Lab Solutions, Version 5. 85, Shimadzu Corporat ion, Tokyo, Japan).
4.3 Results & Discussion
4.3.1 Preliminary study on s urface crystallization
Inherently t he process design desc ribed above necessitates that the dispersion of a
cold aqueous phase in the hot oil stream induces crystallization at the droplet surface.
This could in general be achieved in two manners. In one embodiment the mixing of
the two streams results in a temperature low enough to create the des ired amount of
solids at thermal equilibrium conditions. To achieve this the fraction of the aqueous
stream and its cooling capacity need to be large enough and the mixing time sufficientl y
long to allow for elimination of temperature gradients. Additionally nucleation of
crystals at the surface and mass transfer to the growing crystals has to occur in the same
timeframe. This results in a stable Pickering emulsion. Alternatively, the situation of a
crystalline shell surrounding the water droplets could be transient. This means that even
though the heat balance of the mixed two streams does not result in temperatures low
enough for crystallization, initial crystallization takes place at the cold drop let surface
prior to the system assuming thermal equilibrium. Obviously, the latter proces s has the
advantage to be more en ergy efficient. However, to id entify the desir ed transient state
with sufficient solid material deposited before the interphase region is heated up is
difficult and might prove even impossible.
Some observations of the study on the crystallization of a hardstock (fully
hydrogenated palm oil) out of a warm oil mixture (50 % w/w rapeseed oil, 50 %
hardstock) on to the surface of a cold water droplet are shown in Fi gure 4.4. The
selection of a sequence of high speed camer a images re veals that from the mome nt the
water droplet (4°C) to uches the oil surface it takes ap proximately 1 s to ini tiate
significant crystallization. After addi tional 0.7 s the surface of the drop let (ca. 1 mm

97
diameter) is completely covered with crystals. The crystalline shell stays intact for at
least the next 5 s.

Fi gure 4. 4 : High speed camera images of a cold water droplet (4 °C) injected into a warm oil mix ture
(60 °C, 50 % rapeseed oil, 5 0 % fully hydrogenated pal m oil)

In the interpretation of the different results of cold, dripping droplets it has to be
taken into account that a dramatic excess of hot oil (60 °C) is present and no agitation
takes place. These results indicate that the crystallization at the surface is p ossible fo r
even very asymmetric settings. Furthermore, it was found that the appearance of the
crystal shell is actually rather quick compared to its melt off. The initial results on the
kinetics of crystallization and melting show that even a transient c rystalline state could
allow to separate the water droplets surrounded by fat crystals from the l iquid warm oil
before thermal eq uilibrium is reached.
For further evaluation, a dynamic Pickering emulsion was prepared with an oil
temperature of 75 °C to en sure that the hardstock (fully hydrogenated palm oil) was
completely molten. Hardstock levels of 10 % and 20 % (w/w) were studied t o evaluate
effects of concentration and supersatura tion. The ratio of water to oil stream was se t at
40 to 60 throughout the experiments. The target temperature was set bel ow 35 °C since
the crystallization of th e fully hydr ogenated pa lm oil in the oil mixture was observe d at
35 °C.

98
Assuming the system adiabatic, which in the current setup is certainly not the cas e,
one can calcu late the resulting average temperatures based on the heat capac ities of
water (c p ,H 2O = 4.18 kJ/kgK) and oil (c p,oil = 1.67 kJ/kgK), and the te mperature s of the
both streams. Based on Equation 1 the reference curve (grey circles) in Figure 4.5 is
derived.

T end = Q H 2 O, in + Q oil , in
c p,H 2 O ∙ m H 2 O + c p,oil ∙ m oil

Eq.
12

Figure 4.5 : Predicted oil temperature depending on the amount of water (5°C) injected into oil (75 °C),
experimental data from an oil mixture containing 10 % hardstock (red squ are) and 20 % hardstock
(black triangle).

Figure 4.5 reveals that the ex perimental emulsion temperatu res were lower than
those calc ulated by simple energy balances. Additiona lly it was found that higher levels
of dissolved hardstock and hence high er levels of crystalli ne material result in higher
final temperatu res. This is not surprising. In the first place, the system is not adiabatic
and the heat flux to the environment reduces the emulsion temperatu re. Secondly, the
temperature increase on higher solid levels corresponds well with back of the envelop
calculations that crystallization of 10 % material causes a temperature increase of almost
5 °C.

99
4.3.2 Emulsion Stabil ity and Flocculation
The high speed camera pictures rev ealed that the surface coverage by crystals was
achieved. Next to this, the emulsion stability is important to allow for separation
according to the suggested process. The emulsion stability depends on parameters like
the droplet size, the coverage of the surface by Pickering particles, amount and
conformation of fat crystals, the applied g -force, and the residence time in the
centrifugal field. Therefore, the stability of three different Pickering emulsions against
centrifugal force was e xamined.
4.3.2.1 Centrifuge separation
Dynamic Picke ring Emulsion
For the dynamically prepared emulsions three di fferent volume flows
(100 – 250 ml/min) were tested to vary the mixing process time. The temperature
settings were chosen such that the cryst alline material remained present throughout the
experiment. However, all sa mples resulted in a complete emulsion break up on
centrifugation at 3,000 g for up to 10 min. Hence , no water droplets surrounded by fat
crystals remain ed intact and the emulsions separated into a continuous aqueous phase
topped by a lipid phase. One reaso n might be that the drop lets generated by the T -
piece were not small enough and hence mechanically stable enough to withstand the
compression in the c entrifuge.

Freshly prepared Picke ring Emulsion
The micrograph, recorded with a polarized light microscop e, of the freshly
prepared Pickering emulsion is shown in Figure 4.6. The white areas in the image
represent crystalline material which is located mainly at the surfa ce of water drop lets.
These have a mean size of 7.36  2.07 µm. The micrograph shows that the Pickering
stabilization was established. Figure 4.7 depicts the fresh Pickering emulsion after
centrifugation intervals of 10 min. After 30 min, the maximum separation seemed to
be achieved as further centrifugation does not lead to the release of more free oil. It
has to be noted that after 20 min a small amount of water appeared at the bottom of
the centrifuge tube. This sediment does not increase over time indicating possibly an

100
original fraction of the emulsion no t well stabilized. Overall, the emulsion was
considered stable.

Figure 4.6 : Polarized light microgra ph of the fresh Pickering emulsion. Scale bar r epresents 50 µm.

Figure 4.7 : Freshly prepared Pickering emulsion centrifuged for a) 0 min, b) 10 min, c) 20 min,
d) 30 min, e) 40 min, f) 50 min, g) 60 min.

The DSC thermograms of the freshly prepared Pickering emulsion before and after
centrifugation are sh own in Figur e 4.8. The la rge melting peak at 0 °C in Figur e 4.8a is
associated with water. The melting peaks at - 20 °C and 40 °C, see also insert, can be
assigned to rapeseed oil and fully hydrogenated palm oil, respectively. The top layer,
characterized in Figur e 4.8b, had ne ither a melting peak of fully hydroge nated palm oil
(see insert) nor of wate r. Not surprisingly, this indicates a cle ar oil phase well separ ated
from the water/crystal slurry. The bottom layer showed quite the same melting
behavior as the feed (see Figure 4.8c). A clear p eak of fully hydrogenated palm oil can
be identified in the insert of this figure which also seems slightly highe r in comparison
to the feed. This would lead to the conclusion that fully hydrogenated palm oil was

101
accumulated in the bottom layer after ce ntrifugation. However, the observations
showed that only a limited compaction of the emulsion was achieved during
centrifugation. This manifests itself by the limited amount of supernatant, indicating a
high degree of flocculation. Such a highly porous sediment needs to be avoided in
fractionation processes t o achieve acceptable se paration efficiencies.

Figure 4.8: Freshly prepared Pickering emulsion with 20 % wat er and 80 % oil mixture, be fore (a) and
after centrifugation (top layer (b) and bott om layer (c)).

Table 4.1 gives fatty acid concentrations of the phases during processing the freshly
prepared Pickering emulsion. The GC analysis shows differences between the different
phases. Looking at the data of the supernatant, the upper phase, simple mass balan ces
reveal that approximately 1.5 to 2.0 % of the hardstock was dissolved in the oil. Based
on that, the level of solid fat in the different phases was deduced. This yields 2.5 and
3.5 % of solid fat in the starting emulsion and the slurry, respectively. The ratio of these
values corresponds very well with the compaction of the emulsion phase shown in
Figure 6. Here it was assumed that the crystallized TAGs had a fatty acid composition
of 1 to 1 stearic to palmitic acid. However, the solid levels are small and a large portion
of the high melting TAG’s remains dissolved rendering the process neither deliveri ng
a reasonable yield nor a n acceptable separation efficiency .

102
Table 4.1 : Fatty acid c omposition o f freshly prepared Pickering emulsion before and after 60 min of
centrifugation determined by gas chromatography.

Stored Pickering Emu lsion
Analogue to the freshly prepared emulsion the stored Pickering emulsion was
centrifuged for 60 min. Photos taken every 10 min during centrifugation are shown in
Figure 4.9. For clarity reasons the supernatant is indicated by a dotted line. The volume
of supernatant grows from approximately 2.5 % after 30 min to 5 %, 7.5 %, and 15 %
after 60 min. The observation is hence very similar to the one ma de for the freshly
prepared emulsions. The images however indicate that the stored emulsion had an even
stronger tendency to flocculate and develop a porous sediment phase detrimental to
acceptable separat ion efficiencies.

Figure 4.9 : Stored stabilized Pickering Emulsion centrifuged at 3,000 g for a) 10 min, b) 20 min,
c) 30 min, d) 40 min, e) 50 min, f) 60 mi n.

fatty acid

emulsion (%)

upper part
(%)

lower part
(%)

C 16:0

6. 5  0. 1

5.3  0. 5

7.6  0. 1

C 18:0

4.9  0. 1

3.2  0. 5

6.6  0. 1

C 18:1 cis

54 .6  0.1

54.4  0.4

50 .3  0.6

C 18:2 cis

18.1  0. 1

18.4  0. 3

16 .7  0. 2

C 18:3 alpha

7.2  0. 1

7.1  0. 1

6.5  0.1

103
In Figure 4. 10 the DSC thermograms of the phases from processing the stored
Pickering emulsion are shown. For the interpretation of the data it is impo rtant to
recognize that the hardstock used for this emulsion is fully hydrogenated high erucic
rapeseed oil. The TAG’s related to this hardstock have a different fatty acid
composition (C22:0 and C18:0 at approximately 1 to 1 ratio) resulting in even lower
solubility. All melting thermograms show the overlapping p eaks between - 20 °C and
0 °C. The inserts in Figure 4. 10 a, b, c reveal th at in all phases the high melting TAG’s
of the hardstock had be en crystallized at the low stabilizati on temperatu re, - 50 °C. The
data further reveal that there has been a clear concentration effect of these TAG’s in
the sediment phase (stearin). This was confirmed by the fatty acid analysis and the
thermograms.

Figure 4. 10 : DSC thermograms of th e stored Pick ering e mulsion before and after 60 mi n of
centrifugation at 3, 000 g: a) before c entrifugation, b) top layer after centrifugation, c) bottom layer aft er
centrifugation.

The store d and the fre shly prepared Picker ing emulsions showe d a good em ulsion
stability since sedimentation could be achieved without the occurrence of free water.
However, in both systems the sed iment appeared to b e very porous so that the
compaction of the slurry was insufficient to obtain reasonable separation efficiencies.
In contrast, the dynamically prepared emulsion broke after 10 min at 3000 g yielding a
continuous aqueous phase . This leads to the conclusion that the state of the emulsion,
kinetics of crystallization, stabilization of interphase, and floccu lation are of paramount
importance for a successful process design. Many sub-processes govern the
compromise of stability of emulsion versu s flocculation.

104
4.3.2.2 Separation in a lab scale deca nter centrifuge
The separation in the static centrifuge varied depending on the type of sample
preparation. Both, the stored and the freshly prepared emulsion were quite stable
against centrifugal forces while dynamic emulsions broke up in almost all of the
conducted experiments. The samples studied previously were separated in a decanter
centrifuge as described above to evaluate effect s of this processing step on the
flocculation an emulsi on stability, ultimate ly th e separation efficiency. As a preliminary
test the tem peratures of the streams at the inlet and at the discharges of the decanter
were determined. The temperatures at the discharges were stable around room
temperature indicating some cooling down of the material with inlet temperatures
between 25 and 30 °C. This implies that no melting of crystals should occur in the
decanter due to dissi pation of mechanical energy .

Dynamic Picke ring Emulsion
The mixtures of wate r (4°C) and oil (75 °C) re sulted in an em ulsion te mperature of
approximately 26 °C at the decanter inlet. The ratio of mixing is 4 to 6. The decanter
was operated at an acceleration of 3,000 g and a differential rotational speed of 5 rpm.
The DSC thermogram of the oil mixture, composed of 5 % fully hydrogenated palm
oil and 1 % emulsifier di ssolved in rapeseed oil, is shown in Figure 4. 11 a. In both,
Figure 4. 11 b (liquid discharge) and Figure 4 . 11 c (solid discharge) a water melting peak
is visible. This leads to the conclusion that water and thus hardstock was transported
to both exits and no s eparation occurre d.
Not surprisingly, all thermograms show a very similar oil peak. Looking at the
inserts of different thermograms it becomes apparent that all oil phases contain the
same amount of hardstock. This is also confirmed by the enthalpy values determined,
Table 4. 2 . The data indicate that the hardstock level is equal in both dis charge streams.
The actual value scales with content of water in both streams that appears to be
practically identical as well.

105

Figure 4. 11 : Dynamic prepared Pickering emulsion before and after separation in the decanter
centrifuge (5 rpm differen tial speed, 3,00 0 g); a) oil mixture (5 % fully hy drogenated palm oil, 1 %
emulsifier), b) sample from liquid discharge, c) sample from solid discharg e.

In line with the results reported above on the ce ntrifuge ex periments this indicates
that the dynamically p repared emulsion appears to be of insufficient stability to
withstand t he forces in the centrifugal field applied. Most likely, the emulsion broke up
in the decanter and a three – layered system was established. In this situation possibly
both discharges we re fed from a wa ter layer at the bowl and an inner oil phase wit h an
intermediate layer of fat crystals. Currently the experiments performed did n ot reveal
an operational window of reduced centrifugal force that results in good separation
process while maintaini ng droplet integrity .

Table 4.2 : Specific melting enthalpy  H of hardstock and water of the dynamic Pickering emulsion
separated in the decanter centrifuge (5 rpm differential speed, 3,000 g), products received from the
solid and the liquid d ischarge.

 H hardstock

 H water

Solid discharge

4.1 J/g

71.3 J/g

Liquid discharg e

3.9 J/g

66.4 J/g

Freshly prepared Picke ring Emulsion
The thermograms resulting from a decanter experiment with the freshly prepared
Pickering emulsion are displayed in Figure 4. 12 . The samples taken at the entry a nd at
the liquid discharge of the decanter centrifuge show no difference. This is not surprisin g
since no sample was ex pelled fro m the solid discharge. These results do cument the

106
effect of a reduced centrifugal force, 800 g compared to 3,000 g. Also, this processing
setup does not seem to suit the desired applica tion.

Figure 4 . 12 : DSC therm ograms of the freshly prepared Pickering emulsion before and after the
separation i n a lab scale decanter centrifuge (5 rpm differential speed , 800 g), a) temporary Pickerin g
emulsion, b) product from the exi t for liquids, c) produ ct from the exit for solids.

Possible explanations for the lack of any solid discharge stream m ight be th e
breakup of the emulsion, insufficient friction at the bowl, or insufficient sedimentation.
However, emulsion breakup had most likely occurred during experiments with the
dynamically produced emulsions and led to similar streams at both discharges. In case
the emulsion remains practically unchanged during the p assage of the decanter - no
breakup, no sedimentation - it is understandable that the moveme nt of the sc rew is not
able to transport any material to the solid discharge. Up to this point no combination
of residence time and centrifugal force could be identified that yields a meaningful
separation process for the freshly prepared e mulsions.

Stored Pickering Emulsion
Figure 4. 13 shows t he characte rization of the streams produced from processing a
stored Pickering emulsion. The process parameters used for the sample shown were
3,000 g and a differential speed of 20 rpm. Again Figure s a, b, and c show DSC
thermograms of the o riginal stored Pickering emulsion, the stream from the liquid (b)
and the solid (c) discharge, respectively. The inserts show a magnification of the section
of the thermograms most relevant for the hardstock melting. The data on enthalpy of
melting extracted from the thermograms are given in Table 4.3. In this case the data

107
indicate clearly that the dispersed phase is concentrated in the solid di scharge stream.
Both the thermal signal for the hardstock as well as the water phase are increased
compared to the feed and the liquid discharge. The latter actually appeared to be
depleted of hardstock and water. Applying simple mass balances on the data of water
the yield of solid discharge st ream, the ste arin containing emulsion, was approximate ly
35 %. T his corres ponds to a stearin yield of approximately 24 %. Even though this
combination of feedstock and processing settings appeared to indicate general
feasibility, the separation efficiency is still unacceptably low. However, this was owed
to the low level of solid material in the system and possibly the insufficient drying of
the solid discharge stream. The latter being a function of bowl configuration and
differential speed defi ning the residence time in the drying zone.

Figure 4. 13 : DSC therm ograms of the stored Pickerin g emulsion before and after the separation in a
lab scale decanter centrifuge (20 rpm differential sp eed, 3 , 000 g), a) stored Pickering emulsio n, b)
product from the exit for liquids, c) product from the exit for solids.

Table 4.3: Specific melting enthalpy  H of hardstock and water of the Pickeri ng emulsion and the
products received from the solid and the l iquid discharge of the decanter centrifuge (3 , 000 g, 20 rpm
differential speed).

 H hardstock

 H water

Pickering emulsion

2.0 J/g

24.0 J/g

Liquid discharg e

1.8 J/g

16.7 J/g

Solid discharge

4.8 J/g

37.1 J/g

108
4.4 Conclusion
The goal of this study was to inv estigat e the feasibility of the prop osed emulsion
fractionation process. The conceptual design of the process entails that a cold -water
stream is emulsified into a ho t oil stream such that a Pickering emulsion emerges. Using
the water droplets as means of entrainment the applicat ion of a decante r should yield a
stream of fa t covered water droplets that carry as little adhering oil as possible. It was
established that in general the mixing of the two streams allows to generate Pickering
emulsions. First results on the kinetics of crystallization at the cold droplet surface
could give guidance to further details of the process design. Experiments on the
sedimentation of different emulsions in a l ab centrifuge revealed that an emulsion
prepared dire ctly prior to ex posure to the centrifuga l field, with out a controlled
emulsification process, is of insufficient stability to yield meaningful results.
Sedimentation experiments with different longer stabilized emulsions with droplet sizes
of approximately seven micron showed that the emulsions c an withstand high
centrifugal forces (3,000g) but that the tendency to flocculate result in highly porous
sediments that would cause prohibitively low separation efficiencies. Processing the
different emulsions with a decanter centrifuge at different process settings in essence
reconfirmed the results of the centrifugation experiments. However, in particu lar
stored, and hence stabilized, emulsions could be processed such that significant
differences in the composition of the solid and liquid discharge of the decanter were
observed. A significantly increased level of water droplets in the solid discharge was
accompanied by decr eased levels of hardstock in the liquid lipid phase.
Even though the data presented illustrate the general feasib ility of the emulsion
fractionation process, its prod uct streams were of inacce ptable quality, in particular the
separation efficiency. I f this approach has the potential to ever beco me competitive
cannot be judged currently. Areas that certainly need more detailed attention are
improvement of the drying of the stabilized droplets, by variation of weir height and
differential speed, the effec t of droplet size on crystallization kinetics and emulsion
stability, and the composition of the feed material. Compared to the model systems
studied up to now the fractionation of palm or shea oil will pose much bigger challenges
with among others larg er amounts of solid mat erial to be separate d.

5 D ETERMINATION OF THE C RYSTALLIZAT ION
B EHAVIOR OF L IPIDS BY T EMPERATURE M ODU LATED
O PTICAL R EFRACTOMETRY

Michaela Häupler, Ec khard Flö ter

TU Berlin, Departm ent of Food Proces s Engineering , Seestrasse 13, 13353
Berlin, Germany

Originally published in Food Analy tical Methods (2018), 11 (9), 2347-2359
ht t p s :/ / d o i . o r g/ 1 0 . 10 0 7 / s 12 1 6 1 - 018 - 1217 -y © Springer

The following chapte r is an accepted manuscript and reprinted b y permission from Springer.

110
Abstract
This study was conducted to examine if the new temperature modulated optical
refractometry (TMOR) method is applicable to stu dy the phase behavi or of alkyl based
components. n -Hexadecane, palmitic acid, and glycerol tripalmitate were used as model
components. TMOR was benchmarked against di fferential scanning calorimetry (DSC)
and polarized light mic roscopy (PLM).
For all substances, a good agre ement of the D SC data wit h TMOR was found . For
n -hexadecane a difference of 2.2 °C for the crystallization and 2.6 °C for the melting
temperature was f ound. Consi dering palmitic acid, the cr ystallization temperature
differed by 3.3 °C while the melting varied by 2.8 °C. The crystallization temperature
of tripalmitate identified by TMOR was 2.7 °C higher and the melting temperature
2.3 °C lower compared to the DS C. The crystallization temperature for TMOR was
always higher and the melting temperature always lower if related to DSC. This leads
to the conclusion that TMOR is more accurate and di rect. In addition, the transition
peaks id entified by TMOR were narrower compared to the DSC peaks. This is due to
slower heating and cooling rates leading to a smaller temperature range of phase
transition and less ther mal lag.
The study showed that TMOR is an appropriate method to determine the ph ase
transition temperatures for the three examined substances. The results were
comparable to the DSC data in both melting and crystallization beh avior. Since the
accuracy of TMOR is better at lower heating and cooling rate s it could be a reasonable
extension of the well-know n DSC method in the studies of melting and crystallization.

Key words : crystallization, alkanes, fatty acids, triacylglycerides, temperature modu lated optical
refractometry (TMOR), ph ase behavior

111
5.1 Introduction
Next to carbohydrates and proteins, lipids repre sent a major class of nutrients and
play an important role in food production. In essence, lipids are characterized by their
hydrophobic nature which is also manifested by the existence of aliphatic chains. The
solidification behavior of these materials is strongly influenced by chain-chain
interactions. T herefore, the investigations of alkanes, fat ty acids, partia l glycerides, and
triacylglycerides form a good base to elucidate overriding principles of physicochemical
behavior of alkyl -chain containing molecules. The impr oved understanding of th e
phase behavior is beneficial for substances ranging from petroleum waxes to edible fats.
In this typ e of produ cts, the alkyl components and their composition have a big
influence on qu ality, consumer acceptance as well as appli cation fiel d and range (Sirota
and Herhold, 1999; Sat o, 2001) .
n -Alkanes are hydrocarbon chains composed of carbon and hydrogen atoms and
can align in a defined position to f orm crystals. In addition, even-numbered alkanes
form a triclinic phase while odd-numbered result in an orthorhombic phase (Wentzel
and Milner, 2010). Despite the fact, that even- and odd-numbered alkanes show
different crystallization behavior the autho rs were only interested in even- numbere d
alkanes due to their appearance in naturally derived fats. Work on the phase behavior
of n -alkanes and mixtu res of n -alkanes can be found in abundance (Dorset , 2 005) .
Fatty acids (FA) or carbonic acids are carboxylated hydrocarbon chains. The
aliphatic ch ain is nonpolar while the carboxyl group builds the polar group. FAs are of
big interest because they form the basis for fats and oils as well as for energy st orage
systems or bio fuels (Costa et al. , 2009). The packing of fatty acids into a crystal structure
is determined by the length and angle of tilt of the FAs. Saturated FAs with no double
bond in the hydrocarbon chain allow a denser packing compared to mono- or
polyunsaturate d FAs (Sato, 2001) .
Another important aspect that complicates matters is polymorphism. It is the
ability of a material to crystallize in different crystalline structures. Since the different
structures have spec ific interac tions this leads e.g. to different melting points and x-ray
patterns of the disti nguishable polymorphic forms.

112
Combined with a carboxylic group, n -alkanes form fatty acids, which are the main
components of triacylglycerides (TAGs) after e sterification to a glycerol. For one TAG,
three fatty a cids need to be esterified onto a glycerol backbone. In general, also partial
glycerides with only one or two fatty acid moieties esterified to the glycerol backbone
should be considered in this series. However, for the purpose of this work mono- and
diacylglycerides as investigated by Hernqvist (198 8) will not be considere d.
The investigation of the phase behavior of n -alkanes, FAs or TAGs is often
performed using differential scanning calorimetry (DSC) (Xie et al. , 2008; Costa et al. ,
2009; Ikeda et al. , 2010). The DSC method is bas ed on the variation of the heat released
or absorbed during thermal processing to observe the melting and crystallization events
of a substan ce. For that purpose, the sample is heate d or cooled at a specific rate while
the heat flux is monitored to i nvestigate occurring phase transitions. Ther modynamics,
in particular the Gibb s’ phase rule, di ctates that melting of a pure component should
take place at a fixed temperature. However, depending on the scan r ate of the ch osen
temperature program a limited thermal lag occurs. This implies that the true transition
temperature is actually related to an extrapolation to a sc an rate of zero. To execute this
directly is for obvious reasons, decreasing signal- to -noise ratio and general execution,
not practical.
Studying crystal structures by powder x-ray diffraction ( XRD) yields details on the
geometry of the crystallographic unit cell because the different crystal structures,
polymorphs, have different distances between methyl end planes (small angle) and
neighboring fatty acid moieties (wide angle) and hence scatter differently (Timms, 1984;
D’Souza, DeMan an d DeMan, 1990; Sato, Ue no and Yano, 1999) .
Using table top nuclear magnetic resonance (NMR) to determine the level of
solidified lipid material in a sample is well es tablished since the 197 0s. The solid fat
content (SFC) is an important quality parameter for fats and oils (Van Putte and Van
Den Enden, 1974; Pete rsson, Anjou and Sandström, 1985). The measurement is b ased
on the di fferent relaxation times in of hydrogen nuclei in solid and liquid phases after
excitation in a ma gnetic field.
Further investigations have been cond ucted to validate the poten tial of this
technique to determine po lymorphic forms, which was successful for several
mono- and triacylglyc erides (Van Duynhoven et al. , 2002).

113
All the above-mentioned techniques are u sed for the investigation of fat
crystallization because aliphatic chains are a major build ing block of the respective
molecular structure. Before NMR and XRD were established meth ods, a rather
outdated method called dilatometry was used to character ize phase transitions and
polymorphic forms . It was established for fats in the early 1930’s by Normann (1931)
and well understood in the mid 1940´s (Bailey and Kraemer, 1944; Bailey and Singleton,
1945). This simple method was applied to determine phase transitions of various fats
by investigating differences in the thermal expansion of solid and liquid phases (Bailey
and Kraemer, 1944). The same was applied for saturated fatty acids by Dorinson et al .
in 1942. The so called thermal expansion coefficient describes the thermally induced
density variations and is directly r elated to the derivative of the density regarding
temperature. Hence, a dilatome ter takes advant age of the fact that the liqui d p hase of a
sample is not only charac terized by typically lower densities compared to its crystal
form, but also that the temperature dependence of the said densities changes. Since
density and refractive index have a simple relationship as shown by the Lorentz-Lorenz
equation (Eq. 5.1 (Müller et al. , 2013)), it was a conceivable step to use the r efractive
index as a basis for a new metho d t o determine the density changes and simultaneously
the thermal expansion.
𝐧 𝟐 − 𝟏
𝐧 𝟐 + 𝟐 = 𝐫 ∙ 𝛒

Eq. 5.1

The determination of refractive indices of alkanes, FAs and TAGs has up to now
been limited to exclusively study the liquid state. In these cases, the refractive index is
a sensitive finger print of for example oils that allows to verify the identity of an oil .
Here the differences of refractive indices of oils are prima rily caused by differences in
the level of unsaturated bo nds in th e aliphatic chains. One could cons ider t he refractive
index an optical density which is defined as the velocity of light in vacuum divided by
the velocity of light in the medium. To utilize the refractive index for t he determination
of phase transitions Anton Paar Opt oTec i n cooperat ion with the group around Prof. Dr.
Krüger from the University of Luxembourg developed the temperature modulated
optical refract ometry (TMOR). So far, the application focussed on the isothermal study
of chemically induced glass transitions in polymers (Müller et al. , 2013) . The actual
TMOR prototype used is equipped with a pris m, for which the temperature is precisely

114
controlled (+/- 0.03 °C). The temperature is m odulated with a predetermined period
(30- 12 0 s) and a given amplitude (0.1-1 .0 °C) as shown in Figure 5.1. During the
temperature modulati on, the refractive in dex of the sample i s carefully recorded (n D ).
In the first place the TMOR deter mines the change of the refractive index
depending on the temperat ure. Th is change is expressed as 𝑑𝑛
𝑑𝑇 , which is the basis for
the calculation of the thermal expansion coefficient α . It can be calculated by the
Lorentz-Lorenz-Model us ing the following eq uation
𝛂 = − 𝟔 ∗ 𝐧
( 𝐧 𝟐 − 𝟏 ) ∗ (𝐧 𝟐 + 𝟐) ∗ 𝐝𝐧
𝐝𝐓

Eq. 5.2

where n is the re fractive index and T the te mperature (Müller et al. , 2013) .
If a change in the sample occurs, e.g. glass or phase transition, the und ulation of
the refractive index is trailing the temperature by a phase shift ϕ (see Figure 5.1). This
delay can be expressed by modelling the signal as real and imagi nary part of the therma l
expansion coefficient α (see Eq. 5.3 and Eq. 5.4 ).
𝐑𝐞 ( 𝛂 ) = | 𝛂 | ∗ 𝐜𝐨𝐬 ( 𝛟 )

Eq. 5.3

𝐈𝐦 ( 𝛂 ) = | 𝛂 | ∗ 𝐬𝐢𝐧 (𝛟)

Eq. 5.4

The real part represents the coefficient of thermal expansion (CTE) while the
imaginary part is the loss term. A peak in the curve of the imaginary part vs. temperature
indicates a ph ase transition at the corresponding crystallization/melting temperature
(see Figure 5.2). In turn, the shape of the real part plotted versus temperature is
indicative f or the kin d of observed phase transition. For example, if melting or
crystallization occurre d the real part also displays a peak like the imagi nary part. Other
transitions, such as glass transitions, reveal a step function in the real part of  . This
concept has bee n applied for isothe rmal phase changes induced by c hemical reac tions,
where the derivation of all formulas and variables is also published (Müller et al. , 2013) .

115

Figure 5.1 : Explanatio n of temperature modulation (grey, solid curve) and answer of the refractive
index (black, dashed curve), measured by TMOR.

In addition to the mo dulation around a con stant temperature during isothermal
experiments, the TMOR is also able to perform a modulation of the temperature
around a given temperature ramp. This feature enables a moni toring of dynamic
processes arising from temperature changes and opens the opportunity to realize
investigations lik e thos e execut ed with the help of DSC. Therefore, this stu dy aimed to
apply TMOR instead of the standard DSC me thod to determine crystallizat ion and
melting behavior of alkyl components. Its application would be desirable because
TMOR is potentially an easier and cheaper alternative t o all well-established methods
to monitor phase transitions. In addition, TMOR seem s to have a h igher sensitivity
compared to other methods and thus yields more detailed information. With the
possibility to perform slow scan rates, a reduction of the thermal l ag during alkyl
component investigation can be achieved. In c ontrast to high resolution refractometry,
TMOR yields peaks during the phase transitions instead of a kink in the refractive
index. This should emphasize change of properties of the first order ph ase transition
which becomes in particular important, when mixed systems are going to be studied.
Consequently, this manuscript is concerned with the assessment of the potential of the
temperature modulated optical refractometry to inv estigate the crystallization and
melting behavior of alkanes, fatty acids and triacylglycerides. The chosen model systems

116
represent classes of materials such as waxes, edible fats and oils or biofuels. The
evaluation of the TMOR entangles benchmark ing against conventional methods, even
though this is far from straight forward due to di fferent limitations in temperature range
and scan rates . Additionally, comple mentary benefits were looked for.

Figure 5.2 : Analysis of the loss term (black solid line) and the coefficien t of thermal expansion (CTE,
light grey poi nted line) and the mean refractive index (N mean , dark grey dashed lin e) as a function of
temperature.

To this end, the three substances displayed in Figure 5 .3 were investigated. All of
them have 16 carbon atoms at each chain and only di ffer in their residues, which is a
methyl group for alkanes (hexadecane, Figure 5.3a), a carbo xyl group for fatty acids
(palmitic acid, Figure 5.3b) and a glycerol backbone for triacylglycerides (tripalmitate,
Figure 5.3 c). The purpose of the study was to investigate the applicability of the TMOR
method for the determination of crystallization and melting behavior of alkyl
components.

117

Figure 5.3 : Differences b etween alkane, fatty acid and triacylglycerol ma rked with red boxes: a)
hexadecane (alkane with 16 carbon a toms and two methyl residues), b ) palmitic acid (hexadecane with
acidic group on the one and a methy l group on the other en d), c) tripalmitate (glycerol backbone
esterified with three palmitic acid resid ues).

5.2 Materials a nd Methods
In this study, three different pure substance s ( n -hexadecane, palmitic acid,
tripalmitate) were chosen to evaluate the applicability of TMOR for the investigation
of phase transitions in hydrocarbo n chain based molecule s.
The DSC was chosen as the reference method for the crystallization and melting
range, and polarized light microscopy (PLM) as a p ossibility to determine the
microstructure in the solid state. This is because shape and amount of the crystals
present could influence the re fractive index measure d.
n -Hexadecane and palmitic acid were purchased from Merck (Merck KGaA,
Darmstadt, Germany ) with a specified melting point of 18 °C and 61- 63 °C,
respectively. The glycero l tripalmitate was purchased from Alfa Aesar (ThermoFisher
(Kandel) GmbH, Karls ruhe, Germany) with a melting range between 60- 64 °C.
The temperature modulated optical refractometry (TMOR) measurements were
performed with a prototype from Anton Paar OptoTec GmbH (Seelze -Letter,
Germany). The used modification of the Lorentz-Lorenz model (based on (Beysens
and Calmet tes, 197 7)) is valid if the specific refrac tivity r is constant. Howe ver ,
Aleksandrova et al. (2 014) showed for po lyacrylamide solutions that the specific
refractivity changes if weak bonds are broken (Aleksandrova et al. , 2014). Therefore,

118
the authors assumed like Müller et al. (2014), that the specific refractivity remains
constant in the range of one period of 30 s which leads to a higher c ontribution of the
volumetric changes to the calculated data. The design of the device is described
elsewhere (Müller et al. , 2013). Briefly explained, an Abb e-refractomete r with a precisely
temperature controlled prism is used. The sample is put in a cone -shaped vat directly
on the temperature co ntrolled prism. Both, cooling and heating rate were set at
0.5 °C/min, with a modulation peri od of 30 s and an ampli tude of 0.2 °C. In the scope
of this paper, the reported ramp of 0.5 °C/min is always referred to as the mean ramp
and not to t he local ram p of the modulation. The samples were pressed onto the prism
using a solid stamp to ensure best contact between the sample and the prism and thus
minimizing measurement anomalies. Approximately 100 µL of the sample were molten
at 80 °C on the prism, holding the temperatur e for 10 min to remove all crystal
memory. Su bsequently, the samples were cooled to 4 °C, holding it for 30 min before
heating the sample at the rate of 0.5 °C/min up to 80 °C again. At the beginning of
each day, the device was calibrated with di stilled wa ter. Currently, the procedures
cannot include temperatures of less than 4 °C due to limitations of the prototype
equipment. The data was recorded at all st ages during the meas urement.
The differential scanning calorimetry (DSC) was performed with a device from
Netzsch (DSC 204 F1 Phoe nix, NETZSCH-Geraetebau GmbH, Selb, Germany)
which and indium (T m = 156.6 °C) was used for calibration. The two temperatu re
profiles were dete rmined at a cooling and heating r ate of 2 °C/min and a holding time
for 30 min at 4 °C and for 10 min at - 50 °C, respectively. This was done to check i f
differences in the melting profiles occurred due to different crystallization temperatures
and kinetic effects that might change due to sample size. App roximately 8 mg of sample
were weighed in aluminum pans and sealed hermetically. An empty pan was used as
reference.
The micrographs were taken during crystallization at a 10x magnification using a
Zeiss AxioScope (Carl Zeiss Jena GmbH, Jena, Germany) equipped with a camera
(AxioCam ICm1, Carl Zeiss Jena GmbH, Jena, Germany). The image analysis was done
with the ZEN Software provided by the manufacturer (C arl Zeiss Jena GmbH, Jena,
Germany). The polarized light microscopy (PLM) was combined with a
temperature-control led stage from Linkam Scientific Instruments (Surrey, UK)

119
following essentially the same temperature regime as the TMOR except for the scan
rate of 2 °C/min. The pictures taken were used to investigate the microstructure during
the phase transitions and to generate an asse ssment of the homoge neity of the sample.
TMOR, DSC and PLM measur ements were all perform ed in duplicate. Both the onset
and the peak temperatures determined by TMOR, DSC and PLM we re compared with
each other.
5.3 Results and Di scussion
The aim to investigate all samples with the different methods under the same
processing conditions was hampered by two difficulties. Firstly, TMOR seems to be
more precise at lower scanning rates compared to the DSC, which is normally applied
at high er scan rates. Secon dly, the DSC can cool down to much lower temperatures
than the TMOR prototype. In the evaluation of the applicability of TMOR for the
investigation of crystallization and melting behavior of alkyl components these tw o
issues are addressed as well.
To improve the comparabil ity of the two method s, the DSC data are used to
extrapolate for the determination of the transi tion temperature at scan rates of
0.5 °C/min, where the TMOR showed the best results. The procedure applied in this
approach is well established to determined v alues for zero scan rates (Illers, 1974;
Richardson and Savill, 1975; Vanden Poel and Mathot, 2006). For higher scan rates, the
changes in the refractive index appear to be too fast for the current evaluat ion
algorithms implemented in the prototype equ ipment.
The extrapolation of the experimental DSC data of n -hexadecane, palmitic acid and
tripalmitate investigated at three different heating and cooling rates (2 °C/min,
5 °C/min, and 10 °C/min) is depicted in Figure 5.4 . As expected, the peak
crystallization temperature decrea ses for all three substances with increasing cooling
rate (see in Figure 5.4a).
Table 5.1 gives the crystallization temperature for the different cooling rates
including the value det ermined by ext rapolation to a scan rate of 0.5 °C/min.

120

Figure 5.4 : Extrapolation o f DSC measur ements of n -hexadecane (cir cle), tripalmitate ( triangle black:
first p eak; triangle white: second peak) a nd palmitic a cid (cross) at different cooling (a) and heating
rates (b).

Table 5.1 : DSC peak temperatures at different c ooling rates of n -hexadecan e, palmi tic acid and
tripalmitate, temperatures at 0.5 °C/min are extrapolated and ind icated with italic letters.
cooling rate
(°C/min)

n -hexadecane
( °C)

palmitic acid
(°C)

PPP
(°C)

10

8.3  1.2

53.0  0.5

39.0  0.2

5

10.8  0.1

55.8  0.6

40.4  0.4

2

13.0  0.4

57.5  0.5

41.6  0.2

0.5

13.9

58.5

42.0

The melting curves were recorded after crystallization of th e samples at a scan rate
of 2 °C/min. Figure 5.4b shows that the me asured melting peak temperat ures increase
with increasing heating rate for n -hexadecane, palmitic acid and the first peak of
tripalmitate, as was ex pected. In the ther mogram of tripalmitate a second peak was
identified. This peak, related to the melting of a more stable polymorph, show s neither
an increase nor a decrease in melting temperature , which me ans it se ems not to be
affected by the scan rate. An explanation could be t hat it relates to a transient state
combining the melting of the lower melting polymorph overshadowed by the
crystallization of the more stable polymorph. Consequently, this transition is more
complete at a scan rate of 2 °C/min than at 5 °C/min, or 10 °C/min. The amount of

121
crystallized material and the occurring thermal lag due to different scan rates seem to
counteract each other. This results in the same melting poi nt of the second peak for all
scan rates at 64.3 °C. The calculated melting peak temperatu res for 0.5 °C/min are
listed in Table 5.2.

Table 5.2 : DSC peak temperatures at different heating rates of n -hexadecane, palmitic acid and
tripalmitate (two melting peaks) cr ystallized at 2 °C/min , temperatures at 0.5 °C/min are extrapolate d
and indicated with italic letters.
heating rate
(°C/min)

n -hexadecane
(°C)

palmitic acid
(°C)

PPP
(°C)

10

25.4  0.7

69.4  0.1

49.0  0.5

64.3  0.4

5

22.4  0.5

66.0  0.1

47.1  0.3

64.5  0.4

2

20.9  0.7

65.5  0.1

46.6  0.1

64.3  0.3

0.5

19.4

64.3

46.0

64.3

Before the melting beh avior analysis by TMOR was compared to the DSC results,
the proper modulation of each measurem ent was evaluated as shown in Figure 5.5 .
Owing to the fac t that the piece of equipment and the a nalytical metho d are still in
their infancy, irregularities that occur during the TMOR measurement leading for
example to sharp edges in the curve of the refractive index, the mean refractive index
cannot be dealt with automatically. Consequently, the data gathered needed careful
consideration prior to further analysis. A sinusoidal oscillation of both temperature and
refractive ind ex signal is required for the analysis of the melting behavior which was
done based on Re (  ) and Im (  ), the real and imaginary part, to describe the evolution
of the thermal expansi on coefficient  , re spectively.

122

Figure 5.5 : Evaluation of p roper TMO R measurement , sinusoidal modul ation of temperature (grey
solid curve: mean temperature, grey dashed curve: modul ated temperature) and refractive index (black
solid curve: mean refractive index, bl ack dashed curve: modulat ed refractive index), respectively.

5.3.1 n -Hexadec ane
All results of PLM, DSC and TM OR for n -hexadecane are shown below. For b oth,
PLM and DSC measurem ents, a cooling and heating rate of 2 °C/ min was applied. The
aim was to investigate the microstructure and the crystallization and melting behavior,
respectively. The sampl e tre atment with t he TMOR meth od was performed at a rate of
0.5 °C/min.
In Figure 5.6, a polarized light micrograph shows a dense network of n -hexadecane
at 4.0 °C after cooling the sample at 2 °C/min. The crystallization of n -hexadecane
began at 14.8 °C, which was determined with the PLM, and was completed within a
range of 2.0 °C. This narrow range of phase transitions was also found in the DSC
measurements (see Figure 5.7), where the crystallization starts at 15.0 °C
(T peak = 13.4 °C) and the sample melts at 17.3 °C (T peak = 20.5 °C). The melting po int
is in line with the manufactur er’s specification of 18.0 °C and did not differ between
the two chosen stabilization temperatures of - 50 °C and 4 °C, respectively (see Table
5.3). This was necessary to achieve a truthful comparison of the DSC and the TMOR
method.

123

Figure 5 .6 : Polarized light microscopy of crystallized n -hexadecane at 4 °C; scale bar repr esents
100 µm.

Figure 5.7 : DSC therm ograms of n -hexadecane stabilized at - 50 °C (a) and 4 °C (b); coolin g and
heating rate 2 °C/min.

In Figure 5. 8, the crys tallization (a) and melting (b) of n -hexadecane measured with
TMOR is shown. The curve of the refractive index in Figure 5.8a shows a small kink
15.7 °C during cr ystallization because the change of the r efractive index was not linear
in one period. However, the resulting Re (  ) and Im (  ) were calculated properly and
hence the crystallization behavior of n -hexadecane could be di scussed appropriately.
Such irregularities c an occur since TMOR is still a prototype for w hich the authors are
in the process to develop an experimental protocol, which can be used for different
acyl chain components. If on e considers the crystallizatio n and me lting behavior of
n -hexadecane during t he TMOR me asurements similarit ies to the DSC me thod can be

124
seen. The crystallization occurs at around 16.0 °C and the melting at 18.0 °C,
respectively, ac cording to the TMOR measureme nt.

Figure 5.8 : Crysta llization (a) and melting (b ) of n -hexadecane investigated by temperature modulated
optical refractometry; cooling and h eating rate 0.5 °C/min, 30 s period of modulation, 0. 2 °C
amplitude.

The crystallization begins at 16.0 °C with TMOR, has its peak temperatu re at
15.8 °C an d its offset at 15.6 °C. In comparison, n -hexad ecane starts crystallizing at
15.2 °C in the DSC measur ement and at 14.8 °C if investigat ed with the PLM. The
DSC results show a peak temper ature of 13.6 °C and an of fset temperatur e of 12.6 °C.
For pure substances, the crystallization temperature should be theoretically at one exac t
temperature instead of a range. However, the broader peak in the DSC results
compared to the TMOR can be explained by the emerging thermal la g of both which
should be considered when discussing differences in data obtained by different
methods. This is because the cooling and heati ng rat e of the TMOR measurement was
lower (0.5 °C/min) compared to the one of the DSC (2 °C/min) because current data
evaluation for TMOR is less artifact-pron e at lower rates a nd slower phase changes of
the sample. If one extrapolates the peak temperatures of the DSC measurements during
cooling at 2 °C/min, 5 °C/min and 10 °C/min (see Figure 5.4a), the peak temper ature
at a rate of 0.5 °C/min would be 13.7 °C for n -hexadecane, whi ch is lower than the
16.0 °C determined w ith TMOR.
For the heating of n -hexadecane, an extrapolation would lead to a melting
temperature of 19.4 °C, which is closer but still not matching the TMOR result of

125
18.0 °C. This leads to the conclusion, that TMOR has a higher sensitivity because the
differences in peak temperature can not be explained by only considering the different
scan rates. Both phenomena could be explained by the mentioned difference in the heat
transfer to the measured sample volume, whereby both the crystalliz ation and the
melting on the TMOR prism starts earlier compared to the DSC or the PLM.
Additionally, our observation indicate s that the onset of crys tallization ho wever suffers
less from this phenomenon. The onset temperatures determined with the DSC for
crystallization and melting at 1 5.2 °C and 17.3 °C, respectively, fit better to the TMOR
onset and peak temperature s, which were determined at 16.0 °C and 15.8 °C,
respectively, and 17.4 ° C and 17.8 °C, res pectively.
The experiments of the pure substance n -hexadecane were promising although
other authors state d before that TMOR should only be use d to investigate tra nsparent
material. However, this study showed that opaque mat erial like n - hexadecane in the
crystalline state can be examined. Subsequently, the next step was to inve stigate a
substance with a slig htly more complicated struc ture compared to n- hexadeca ne.
5.3.2 Palmitic aci d
The sample of palmitic acid showed a di fferent microstructure under th e polarized
light microscope compared to the other sa mples (see Figure 5.9). No clear crystal
structure was visible but a change of struc ture occurre d at around 60.6 °C. The sample
got rigid immediate ly and changed from translucent to a very bright white color.
Therefore, the generation of a proper TMOR signal was not as easy as for the other
samples which sho wed a crysta l structure.

126

Figure 5 .9 : Polarized light micrograph of palmitic acid during crystallization at 60 .6 °C; scale bar
represents 100 µm.

In Figure 5. 10 , the two DSC thermograms for different stabilization
temperatures are di splaye d. There was no difference observed whether palmitic acid
was stabilized at - 50 °C (Figure 5. 10 a) or at 4 °C (Figure 5. 10 b). The crystallization and
melting of palmitic acid investigated with TMOR is depicted in Figur e 5. 11 . During
crystallization, the peak temperature was found to be 57.9 °C in the DSC (Figure 5. 10 a)
and 61.2 °C in the TMOR measurement (Figure 5 . 11 a). The sample beg an to crystallize
at 59.9 °C in the DSC and at 61.4 °C on the T MOR prism.

Figure 5. 10 : DSC t hermograms of palm itic acid stabilized at - 50 °C (a) and 4 °C (b); cooling and
heating rate 2 °C/min.

127

Figure 5. 11 : Crystallization (a) an d melting (b) of palmitic acid investigated by temperature modulated
optical refractometry; cooling and heating rate 0.5 °C/min, 30 s period of modulation, 0.2 °C
amplitude.

For the melting behavior, 65.1 °C was the peak temperat ure of palmitic acid
investigated by the DSC ( Figure 5 . 10 b) whereas the peak melting temperature was
62.3 °C in t he TMOR measurement (Figure 5. 11 b). The DSC o nset temper ature of the
melting peak was at 61.7 °C and the onset determined by TMOR at 61.8 °C. As seen
for n -hexadecane, the temperatures determined by TMOR are closer to the onset
temperature of the DSC than the peak te mperature. The supporting data is given in

128
Table 5. 3. The extra polated values for the peak temperatures during crystallization
and melting at 0.5 °C/min with the DSC are 58.5 °C and 64.3 °C, respectively (see
Table 5.1 and Table 5.2). These values are closer to the peak temperatures determined
with TMOR but also after heating rate correction not in agreement. Again, the higher
crystallization and lower me lting temperatures cannot be explained by only consider ing
the different s can rates.
The experiments showed that it was also possible to study the phase behavior of
palmitic acid as a highly pure substance even though it ha s a slightly more complicated
phase behavior compared to n -hexadecane. These results let hope that also even mor e
complicated samples can be analyzed successfully. Therefore, the next step was to
analyze tripalmitate to incre ase the complexity of material.
5.3.3 Tripalmitate
The polarized light mic rograph of tripalmitate in Figur e 5. 12 shows the spherulitic
structure o f the formed solids. In contrast to n -hexadecane and palmitic acid,
tripalmitate showed small crystals, which can be identified as white areas. Furthermore,
in the solid state at 4 °C also dark areas re mained which lead to the con clusion that the
tripalmitate crystals formed a network and did not align as properly as the other two
substances.
The crystallization peak temperatu re in the DSC measurement was determined at
41.9 °C and the on set temperature at 44.2 °C (see Figure 5. 13 ). Again, as shown for the
other two pure substances, the peak temperature of TMOR (see Figure 5. 14 a), which
is 44.2 °C, fits better to the onset temperature of the DSC me asurement instead of the
peak temperature (see Table 5.3 ).
Both DSC thermograms in Figure 5. 13 show a recrystallization during melting for
both stabilization procedures at - 50 °C (Figure 5. 13 a) and 4 °C (Figure 5. 13 b),
respectively. This indicates that during crystallization a less stable po lymorph was
formed. The recrystallization into a more stable polymorph appears to be melt
mediated. At least the melting of the less stable po lymorph is clearly detectable prior to
the overlapping crystall ization of the more stable polymorph. On further temperature
increase the more stable polymorph melts.

129

Figure 5. 12 : Polarized light micrograph during the crys tallization of glycerol tripalmita te at 51.0 °C;
scale bar represents 100 µm.

Figure 5. 13 : DSC thermograms of tripalmitate stabiliz ed at - 50 °C (a) and 4 °C (b); co oling and heating
rate 2 °C/min.

Also, the signal ob tained during melting by TMOR indicates an event at
temperatures between 43 .0 °C and 47 .0 °C. A small bump at 45.0 °C in the imaginary
part as well as in the refractive index is visible in the enlarged section in Figure 5. 14 b.
This is an indication tha t TMOR also detect ed the melting of the unstable polymor ph.
A po ssible explanation for the much less pronounced signal of this melt -mediated
polymorphic transition are the slower scan rate and the temperature undulation both
promoting a simultaneous progression of melting of the less stable polymorph and
crystallization of the more stable polymorph. This overlap of melting and crystallization
causes that the net effect appears to be minute compared to separate distinct melting
or crystallization events. As the insert in Figure 5. 14 b reveals, this change of the

130
polymorphic form is accompanied by a change in physical prop erties. The slopes of the
refractive index over temperatu re (grey) are different before and afte r the polymorphic
tr ansition. In addition, the thermal expansion coefficient shows a minute differe nce
before (lower arrow) and after (upper arrow) the transition from a less to a more stable
polymorph. B oth observations trigger dee per investigations of the ability of TMOR to
determine polymorp hic transitions or polymor phic forms.

Figure 5. 14 : Crystallization (a) and melting (b) of tripa lmitate investigated by temperature modulated
optical refractometry; cooling and h eating rate 0.5 °C/min, 30 s period of modulation, 0. 2 °C
amplitude.

The onset temperatures of melting were determined at 45.0 °C and 59.7 °C and the
peak temperatures at 46.5 °C and 64.3 °C, respectively. For the first melting peak the
TMOR peak temperature fits exactly the onset temperature determined by DSC. The
second mel ting peak was found at 61.7 °C, which was lower compared to the DSC
onset temperature. Even though the temperature of the second melting pea k of
tripalmitate does no t depend on the crys tallization of the original material bu t
recrystallization, both peaks can be considered for the extrapolation of the peak
temperatures at 2 °C/min, 5 °C/min and 10 °C/min (Figure 5.4b). At a rate o f
0.5 °C/min a crys tallization pea k temperature of 42.0 °C and a melting peak at 46.0 °C
were calculated for tripalmitate . This is slightly higher than the determined values with
TMOR.

131
In addition to n -hexadecane and palmitic acid, it was also successfu l to study the
crystallization and melting behavior of tripalmitate, a material with an even more
complex ph ase behavior, by TMOR. In summary, all results sh ow that this new
technique could be a promising tool to investigate the ph ase behavior of lipid
components inde pendent of the struc ture or the optical appearanc e.
5.3.4 Comparison of TMOR and DSC
The collected DSC data is summarized in Table 5.3. For the three substances under
investigation, the differe nces between the stabilization at - 50 °C for 10 min and at 4 °C
for 30 min are minute, which makes them suitable as refer ence materials to prove the
suitability of t he new TMOR meth od. It was al so important t o show that the collected
data can be compared to bo th the standard DSC procedure, in which the samples are
cooled down to - 50 °C, and to l iterature data collecte d with this stan dard method.
The differences between the determined temperatures with bo th methods cannot
be completely explained by the variation in the scan rates because otherwise the TMOR
data would have f it the ex trapolation of the DSC data. The thermal lag is not only
dependent on the scan rate but also on the sample volume which is considered for
measurement. This means that not the whole sample volume is important but the
volume at the point of measurement. If one considers the heat transfer inside a DSC
pan, a volume of approximately 6 mm 3 needs to be heated. In contrast, the
refractometer is eq uipped with a 1 mm 2 photo diode arra y and its beam enters not even
1 µm. This results in a volume considered for measurement in TMOR whi ch is 6000
times smaller than the DSC sample volume. The t hermal lag is a proble m which nee ds
to be addressed if phase transitions due to tempera ture changes are di scussed.
However, this problem is less pronounced using TMOR because the lower sample
volume makes the measurem ent even more direct than the DSC.
In addition to the thermal lag, it is advantageous that an instantaneous temperature
change occurs at the measurement point. Therefore, a very direct measurement is
expected. At the same ti me this coinciding of the heating or cooling surface with the
point of measurement is the origin of possible artifacts when measuring
multicomponent sy stems. In thes e cases, a se lective crystallizati on on the prism,

132
fractionation of high melting compounds, would cause a dramatic inhomogeneity of
the sample and hence affect the measu rement results.

133
Table 5.3 : O nset, offset and peak temperatures during the phase transitions of n -hexadecane, palmitic
acid and tripalmitate determined via DS C measurements at 2 °C/min and TMOR measurements a t
0.5 °C/min.

DSC (2 °C/min)

TMOR (0.5 °C/min)

T stab.

phase transition

T onset
(°C)

T peak
(°C)

T offset
(°C)

T onset
(°C)

T peak
(°C)

T offset
(°C)

n -hexadecane

- 50 °C

crystallization

15.1

13.5

12.6

-

-

-

melting

17.3

20.4

21.5

-

-

-

4 °C

crystallization

15.2

13.6

12.6

16.0

15.8

15.6

melting

17.3

20.4

21.5

17.4

17.8

18.1

palmitic acid

- 50 °C

crystallization

60.6

57.8

55.9

-

-

-

melting

61.9

65.5

67.8

-

-

-

4 °C

crystallization

59.7

57.9

55.9

61.4

61.2

61.0

melting

61.7

65.1

67.3

61.8

62.3

62.5

tripalmitate

- 50 °C

crystallization

44.2

41.9

41.1

-

-

-

melting 1 st peak

45.0

46.5

47.2

-

-

-

melting 2 nd peak

59.6

64.0

64.9

-

-

-

4 °C

crystallization

44.2

41.5

41.1

44.4

44.2

44.0

melting 1 st peak

45.0

46.5

47.2

43.6

45.0

46.0

melting 2 nd peak

59.7

64.1

64.9

60.0

61.8

62.0

The surfac e sensitivity and the directness of measurem ent is one of the advantages
of the new TMOR method. How ever, one might be conc erned about the birefringence
of the crystal structu re affecting the measurement. If it is assumed that the crystals are
randomized, the local birefringence should be averaged in the plane without taking
influence on the measurement. Due to the tempering from only one si de of t he sample
an orientation of the crystals could take place. Since s -polarization is used for TMOR
the o ri entation takes place in the z -direction and an ordinary beam is measured. Hence,
because the calculation of the TMOR data is based on the measurement of the ordinary
beam, the concerns due to potential orientation of the crystals are negligible. Anyhow,
on going experiments to benchmark the results obtained by TMOR against
conventional methods aim to evaluate the se effects.

134
5.4 Conclusion
The new method of temperature modulated optical refractometry (TMOR) is
comparable to the DSC in determining crystallization and melt ing events of pure
substances. In evaluation of differe nt crys tallization regimes prior to DSC analysis only
insignificant variations were encountered. This indicates that possible di fferences in
DSC and TMOR are not due to the crystallization proc edure for the material studied.
The comparison of DSC onset, offset and peak temperatures with data ob tained by
TMOR show good agreement. One disadvantage is the limited cooling of the current
prototype to minimum 4 °C. Hence, TMOR can be used as a possible device to
determine phase transitions of the three investigated substance classes in a te mperature
range between 4 °C and 85 °C at the present moment. Nevertheless, if the cooling
procedure can be extended to temperatures below 0 °C, TMOR could become even
more competitive to DSC.
The melting and crystallization peaks measured by TMOR are narrower and thus
the ph ase transitions occur in a shorter temperature range than the ones from DSC
measurements and the response of the sample is more direct. This might be due to the
slower heati ng and cooling rate because the peaks ge t narrower with decreas ing rate of
temperature change. Furthermore, the results obtained by TMOR could be more
accurate due to the smaller sample size considered for measurement and less thermal
lag because of more direct heat transfer. Additionally the better exploitation of th e
signal relating to the thermal expansion coefficient is believed to give rise to more
accurate tempera ture determinatio ns.
Supplementary , TMOR showed accurate da ta considering phase transitions. The
provided time for the transitions is longer at slower scan rates. In addition, the
undulation promotes the transition. If one considers the signal for tripalmitate, it could
be that it a lmost diminished due to the overlapping of the melting of the unstable
polymorphic form and the crys tallization of th e more stable polym orph. To determine
properties like dn/dT or the therm al ex pansion coefficien t could be a possible route to
exploit accurate phase transition temperatures. Also, utilizing the undulation at low scan
rates can help to increase the accuracy of the measurement. Besides the investigation
of phase transitions, TMOR could be used to study kinetic effects. In additi on, it could

135
be applied to measure suspension sy stems a nd calculating therefrom the solid fat
content, which is an important paramete r in the oils and fats industry. However, while
such measurements of inhomogeneous systems and phase transitions are challenging
these possibilities are still under invest igation at this point.
Apart from the high sensitivity the sample prep aration is easy and TMOR has low
acquisition costs. The isothermal and dynamic measurements could yield more
information about the microstructure and phase behavior of n- alkanes, fatty acids and
triacylglycerides. To extend the applicability of TMOR, investigations of the phase
behavior of edible fats and oils are currently conducted by the authors. This ste p is
desirable because TMOR could be a helpful tool in quality ass urance, thermal
characterization, an d phase transition monitoring in the food industry.

Acknowledgements
The authors would like to thank Anton Paar Op toTec GmbH for the support
providing the prototype and room for discussion. In addition, the authors are gratefu l
for the financial support of Anton Paar OptoTec and the Elsa Neumann scholarship
of the federal state of Berlin.

6 E VALUATION OF TMOR PARAMETERS FOR THE
INVESTIGATIO N OF FATS

137
6.1 Theoretical b ackground
The temperature modulated optical refractometry (TMOR) is a novel method
based on refractome try with an additional te mperature undulati on. The device a nd the
method are explained in detail in chapter 3.2. TMOR was for the first time applied
investigating the crystallization of fats and lipid mixtures. As mentioned, TMOR is
based on refrac tometry. From this some challenges for the measurements arise suc h as
that the reflection of the fats turning nearly white during crystallization and that fats
become brittle during o r after crystallization.
In this work, the applicability of TMOR for the determination of li quid-solid
transition (crystallization / melting) of fats was studied. Therefore, parameters which
could be adjusted for the TMOR measure ments were varied. Three di fferent scan rat es
for three distinct fats were investigated before the influence of the temperature
amplitude and the period was examined. The overall goal for these preliminary tests
was to determine the best applicable procedure for the investigations of fats using
TMOR by the comparison with the established differential scanning calorimetry (DSC).
In addition, different fats w ere used to see if and how the triacylglyceride (TAG)
composition impacts the appl icability of TMOR. For a reliable comparison, the samples
need to be t reated equally before each meas urement so t hat the t emperature history of
the sample is t he same.
Beside the fat composit ion and the scan rate, also the period and ampli tude of the
modulation during TM OR measur ements influe nce the results. Care needs to be taken
because the sc an rate and both, the amplitude and the period, can interfere. Therefore,
a proper combination of all three parameters needs to be chosen and, thus, the
influence of amplitude and period i s elucidated.
The graphical explanation of period and amplitude is given in Figure 6.1. The
period of a sinus function is the time between two maxima of the wave, also referred
to as wave length. The amplitude is the height of the signal. In this study, three different
periods and three different amplitudes we re investigated.

138

Figure 6.1 : Explanation of amp litude an d pe riod of a modulated wave; the amplitude is referred to the
height of the signal (°C) and the period to the time between two zero -crossings (s).

For the study on the influence of period and amplitude, fully hydrogenated palm
oil was used because it shows a very narrow melting and crystallization range,
respectively. The peak temperatures are only given for the graphs presented even
though the analy sis was performed in triplicate. The experiments were conducted at a
scan rate of 2 °C/m in.
The prop er combination of scan rate, period, and amplitude might needs to be
changed for other materials. A short explanation is given in Figure 6.2, where two
different scan rates with the same modulation amplitude are show n. The red dashed
curve represents a scan rat e of 3 °C/min while the black dashe d line symbolizes a scan
rate of 0.75 °C/min. Both cu rves have the same amplitu de of 0.5 °C. For the scan rate
of 3 °C/min the modulation and the scan rate do not interfere whi ch is why the
temperature of 31.0 °C (upper grey solid line ) is only achieved once during this
measurement indicated by the yellow diamond. In contrast, the modulation comes
across 29.5 °C (lower grey line) for nine times at a scan rate of 0.75 °C/min with the
same amplitude. Only the red diamond represents the determination point at which the
mean refractive index and the modulated one have the same value. The frequent
achievement of one temperature can be crucial if the phase transition is happening at
this temperature . Hence, care must be taken if the predefined amplitude imped es a
proper measurem ent with the applied scan rat e.

139

Figure 6.2 : Interference of s can rate (red 3 °C/min, black 0.75 °C/min) and amplitude (0.5 °C ) during
temperature modulation.
6.2 Materials a nd Methods
6.2.1 Material pr operties
For the experiments, fo ur different fats (shea butter, coconut oil, palm oil, and fully
hydrogenated palm oil) were used and provided by ADM (Hamburg, Germany). Shea
butter is mainly composed of stearic acid (C18:0) and oleic acid ( C18:1) with minor
amounts of palmitic (C16:0) and linoleic acid (C18:2) (Adomako, 1977) . The TAG
composition of coconut oil is characterized by medium-chain length TAGs (MCT)
consisting of e.g. lauric acid (C12:0) and myristic acid (C14:0) (Salas et al. , 2009;
Chaleepa, Szepes and Ulrich, 2010). These MCTs show a melting range typically around
room temperature. Palm oil consists of a mixture of various TAGs, mainly POP
(30.0 %), POO (25.0 %), POL (1 0.3 %), POS (5.9 %), PPP (5.3 %), and OOO (4.6 %),
wi th palmitic (P), oleic (O), stearic (S), and linoleic acid (L) (Braipson-Danthine and
Gibon, 2007) . This variety causes a complex phase behavior and, thus, palm oil shows
many different melting points.
The data collected in Table 6.1 is a summary of values for the melting and
crystallization temperatures of the investigated fats determined by DSC. These values
were used to e valuate the resulting T MOR data.

140
Table 6.1 : Literature data of the crystallization (T c ) a nd melting (T m ) temperatures of coconut oil, shea
butter and palm oil derived from literatur e.
sample

scan rate

T m

T c

reference

shea butter

-

32.0-45.0 °C

-

Firestone, 1999

shea butter

2 °C/min

38.0-39.5 °C

26.5-30.0 °C

Adomako, 1977

shea butter

5 °C/min

37.0 °C

-

Hajj Ali et al. , 2016

coconut oil

-

23.0-26.0 °C

Firestone, 1999

coconut oil

1 °C/min

11.4 °C
21.1 °C

0.7 °C
2.7 °C

Tan and Che Man,
2002b

coconut oil

5 °C/min

12.4 °C
22.5 °C

-0.7 °C
-7.9 °C

Tan and Che Man,
2002b

coconut oil

10 °C/min

7.9 °C
23.6 °C

-2.9 °C
-16.5 °C

Tan and Che Man,
2002b

palm oil

-

33.0-40.0 °C

-

Firestone, 1999

palm oil

1 °C/min

0.9 °C
10.8 °C
26.8 °C
41.5 °C

18.4 °C

Tan and Che Man,
2002b

palm oil

5 °C/min

0.2 °C
5.3 °C
21.9 °C
35.4 °C

15.4 °C
6.1 °C
-2.9 °C

Tan and Che Man,
2002b

palm oil

10 °C/min

2.9 °C
6.4 °C
27.2 °C
35.8 °C

12.9 °C
-5.7 °C

Tan and Che Man,
2002b

141
6.2.2 Devices
Three di fferent TMOR parameters were investigated: the scan rate (2 °C/min,
5 °C/min, 10 °C/min) , the amplitude (0.25 °C, 0.5 °C, 0.75 °C), and the period (30 s,
60 s, 120 s).
The sample preparation for TMOR measurements was done in preliminary tests
(data not shown). Approximately 100 µl of sample were placed directly on the prism.
The samples were poured into a cylindrical geometry to generate a crystallization/
melting which occurs only vert ically. This ge ometry was applied for t he fats with a low
degree of sa turation lik e palm oil. For fats with a narrow composition range (e.g. fully
hydrogenated palm oil), which means mostly saturated fatty acids in this case, it is
recommended to use a stamp to ensure proper contact between the sample and the
prism during the whole measurement. These samples which are too hard or brittle
needed to be exam ined carefully.
The samples were heate d at differ ent scan rates, 2 °C/min, 5 °C/min, and
10 °C/min, respectively, until 80 °C were reached. This temperature was held for
10 min before the same cooling rate as the heating rate was applied to investigate the
crystallization behavior. Due to limitations of TMOR the minimu m temperature a t
which data can be obtained is 5 °C. The parameters for the TMOR measurements were
a period of 60 s and an amplitude of 0.25 °C. The sample was placed directly on the
prism. The peak temperature analysis of the TMOR data was done for the va lues of
abs (  ).
To evaluate the data obtained by TMOR, differential scanning calorimetry (DSC)
was used as the reference method. A device from Netzsch (DSC 204 F1 Phoenix,
NETZSCH-Gerae tebau GmbH, Selb, Germany) was used. The temperature profiles
were executed in the same manner like the TMOR measurements. Approximately 8 mg
of sample were weighed in aluminum pans and se aled hermetically. An em pty pan was
used as reference .
All DSC measurements were performed in duplicate while the TMOR
measurement was done at least in a triplicate to prove its applicability to properly study
fat crystallization.

142
Polarized light microscopy (PLM) was used to determine the microscopic structure
of the samples at various temperatures. A Zeiss AxioScope ( Carl Zeiss Jena GmbH ,
Jena, Germany) equipped with a camera (Axi oCam ICm1) was used. A scan rate of
2 °C/min was applied using a temperature controlled stage ( Linkam Scientific
Instruments , Surrey, UK ). The micrographs were captured at di fferent temperatures
where the samples were in transient stat e.
6.3 Influence of fat comp osition and scan rate
The crystallization and melting behavior of lipid mixtures is influenced by its
composition and the applied scan rate. Therefore, the effect of these factors on the
TMOR measurem ents was examined.
The thermogram of shea butter determined by DSC is depicted in Figure 6.3a. The
increase of the scan rate results in a crystallization peak te mperature which is shifted to
lower temperatu res and a melting temperature whi ch is detected at higher temperatures.
A scan rate of 2 °C/min resulted in a melting peak at 38.9  0.1 °C and a crystallization
peak at 13.7  0.1 °C , respectively. The melting peak temperature at a heating scan rate
of 5 °C/min was determined at 35.6  0. 5 °C by DSC while the crystallization peak was
detected at 13.5  0.2 °C . With a s can rate of 10 °C/min, the melting temperature was
determined at 29.9  1.3 °C and the cr ystallization peak temperatu re at 10.5  0.3 °C .
Figure 6.3b depicts the DSC therm ogram of coconut o il crystallized and me lted at
a rate of 2 °C/min (black line). The crystallization peak temperature was found at
9.8  0. 1 °C and the melting peak at 27.1  0.1 °C. Taking a look at a scan rate of
5 °C/min (dark grey line), the melting peak temperature was determined at
26.4  0.6 °C. The crystallization could not be detected because the minimum
temperature which could be achieved with TMOR was higher than the temperature at
which the coconut oil is complet ely solid. The refore, the DSC temperature profile was
only conducted until 5 °C. The increase of the scan rate up to 10 °C/min was
investigated because it is one of the mostly used scan rates in fat techn ology. The DSC
thermogram shows a melting p eak detect ed at 28.1  0.1 °C while there was no
crystallization peak visi ble for the experiment conducted at 10 °C/min (light grey line).

143
The investigations by DSC of palm oil at a scan rate of 2 °C/ min showed melting
peak temperatures at 11.7  0.9 °C, 27.1  2.7 °C, 37.2  1.8 °C, and 43.7  2.7 °C,
respectively (see Figure 6.3c, black line). These various melting points occurred because
palm oil has a diverse composition of TAGs whi ch all melt at different temperatures
(Braipson-Danthine and Gibon, 2007) The crystallizatio n temperature was detected at
19.8  0.7 °C. At 5 °C/min the peak melting temperatures were detected at
10.1  1.0 °C, 33.5  1.1 °C, and 43.9  0.8 °C, respectively (s ee Figure 6.3c, dark grey
line). The crystallization took place at 18.6  0.3 °C. The thermogram at a scan rate of
10 °C/min is indicated by the light grey line in Figure 6.3c. The melting peaks were
determined at 11.9  0.5 °C, 35.7  1.7 °C, and 44.0  0.5 °C, respectively while the
crystallization peak was detecte d at 15.9  0.1 °C.

Figure 6.3 : DSC thermogram of shea bu tter (a), coconut oil (b), and palm oil (c) a t 2 °C/min (bl ack
line), 5 °C/min (dark grey li ne), and 10 °C/min (ligh t grey line) during melting (positive heat flow)
and crystallization (negative heat flow).

The analysis of TMOR graphs was done using the values of the absolute thermal
expansion coefficient  (abs (  )). These values were co nsidered because they were
always in a po sitive range while the values of the real and the imaginary part showed
sometimes negative values. This was caused by a not suitable combination of the
applied scan rate and amplitude leading to calculating artefacts. Hence, these
combinations were later excluded as not -suit able for the investigation of the phase
behavior of lipid mixtur es.

144
Investigated by TMOR, the melting peak temperature of shea butter was detected
at 34.9  1.9 °C (see Figure 6.4a, black line) and the crystallization occurs at a peak
temperature of 14.3  0.2 °C (se e Figure 6.4b, black line) when a scan rate of 2 °C/min
was ap plied. For 5 °C/min, the crystallizati on peak was determined at 13.3  0.3 °C
(see Figure 6.4a, dark grey line). Th e melting graph showed three peaks in the thermal
expansion coeff icient  located at 32.5  2.0 °C, 35.6  2.0 °C, 38.2  2.1 °C (see
Figure 6.4b, dark grey line).
The application of a scan rate of 10 °C/min lead to the detection of a crystallization
peak temperature at 14.5  0.4 °C (see Figure 6.4a, light grey line). The melting peak
temperatures were detected at 31.5  1.2 °C, 37.4  0.9 °C, and 42.5  0.4 °C,
respectively (se e Figure 6.4b, light gr ey line).
In comparison, Figure 6 .4c and Figure 6.4d show the TMOR measurem ents o f
coconut oil during crystallization and melting, respec tively. The crystallization
temperature with a scan rate of 2 °C/min was found at 5.2  0.3 °C and the melting
occurred at 13.5  0.4 °C. TMOR seems to detect the melting behavior at a lower
temperature, whi ch means more direct, compared to the DSC. In con trast, the
crystallization was dete cted later which is in co ntradiction to the results found for shea
butter. The melting peak temperature at 5 °C/min was determined at 14.7  2. 1 °C
while the crystallization occurred at 7 .7  0. 1 °C . The shift of the melting temperature
to a higher temperature compared to a scan rate of 2 °C/min can be explained by the
thermal lag. Smaller rates lead to a decrease of the thermal lag while faster rates increase
the measured melting te mperature and lead to a broadening of the peaks (T an and Che
Man, 2002a; Chiu an d Prenner, 2011; Peyronel and Marangoni, 2014a). In comparison
to the DSC, a crystallizat ion temperature could be detected by TMOR. T his could be
an indicati on that the de vice indee d has a higher sensitivity compare d to the DSC. The
TMOR graphs obtained at a scan rate of 10 °C/min show a crystallization peak at
8.3  0.3 °C and two kinks in the refractive index during melting at 15.5  0.3 and
20.5  0.7 °C, res pectively.

145

Figure 6.4 : TMOR graphs of crystallization (left column) and melting (right column) of shea butter (a
and b), coconut oil (c and d), and palm oil (e and f) at 2 °C/min (black line), 5 °C/min (dark grey line),
and 10 °C/min (light grey line), peri od 60 s, amplitude 0.25 °C .

Using TMOR, the melting temperatu res using a scan rate of 2 °C/min were
determined at 9.9  2.0 °C, 25.3  0.7 °C, 35.9  1.3 °C, and 43.7  0.4 °C for palm oil ,
respectively (see Figure 6.4e and f, black line). The distinct peaks can be explained by
the variety of TAGs in palm oil , each contributing to the complex melting behavior.
Bes ide the last peak, all melting temperatures were lower compared to the DSC

146
measurement. The higher crystallization peak tem perature of 20.7  1.0 °C detected by
TMOR could as well as the lower melting temperatures be r elated to the more direct
heat transfer . At a scan rate of 5 °C/min, TMOR detect ed phase tra nsitions during
heating at 7.5  0.9 °C, 23.0  0.9 °C, 34.1  0.3 °C, and 41.6  0.4 °C, respectively,
while the crystallization was determined at a peak temperature of 21.0  0.4 °C (see
Figure 6 .4e and f, dark grey lin e). The crystallization was determined at a high er
temperature by TMOR compared to DSC. In addition, more melting peaks were
detected. Both findings support the conclusion that TMOR is more sensitive to phase
transitions in comparis on to the DSC me thod.
However, a scan rate of 5 °C/min could be too fast for the investigation of the
crystallization behavior of pal m oil by TMOR because the applied modulation did not
fit to the scan rate properly. This was also visible when TMOR data was recorded at
10 °C/min, which is indicated with t he light grey lines in Figure 6.4e and f. Melting
peaks were determined at 11.1  0.1 °C, 35.7  0.3 °C, and 41.4  0.2 °C, respectively.
It seems that the modulation did not fit properly to the ap plied scan rate. This beco mes
even clearer if the crystallization is considered. The peak temperature was detected at
15.9  0.1 °C by DSC and at 18.6  0.1 °C by TMOR. Even though this tendency
supports the assumption that TMOR is more sensitive and dete cts the phase transition
earlier compared to DSC, the modulation was not suita ble to the applied scan rat e. Th e
combination resulte d in partially negative peaks wh ich make the data not reliable .
The microscopy pictures were taken to obtain a visualization of the experimental
data from DSC and TM OR measurements. The polarized light micrographs at transient
states of the thre e investigated fats are shown in Figure 6.5. The micr ographs a, b, and
c show the crystallization of shea butter at 21.0 °C, 16.0 °C, and 8.0 °C, respectively.
Coconut oil crystallization is depicted in inserts d, e, and f at 11.0 °C, 8.0 °C, and 5.0 °C,
respectively. The cry stallization of palm oil is shown in the micrographs g-i at 24.0 °C,
19.0 °C, and 5.0 °C, res pectively.

147

Figure 6.5 : Polarized light micrographs of shea butter (a-c), coconut oil (d-f), an d palm oil (g-i) during
cooling at 2 °C/min, micrographs captured at transient states at 21 .0 °C (a), 16 .0 °C ( b), and 8 .0 °C (c),
the scale bar represents 100 µm.

Since shea butter is composed half of unsaturated and half of saturated fatty acids
the di stribution of the crystals and th e dark areas is not surprising. It is surprising that
the PLM reveals that the sample was already crystallized at 16.0 °C (see Figure 6.5b)
while both, DSC and TMOR, detected the phase transition at a lower temperatures.
However, the small visible crystals in Figure 6.5a at 21.0 °C indicate the on set of
crystallization and could be associated with small bump shown in the DSC
thermograms (see Fi gure 6.3). An explanation for the crystallization at higher

148
temperatures could be that small particles on the microscope slide acted as nuclei
helping inducing secondary nucleation. This could explain the higher crystallization
temperature.
The crystals of coconut oil formed at a cooling rate of 2 °C/min are depicted in
Figure 6.5d-f. The crystallization starts at around 8 .0 °C (see Figure 6.5 e). It can be seen
that at 5 .0 °C, in contrast to shea butter, many crystals are formed of about 25 µm in
diameter (see Figure 6.5f). This supports the detected crystallization peak temperatures
of both DSC and TMOR. The temperature profile during cooling seems to pr omote
the formation of single crystals.
The PLM micrographs di splayed in Figure 6.5g-i show the microstructure during
crystallization of palm oil which is initiated at around 24 .0 °C (see Figure 6.5g). In the
micrograph at 5 .0 °C (see Figure 6.6i) dark areas are visible which can be referred to
still liquid oil. This means that not the complete palm oil was crystallize d at the
displayed temperatu res and it can be concluded that the fat should have been cooled
down further to guarantee complete crystallization. However, the given temperature
range of TMOR limited the application. This may lead to the assumption that TMOR
should be predominantly applied for fats and oils which are completely solid at 5 .0 °C
to guarantee the proper analysis of the crystallization and melting beh avior.
During heating micrographs were captured at 11 .0 °C, 28 .0 °C, and 32 .0 °C (see
Figure 6.6). The dark areas represent the liquid TAGs with mostly unsaturated fatty
acids. The crystals have a size of less than 1 µm and are uniformly distributed over the
sample.
The micrographs captured during melting show that some areas are already liquid
at 28 .0 °C (see Figure 6.6b) indicated by the dark parts. The melting is nearly completed
at 32 .0 °C (see Figure 6.6c). This is lower than the values determined by DSC and
TMOR and could be explained by t he differenc es in heat transfer.
The vanishing of the coconut crystal network starts at around 20 .0 °C during
heating (see Figure 6.6e). Since there are crystals still visible at 20 .0 °C during heating,
with TMOR probably not all crys tals were detected but on e me asured a partial fraction
melting. The kink in th e re fractive index at around 27 .0 °C might be th e me lting of the
last remaining cry stals which can also be se en in the micrograph duri ng melting.

149
The melting transition states of palm oil are shown in Figure 6.6g-i. The melting
starts at 42 .0 °C (see Figure 6.6h) and is almost finished at 47 .0 °C (see Figure 6.6i).
The temperatures are slightly higher than detected by DSC and TMOR. This could be
explained by the potentially l ower heat transition through the glass microscope slide in
comparison to the alum inum DSC pan or the TMOR prism.

Figure 6.6 : Polarized light micrographs of shea butter (a-c), coconut oil (d-f), an d palm oil (g-i) during
heating at 2 °C/min, micr ographs captured at transien t states at 11 .0 °C (a), 28 .0 °C (b), and 32 .0 °C
(c), the scale bar represents 100 µm.

150
The comparison of all three scan rates investigated and the literature data of shea
butter are listed inTable 6.2. The thermal lag is visible for the DSC measurements for
the used scan rates which means that a higher cooling rate leads to a lower
crystallization peak temperature. However, during melting, a higher heating rate
normally results in a higher melting peak temperature which was not found for shea
butter. In contrast, the TMOR data depending on th e scan rate progres ses in
accordance to the t heory and shows some thermal lag.

Table 6 .2 : Melting and crystalliz ation peak temperatures of shea butter determined at different rates
(2 °C/min, 5 °C/min, 10 °C/min).

scan rate

DSC

TMOR

literature

melting

2 °C/min

38.9  0.1 °C

34.9  1.9 °C

38.0-39.5 °C

5 °C/min

35.6  0. 5 °C

35.6  2.0 °C

37.0 °C

10 °C/min

29.9  1.3 °C

37.4  0.9 °C

crystallization

2 °C/min

13.7  0.1 °C

14.3  0.2 °C

26.5-30.0 °C

5 °C/min

13.5  0.2 °C

13.3  0.3 °C

10 °C/min

10.5  0.3 °C

14.5  0.4 °C

Compared to the DSC, the melting temperature is lower and the crystallization
temperature is higher if determined by TMOR. This could be due to the direct heat
transfer into the sample when using TMOR because the sample is placed directly on
the prism whereas during the DSC measurement the sample is placed in a sealed
aluminum pan. In addition, the sample volume considered for TMOR is smaller than
for DSC (around 0.001 mm 3 vs. 10 - 20 mm 3 ). At 5 °C/min, the crystallization
temperature is in accordance with the DSC measurement, while during the heating
more melting peaks were observed using TMOR. The same results were found for a
scan rate of 10 °C/min.

151
For the melting behavior, the detecte d peak temperatures are in the same range as
the literature. However, the crystallization point was determined at much lower
temperatures in this study. This could be due to the fact that in the cite d literature
another method to determine crystallization was used. In addition, a small peak is visible
during the crystallization of shea butter at around 22 .0 °C, which could be an indication
that already at higher te mperatures a part of t he sample crystallizes. This is also s hown
in the PLM micrographs in Figure 6 .5a. Sin ce the crystallization point determined by
DSC and by TMOR are quite similar it could also be that the shea butter used in the
cited study has a differ ent composition.
I n Table 6.3, the peak melting and crystallization temperatures of coconut oil a t
different scan rates a re listed and compared to literature data. It can be seen, like also
shown for shea butter, that the melt ing is detected at lower temperatures when TMOR
is used compared to the DSC. The c rystallization was detected by TM OR also at higher
rates when there was no phase transition measurable by the DSC. This phenomenon
co uld be a hint that TMOR is a more sensitive method. The comparison to the literature
data shows that the data recorded with the DSC in this study always resulted in a high er
melting peak temperature. It was confirmed that it was not possible to detect the
crystallization since all given crys tallization temperatures are below 5 .0 °C. However at
2 °C/min there was a p eak detected. One explanation could be a diffe rent composition
of the examined shea butter in comparison to the literature data. The melting pea k
temperatures measured by TMOR are close to th e literature data and show a slight shift
to higher temperature s with increasing scan rate. The high accuracy of TMOR made it
possible to determine a cryst allization temperatu re for all rates eve n though the
temperature was shifted to higher temperatures. This is rather illogical and could be a
result of the poor fit of the modulation and the scan rate.

152
Table 6.3 : Melting and crystallization peak temperatures of coconut oil determined by DSC and
TMOR compared with literature data .

scan rate

DSC

TMOR

literature

melting

2 °C/min

27.1  0.1 °C

13.5  0.4 °C

11.4 °C
21.1 °C

5 °C/min

26.4  0.6 °C

14.7  2. 1 °C

12.4 °C
22. 5 °C

10 °C/min

28.1  0.1 °C

15.5  0.3 °C
20 .5  0.7 °C

7.9 °C
23.6 °C

crystallization

2 °C/min

9.8  0.1 °C

5.2  0.3 °C

0.7 °C
2.7 °C

5 °C/min

n.d.

7.7  0. 1 °C

-0.7 °C
-7.9 °C

10 °C/min

n.d.

8.3  0.3 °C

-2.9 °C
-16.5 °C

The comparison of the peak temperatures of phase transitions of palm oil
determined with TMOR, DSC, and the associated literature data are listed in Table 6.4 .
Both, DSC and TMOR detected the melting transition peaks at higher temperatures
compared to the literature. This could be explained by possible different handling
before the measurement which influences the melting beh avior or by distinct origins of
the samples which cause varieties in the composition. In addition, more peaks were
detected at lower scan rates which is in acc ordance to the literat ure (Tan and Che Man,
2002b). The thermal lag for the determination of the crystallization temperature was
seen for the DSC and the literature data but did oc cur to a smaller extend for TMOR
data.

153
Table 6.4 : Palm oil peak temperatur es measured by DSC and TMO R during heating and cooli ng at
different scan rates (2 °C/m in, 5 °C/min, 10 °C/min) comp ared to literature data.

scan rate

DSC

TMOR

literature

melting

2 °C/min

11.7  0.9 °C
27.1  2.7 °C
37.2  1.8 °C
43.7  2.7 °C

9.9  2.0 °C
25.3  0.7 °C
35.9  1.3 °C
43.7  0.4 °C

0.9 °C
10.8 °C
26.8 °C
41.5 °C

5 °C/min

10.1  1.0 °C

33.5  1.1 °C
43.9  0.8 °C

7.5  0.9 °C
23.0  0.9 °C
34.1  0.3 °C
41.6  0.4 °C

0.2 °C
5.3 °C
21.9 °C
35.4 °C

10 °C/min

11.9  0.5 °C
35.7  1.7 °C
44.0  0.5 °C

11.1  0.1 °C
35.7  0.3 °C
41.4  0.2 °C

2.8 °C
6.4 °C
27.2 °C
35.8 °C

crystallization

2 °C/min

19.8  0.7 °C

20.7  1.0 °C

18.4 °C

5 °C/min

18.6  0.3 °C

21.0  0.4 °C

15.4 °C
6.1 °C
-2.9 °C

10 °C/min

15.9  0.1 °C

18.6  0.1 °C

12.9 °C
-5.7 °C

In summary, both scan rates of 5 °C/min and 10 °C/min seem not to fit the
applied amplitude and period for all samples. On the one hand, the peak temperatures
are too high for the melting process. On the other hand , negative values occurred which
indicate that the calculation based on the m odulation was no t done appropriately. This
results in a calculation error giving negative values which cannot be used. Thus, the
application of a these scan rates for TMOR for the investigation of the inv estigated oils
is not recomme nded in combinati on with an amplitude of 0.25 °C and a period of 60 s.

154
Therefore, a smaller ra te should be applied. I t was shown that the TMOR me thod
is well applicable for the inv estigation of the phase behavior of the examined fats at a
scan rat e of 2 °C/min . S ubsequently, the influence of amplitude and period depending
on the scan rat e needs to be investigat ed.
6.4 Influence of amplitu de and p eriod
6.4.1 Amplitude
The consideration of the amplitude can be crucial because it could impact the
determination of the phase transition due to the temperature variation due to the
modulation. If a phase transition occurs within a small temperature range a large
amplitude can give a too low transition te mperatu re. Since the modulation is required
for the calculation of the real and imaginary part of the thermal expansion coefficient
it is necessary to determine the suitable amplitude which should be large enough to
ensure a pr oper calculation but not too large to impair the applied scan rate. Three
different amplitudes (0.25 °C, 0.5 °C, 0.75 °C) were investigated while the modulation
period was kept at 30 s because this was the smallest perio d which c ould be applied by
the device. For all measurements, the modulation was first checked on its regularity.
Second, the calculations of the TMOR peaks during cooling and heating were
evaluated.
A modulation amplitude of 0.25 °C was applied on the sample shown in Figure
6.7. Both, the refractive index and the temperature are plotted over time ( Figure 6.7a).
A deeper insight into th e modulation during me lting is shown in Figure 6.7b. The grey
line refers to the actual temperature while the black dashed line is the mean temperature
of one period of modulation. The solid black line represents the determined refractive
index depending on the modulated te mperature. The red dashed line represents the
mean refractive index.
It can be seen that the modulation was not smooth since the solid black line s hows
spikes and lacks a sinusoidal mo dulation, like explained in chapter 3.2. Considering the
calculated peaks during cooling (Figure 6.8a) and heating (Figure 6.8b) it was shown
that despite the fact that the chosen amplitude did not suit the applied scan rate the
calculation basically worked since positive peaks are visible in the thermal ex pansion

155
coefficient  . The crystallization peak te mperature is at 46.7 °C while the melting peak
temperature was detected at 50.0 °C and 58.7 °C, respectively. However, the amplitude
of 0.25 °C is not recommended in comb ination with a scan rate of 2 °C/min and a
period of 30 s for the investigation of the phas e tra nsition of highly saturate d fats such
as fully hydrogenate d palm oil.

Figure 6.7 : Temperature/ mean temperature a nd refractive index/ mean refract ive index of fully
hydrogenated palm oil plotted against temperature, 2 °C/min, 0.25 °C amplitude, 30 s period;
a) complete time range b) zoom.

Figure 6.8 : TMOR graphs of fully hydrogenated palm oil, 0.25 °C amplitude, 30 s pe riod, a) cooled at
2 °C/min, b) heated at 2 °C/ min.

156
Since the modulation of 0.25 °C prove not to be suitable, the modulation amplitude
was increa sed to 0.5 °C. In Figur e 6.9, the temperature and refractive index are plotted
over time. The spikes in the refractive ind ex are almost diminished in comparison to
the results when using an amplitude of 0.25 °C. The calculated peaks from the
mo dulation are shown in Figur e 6. 10 . The crystallization (a) occurred at a peak
temperature of 46.7 °C while the melting point was dete rmined at 49.6 °C and 58.3 °C
(b) probably referring to two polymorphic forms. At an amplitude of 0.5 °C the
calculation of the peaks seemed to work better than at 0.2 5 °C.

Figure 6.9 : Temperature/ mean temperature a nd refractive index/ mean refract ive index of fully
hydrogenated palm oil plotted against temperature, 2 °C/min, 0.5 °C amplitude, 30 s period;
a) complete time range b) zoom.

Figure 6. 10 : TMOR graphs of fully hydrogenated palm oil, 0.5 °C amplitude, 30 s period, a) cooled at
2 °C/min, b) heated at 2 °C/ min.

[Document text truncated for crawler view.]

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