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Katholieke

Universiteit
Leuven

FACULTY OF BIOSCIENCE ENGINEERING

INTERUNIVERSITY PROGRAMME (IUPFOOD)


MASTER OF SCIENCE IN FOOD TECHNOLOGY
Option Food Science and Technology

Academic year 2010-2011

Formulation and characterization aspects of low fat whipping cream by


Water/Oil/Water technology

by Lien Vermeir

Promotor : Prof. dr. ir. Paul Van der Meeren

Master's dissertation submitted in partial fulfilment of the requirements


for the degree of Master of Science in Food Technology
The author and promoters give the permission to consult and copy parts of this work for
personal use only. Any other use is under the limitations of copyrights laws, more specifically
it is obligatory to specify the source when using results from this thesis.

Gent, June 2011

The promotor the author

Prof. dr. ir. Paul Van der Meeren Lien Vermeir


Acknowledgements

I would like to thank my promotor, supervisor and mentor, Prof. dr. ir. Paul Van der Meeren,
whose guidance, insights and stimulating suggestions helped me in all the time of research.
Deep gratitude is expressed to the members of the department of Applied Physical Chemistry:
Eric, Marios, Maryam, Paolo, Quenten, Saskia and Zhu.
Lastly, I offer my regards to all of those who supported me in any respect during the
completion of my thesis: members of the lab of FTE, the staff of IUPFOOD in Gent and
Leuven and last but not least, my family.
Table of contents

Table of contents

Abstract 1

Chapter 1: Literature review 2


1.1 Water-in-oil-in-water emulsions 2
1.1.1 Water-in-oil-in-water technology 2
1.1.2 Preparation of a w/o/w-emulsion 3
1.1.3 Encapsulation efficiency 4
1.1.4 Instability of w/o/w-emulsions 6
1.1.4.1 Thermodynamic instability of a w/o/w-emulsion 6
1.1.4.2 Water transport between the w1 and w2-phase 9
1.1.4.3 Gravitational instability of a w/o/w-emulsion 11
1.1.5 Desired characteristics of a whippable w/o/w-cream 12
1.2 Traditional dairy whipping cream 12
1.3 Changes during whipping of cream 13
1.3.1 Three stages during whipping of cream 13
1.3.2 Foam stabilization by partial coalescence 15
1.3.2.1 Determination of partial coalescence 17
1.3.2.2 Distinction between partial coalescence and complete coalescence 17
1.3.3 Alternatives to partial coalescence 18
1.4 Mimicing whipping cream 19
1.5 Quality characteristics of whipped cream 21
1.5.1 Overrun 22
1.5.2 Whipping time 23
1.5.3 Textural analysis 24
1.5.4 Physical stability 24
1.5.4.1 Coarsening of foam 24
1.5.4.2 Drainage 25
1.6 Factors determining functional properties of whipping cream 26
1.6.1 Temperature 26
1.6.1.1 Tempering 27
1.6.1.2 Heat treatment 28
1.6.2 Fat content 28
1.6.3 Homogenization 28
1.6.4 Miscellaneous factors 29
Table of contents

Chapter 2: Materials and methods 31


2.1 Commercial butters 31
2.1.1 Preparation of butter samples 31
2.1.2 Analysis of butter samples 32
2.2 Water-in-oil emulsions 32
2.2.2 Materials needed for the preparation of w/o-emulsions 32
2.2.3 Composition of the w/o-emulsions 33
2.2.4 Preparation of the w/o-emulsions 34
2.3 Quantitative particle size analysis of water in w/o-emulsions 36
2.3.1 Pulsed field gradient-Nuclear Magnetic Resonance 36
2.3.2 The pfg-NMR experiment 37
2.3.2.1 Calibration procedure 39
2.3.3 Restricted diffusion in w/o emulsions 39
2.3.3.1 Data procesing by the Droplet Size application 41
2.3.3.2 Data processing by Excel 42
2.3.3.3 Data processing by Matlab 46
2.3.4 Statistical methods 48
2.3.5 Important issues during pfg-NMR analysis 49
2.3.5.1 Temperature of the emulsion during pgf-NMR analysis 49
2.3.5.2 Assumption of a lognormal size distribution 50
2.3.5.3 Advantages of pfg-NMR analysis for determination of water droplet size 50
2.4 Qualitative particle size analysis of water in w/o-emulsions 51
2.4.1 Fluorescence microscopy 51
2.4.2 Fluorescence 52
2.4.3 EosinY 53
2.4.4 Fluorimetric analysis of w/o emulsions 54
2.4.4.1 Fluorimeter 54
2.4.4.2 Determination of a suitable concentration of eosinY 54
2.4.4.3 Determination of maximum excitation and emission wavelength 55
2.4.5 Confocal laser scanning microscopy 58
2.5 Water-in-oil-in-water emulsions 59
2.5.1 Composition of the w/o/w-emulsions 59
2.5.2 Preparation of the w/o/w-emulsions 59
2.5.2.1 Method A 59
2.5.2.2 Method B 60
Table of contents

2.5.2.3 Method C 61
2.5.2.4 Method D 64
2.6 Quantitative analysis of the enclosed water volume and yield of water-in-oil-in-water 64
emulsions
2.6.1 The CPMG-experiment 64
2.6.2 Determination of the enclosed water volume 70
2.6.3 Determination of the yield of a double emulsion 71
2.6.4 Statistical methods 72
2.7 Fat globule analysis by laser diffraction 72
2.8 Profilometric analysis of double emulsions 72
2.9 Whipping of a commercial dairy cream and w/o/w-emulsions 73
2.9.1 Commercial dairy cream 73
2.9.2 W/o/w emulsions 73
2.9.2.1 First attempt to prepare whippable double emulsions 73
2.9.2.2 Second attempt to prepare whippable double emulsions 73
2.9.2.3 Third attempt to prepare whippable double emulsions 74
2.9.2.4 Fourth attempt to prepare whippable double emulsions 74
2.9.3 Determination of the whipping time 74
2.9.4 Determination of the overrun of a whipped emulsion 74
2.9.5 Physical destabilization (drainage) of whipped emulsions 75

Chapter 3: Results and discussion 76


3.1 Optimization of wayer droplet size analysis 76
3.1.1 Temperature experiment by using a thermocouple 76
3.1.2 Repeatability experiment 78
3.2 Quantitative particle size analysis of water droplets in commercial butters 80
3.3 Quantitative particle size analysis of water droplets in w/o-emulsions 85
3.3.1 Analysis of emulsions with composition H0/1, H0.5/1, M0/1 and P0/1 85
3.3.2 Analysis of emulsions with composition H0/1. H0.5/1, M0/1, P0/1, P0.5/1 and 88
P0.5/2
3.3.2.1 Statistical analysis of emulsions H0/1, H0.5/1, P0/1, M0/1, P0.5/1 and 89
P0.5/2
3.3.2.2 Difference between different data processing methods 91
3.3.2.3 Influence of the type of fat on the mean water droplet size 91
3.3.2.4 Influence of the emulsifier on the mean water droplet size 92
Table of contents

3.3.3 Elevation of the water fraction of emulsions based on Hozol 93


3.3.4 Effect of the decrease of the homogenization pressure of the Microfluidizer M110S 96
on the water droplet size in a w/o emulsion
3.3.5 Analysis of emulsions with the hydrophilic surfactant whey protein isolate 98
3.4 Qualitative particle size analysis of water droplets in w/o-emulsions by fluorescence 99
microscopy
3.4.1 Preliminary investigation of eosinY-solutions by fluorimetry 99
3.4.2 Determination of the maximum excitation and emission wavelength of eosinY 99
(5µg/mL) in water
3.4.3 Determination of the maximum excitation and emission wavelength of eosinY 101
(5µg/mL) in an aqueous phosphate buffer (pH6.7)
3.4.4 Determination of the maximum excitation and emission wavelength of eosinY 102
(5µg/mL) in aqueous phosphate buffer (pH6.7) with sodium azide (0.02%, w/v)
3.4.5 Determination of the maximum excitation and emission wavelength of eosinY 103
(5µg/mL) in aqueous phosphate buffer (pH6.7) with sodium azide (0.02%, w/v) and
sodium caseinate (1.25%,w/v)
3.4.6 Selection of the filter block in the fluorescence microscope 104
3.4.7 Fluorescence microscopic images of water droplets in a w/o-emulsion 105
3.4.8 Imaging of water droplets in water in oil emulsions by confocal laser scanning 106
microscopy
3.5 Determination of the enclosed water volume and yield of w/o/w-emulsions 107
3.5.1 Method optimization 107
3.5.1.1 Optimization of the preparation method 107
3.5.1.2 Finding an appropriate concentration of MnCl2 110
3.5.1.3 Investigation of the permeability of the oil phase by a temperature 110
experiment
3.5.1.3.1 Analysis of the area under the curve at different temperatures 113
3.5.1.3.2 Analysis of the signal amplitude at different temperatures 116
3.5.1.3.3 Analysis of the relaxation time at different temperatures 117
3.5.1.4 Variation of the duration of mixing of the double emulsions 121
3.5.1.5 Effect of the reduction of the duration of mixing with an Ultraturrax S25- 122
10G
3.5.1.6 Effect of quick cooling on the percentage of enclosed water volume 123
3.5.2 Method application 124
3.5.2.1 Variation of the composition of the fat phase 124
3.5.2.1.1 Effect of the fat composition on the enclosed water volume and 125
Table of contents

yield of double emulsions made by Method B


3.5.2.1.2 Effect of variation of the fat phase on the T2-distribution of 125
double emulsion made by Method B
3.5.2.1.3 Effect of the fat composition on the enclosed water volume and 126
yield of double emulsions made by Method C
3.5.2.1.4 Effect of the variation of the fat phase on the T2-distribution of 128
double emulsion made by Method C
3.5.2.1.5 Effect of the fat composition on the enclosed water volume and 129
yield of double emulsions made by Method D
3.5.2.2 Effect of the concentration of the hydrophilic emulsifier in the external 130
water phase on the enclosed water volume
3.6 Fat globule analysis by a Malvern Mastersizer S 130
3.7 Visualization of double emulsions by light microscopy 131
3.8 Research on the thickness of the separated cream layer of double emulsions 131
3.8.1 Determination of the thickness of the separated creamy layer with a ruler 131
3.8.2 Profilometric analysis of double emulsions 132
3.9 Whipping of a commercial dairy cream 136
3.9.1 Whipping time of a whipped commercial dairy cream 136
3.9.2 Overrun of a whipped commercial dairy cream 136
3.9.3 Physical destabilization (drainage) of a whipped commercial dairy cream 136
3.10 Whipping of w/o/w-emulsions 136
3.10.1 First attempt 136
3.10.2 Second attempt 137
3.10.3 Third attempt 138
3.10.4 Fourth attempt 138

General conclusions 139


List of references 141
Abstract

Abstract

A proper foundation is established with the prospect of manufacturing a food grade


recombined whipping cream by water/oil/water technology. The w/o/w technology can offer
the opportunity to produce a cream in which the oil droplets are filled with water droplets and
hence a low-energy-dense cream can be created.
In this thesis three main parts can be distinguished. In the first section, as a water-in-oil-
in-water emulsion requires an appropriate water-in-oil emulsion, research was performed on
w/o-emulsions. The composition of an emulsion is determined by the type of fat, the
emulsifier and the water content. The water droplet size was affected by the type of fat, the
emulsifier concentration and the driving air pressure during preparation. Visualization of
water droplets might be possible by light microscopy and the use of the fluorescent dye
eosinY. In the second section, the manufacturing of w/o/w-emulsions is optimized by various
ways of manufacture and characterized by T2-analysis, laser diffraction and profilometry. A
yield of about 42 to 62% could be obtained if the double emulsion was made by intensive and
less intensive homogenization during the first step of the preparation of double emulsions,
respectively. Moreover the homogenization intensity during the second step needed to be
sufficiently small. The duration of mixing in the second step and the concentration of sodium
caseinate in the external water phase affected the yield. In a third section, whipping of w/o/w-
creams was attempted. Although by the use of xanthan gum in the external water phase a
gravitational stable cream was obtained, the desired textural change during whipping was not
observed.
In the whole process, due attention is paid to proper characterization techniques, with
special focus on low-resolution NMR as a non-invasive and non-destructive method. This
technique not only enabled water droplet analysis in the primary w/o-emulsion (based on
diffusometry), but also allowed the discrimination between internal and external water in the
multiple emulsion (based on T2-analysis) and determination of the thickness of the separated
cream layer and its water content in double emulsions (based on profilometry).
Overall, it was shown that multiple emulsions with about 30wt% of dispersed phase could
be obtained with only 20wt% of fat using food-grade emulsifiers (i.e. PGPR and sodium
caseinate). In order to ensure good whipping properties, further research will be needed.
Hereby, the composition of the interfacial layers, as well as fat crystallization will be
important issues.

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Chapter 1 Literature review

Chapter 1

Literature review

1.1 Water-in-oil-in-water emulsions

1.1.1 Water-in-oil-in-water technology

A w/o/w emulsion, which is a double emulsion, consists of two non-miscible liquids, water
and oil. Small water droplets are dispersed in larger oil globules, which are themselves
dispersed in an aqueous continuous phase. Three potential benefits are the possibility of
lowering the fat content, entrapping (and releasing) therapeutic, nutritional or odourous
compounds in the internal water droplets and separation of incompatible substances (Márquez
and Wagner, 2010). In this thesis, emphasis lies on the potential of w/o/w-emulsions to reduce
the fat content. The accelerated increase in the incidence of cardiovascular diseases at
worldwide level has led consumers demand healthier low-fat food that reduces or helps to
maintain triglyceride blood levels (Lobota-Calleros et al., 2009).
Most double emulsions are very polydispersed. The range of the size of double emulsion
droplets can be quite extensive: the size of multiple droplets can be between 2-5 µm and 15-
50µm, containing respectively a few and 50-100 water droplets. In this regard, three classes
can be distinguished: type A, B and C (Figure 1.1), which differ from each other in the
amount of water droplets entrapped in the multiple droplet (Garti and Bisperink, 1998).

Figure 1.1 Three different types of entrapment of


water droplets in the multiple droplet (Garti and
Bisperink, 1998).

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Chapter 1 Literature review

1.1.2 Preparation of a w/o/w-emulsion

Multiple emulsions can be produced in a one-step or a two-step process. With regard to food
applications, a two-step process (Figure 1.2) is more common and consists of a first
emulsification step at higher shear forces than the second step, resulting in a w1/o-emulsion
and w1/o/w2-emulsion, respectively.
The second emulsification step is a critical step. A too high homogenization pressure results
in swelling and rupture of the internal water droplets (w1) and coalescence of the w1-droplets
with the outer water phase (w2) (Hindmarsch et al., 2005). A lower homogenization pressure
might result in more coalescence in virtue of too large multiple emulsion droplets.
Besides the homogenization pressure, the amount of homogenization cycles and the
temperature are important. High temperatures and a high amount of homogenization cycles,
assuming sufficient emulsifier, result in smaller multiple droplets, which might compromise
the encapsulation of water (Lindenstruth and Müller, 2004).

Figure 1.2: Scheme of the preparation of a w/o/w- emulsion


(Remon & Vervaet, 2003)

Mostly, the mixing of the w1/o-emulsion and the w2-phase is done at a lower temperature than
the preparation of the w1/o-emulsion. Water-in-oil emulsions are made at 50 to 70°C, whereas
w/o/w-emulsions are prepared at room temperature (Min et al., 2010; Lutz et al., 2009) or in
an ice bath (Frasch-Melnik et al., 2010b). O’Regan and Mulvihill (2010) prepared the w/o/w-
emulsions at 60°C.

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Chapter 1 Literature review

Emulsification can be performed by homogenization, membrane emulsification and micro-


channel emulsification.

Evaluation of the type of double emulsion can be done by dilution with water. Water will not
be miscible with an o/w/o-emulsion, whereas the reverse is true for an w/o/w-emulsion.
Alternatively, a water-soluble compound, e.g. methylene blue, can be added to the double
emulsion, followed by visual evaluation of the miscibility and/or microscopical investigation.
Methylene blue in an w/o/w-emulsion makes the sample turn blue, whereas in an o/w/o-
emulsion, this won’t have a profound effect on the color (Tirnaksiz and Kalsin, 2005).
An overview of different compositions of double emulsions in literature is given in Table 1.1.

1.1.3 Encapsulation efficiency

The encapsulation efficiency is determined by the conditions during emulsification (e.g.


homogenization pressure and temperature) and the factors affecting the release of internal
water. Regarding the latter, shear action or the presence of a concentration gradient influences
the encapsulation of the w1-phase. Depending on the direction of the osmotic gradient
swelling or shrinkage of the internal water droplets can occur (Lutz et al., 2009).
The yield of encapsulation after emulsification can be determined by the entrapment of a
marker compound in the w1-phase and its detection in the w2-phase. An overview of markers
and their detection is given in Table 1.2. For example, the entrapment or yield percentage can
be determined by conductometry and is determined by the formula:
Yield percentage= (Ci-Ct)100% / Ci
where Ci and Ct are the conductivity of the internal aqueous phase and of the multiple
emulsion at a given time t (Tirnaksiz and Kalsin, 2005).
The release of marker compounds from the inner to the outer water phase depends on its
diffusion coefficient, its initial concentration, the sphere radii (surface area of oil globule), the
viscosity of the oil phase and the solubility of the marker in the oil phase (Sela et al., 1995).

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Chapter 1 Literature review

Table 1.1: Overview of compositions of double emulsions in literature.


Lipophilic surfactant in the Concentration of Concentration Compounds in Concentration of Ratio of
Reference Oil phase oil-phase lipophilic Compounds in the w2-phase of the the w1-phase compounds in the the
surfactant in the hydrophilic w1-phase w1/o/w2-
oil phase surfactant in emulsion
w2-phase

Muschiolik et al., 2006 Sunflower oil PGPR 4%(w/v) WPI 1%(w/v) NaCl or gelatin 0.6 or 3%(w/v) 3/12/85

Su et al., 2006 Soybean oil PGPR 2%(w/v) Na caseinate 0.5%(w/v) Na caseinate 0.5%(w/v) 4/16/80

O'Regan and Mulvihill, Median chain triglyceride Na caseinate/


2010 (MCT) PGPR 2wt% Na caseinate-maltodextrin 1wt% protein gelatin and NaCl 5wt% and 0.6wt% 8/32/60

Median chain triglyceride 4% and


Lutz et al., 2009 (MCT) PGPR 10wt% WPI and pectin 0.5wt% - - 12/18/70

Frasch-Melnik et al., 2010 Sunflower oil Saturated MG, tripalmitate and 0.44wt%+0.88wt% Na caseinate 1wt% - - 3/17/80
PGPR 0.35wt%

GMO and PGPR 2.2wt% and 8.9wt% WPI 6.7wt% Glycerol 3wt% 4/16/80
Benichou et al., 2007 Median chain triglyceride
(MCT)
WPI and xanthan (4/0.5) 6.7wt% Glycerol 3wt%

Mun et al., 2010 Soybean oil PGPR 4; 6; 8wt% WPI 2/4/6wt% - - 8/32/60

Márquez and Wagner, 2010 Sunflower oil PGPR 0.5 to 2wt% xanthan gum in soy milk 0.2wt% CaCl2 0.38-1.5wt% 8/32/60
CaCO3 1.5wt%
Ca lactate 1.5wt%

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Chapter 1 Literature review

Table 1.2: Overview of different markers and techniques of detection


Reference Marker Measurement
Sela et al., 1995 2wt% NaCl/NaF/NaBr/NaI in w1-phase Conductometry of serum phase
1wt% ephedrine HCl in w1-phase

2wt% KNO3 in w1-phase

Lutz et al., 2009 4.4wt% NaCl in w1-phase Conductometry of the serum phase
15wt% Na ascorbate in w1-phase

Lindenstruth and Müller, 2004 0.5%(w/v) Na diclofenac in w1-phase HPLC after ultrafiltration

Frasch-Melnik et al., 2010b 1.6wt% KCl in w1-phase Conductometry of the serum phase

Benichou et al., 2007 2wt% glucose in w1-phase Glucometry


0.33-3g/100g emulsion vitamine B1 Differential pulse polarography

Tirnaksiz and Kalsin, 2005 1.5wt% caffeine + 0.03wt% NaCl Spectrophotometry (271nm) after dialysis
0.3wt%NaCl in w1-phase Conductometry of the serum phase

Spectrophotometry (361nm) after centrifugation


Wolf et al., 2009 0.02wt% vitamin B12 in w1-phase and filtration of the serum phase

1.1.4 Instability of w/o/w-emulsions

1.1.4.1 Thermodynamic instability of a w/o/w-emulsion

Applicability of multiple emulsions is limited by their thermodynamic instability


(Ursica et al., 2005), which is caused by the large surface free energy between fat and
water. Hence, emulsions will strive to lower the interfacial free energy by
minimization of the contact area between the phases until water is completely
separated from the fat (Jiao and Burgess, 2008; Goff, 1997). Destabilization of a
double emulsion can occur by coalescence of internal water droplets or multiple
droplets and water diffusion between the two water phases, as represented in Figure
1.3.

Muschiolik et al. (2006) defined long–term stability of double emulsions as a period


of at least 12 months. A high yield of the w1-phase, no phase alteration and no phase
separation during 8 month at +7°C are the characteristics of a high storage stability
emulsion.

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Chapter 1 Literature review

Figure 1.3: Overview of the possible


destabilization mechanisms in w/o/w
emulsions (Mezzenga et al., 2004)

Coalescence involves rupture of the film between two adjacent water or oil droplets: a
hole is formed that grows under the action of surface tension and results in the fusion
of two adjacent droplets. Rising the temperature might activate coalescence and
decrease the entrapment of water in the multiple droplets. Larger inner water droplets
promote coalescence between the interfaces of the inner water droplets and the
multiple droplets (Bibette et al., 1999). The kinetics of coalescence of inner water
droplets with the multiple droplet interface is related to the concentration of the
hydrophilic surfactant in the w2-phase phase, whereas the kinetics of the coalescence
between inner water droplets is related to the type of hydrophilic emulsifier in the w1-
phase (Garti and Bisperink, 1998).

Coalescence can be measured by measuring the turbidity. Since the larger droplets
scatter light less effectively than smaller ones, the emulsion may appear less turbid. It
can also be measured by particle size analysis. A more time consuming method is the
following method: an oil droplet can be released from a capillary tube, placed on the
bottom of an aqueous phase with at the top a planar oil-water interface. The droplet
moves upwards, reaches the oil-water planar interface and the time to merge with the
planar oil-water interface is determined with an optical microscope (McClements,
2007).
Water diffusion or Ostwald ripening from the outer to the inner water phase can result
in a bigger average inner water droplet diameter (coarsening) and a reduction in
number and is due to diffusion of water molecules across the oil layer in both
directions, which doesn’t disrupt the interfacial film (Bibette et al., 1999).

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Chapter 1 Literature review

The composition of a double emulsion can affect its stability. Garti and Aserin (1996)
reported that stable double emulsions are obtained by the use of an inner hydrophobic
emulsifier in great excess (10-30wt% of the w/o-emulsion) and an external
hydrophilic emulsifier in lower concentrations (0.5-5wt%). Increasing the latter
concentration above a certain threshold concentration resulted in a lower
encapsulation efficiency, due to an excess of osmotic pressure in the outer water
phase and the formation of reversed micelles that pumps the hydrophobic surfactant
outside into the w2-phase (Shima et al., 2004; Bibette et al., 1999). Consequently, the
w/o/w-emulsion turns into a o/w-emulsion. However, absence of hydrophilic
surfactants in the w2-phase, might not be able to prevent flocculation and coalescence
(Shima et al., 2004).

Other approaches to influence the stability of a double emulsion are the increase of
the viscosity of the inner water or oil phase, strengthening and rigidifying the
interfaces with polymeric emulsifiers and reduction of the inner water droplet size
(Garti and Aserin, 1996; Leal-Calderon et al., 2007). The viscoelasticity of the film
can be the result of the interaction between surface active lipids and folded and
unfolded proteins at the (w/o)/w-interface (Rousseau, 2000). Addition of sodium
caseinate to the w1-phase improves the stability of PGPR-based double emulsions,
which might be due to its effect on the water-oil interface (Hindmarsch et al., 2005).
The combination of a polymeric surfactant (BSA) and monomeric lipophilic
surfactant (Span 80) formed a thick interfacial layer w1/o, which improves its
elasticity and resistance to rupture (Garti, 1997).

Stability testing of a multiple emulsion can be performed by video microscopy,


whereby multiple emulsions are covered on micro slides, which makes the inner
water droplets to coalesce inside the oil droplet. This results in a dimpled structure
(Figure 1.4), because inner water droplets are pushed to the edge of the multiple
droplets, whereby the inner water droplets are still separated from the oil phase by a
thin film as long as the interfacial film is strong enough. Hence, a double emulsion is
more stable if there is a larger resistance to coverslip pressure (Jiao et al., 2002).

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Chapter 1 Literature review

Figure 1.4: Schematic representation of typical changes occurring to a multiple droplet on


application of a cover slip pressure (Jiao et al., 2002).

1.1.4.2 Water transport between the w1 and w2-phase

Benichou et al. (2004) reported three mechanisms of water transport in double


emulsions: transport through thin lamellae of surfactants, transport in reverse micelles
and transport via hydrated surfactants (Figure 1.5).
Reverse micelles are formed in the oil phase of a w/o/w-emulsion in the presence of
monomeric hydrophobic and hydrophilic surfactants, such as Span and Tween,
respectively, whereas for polymeric surfactants (e.g. BSA) this is unknown in
literature as they serve as a mechanical barrier for the transport of solutes (Sela et al.,
1995; Benichou et al., 2007). Hence, the release can be slowed down by the complex
formation of biopolymers and hydrophobic surfactants at the inner w/o-interface,
which reduces the rate of solubilization by reverse micelles (Bibette et al., 1999).
Reverse micelle formation can occur without the presence of an osmotic gradient and
doesn’t disrupt the emulsion, whereas transport through thin layers of oil destroys the
interfacial films and releases entire inner water droplets (Garti, 1997; Lutz et al.,
2009). Especially when there is an osmotic pressure difference between the two
aqueous phases, thin layer transport of water happens (Florence and Whitehill, 1982).
The stability of the oil film can be improved by increasing the concentration of the
hydrophobic surfactant (Bibette et al., 1999).
Transport via hydrated surfactants occurs by slow emulsification of water droplets in
the oil phase (Wen and Papadopoulos, 2000).

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Chapter 1 Literature review

Figure 1.5: Schematic


representation of three
transport mechanisms of
water in double emulsions.
(a) transport via reverse
micelles. (b) transport
through thin lamellae. (c)
water transport via
hydrated surfactants
(Benichou et al., 2004).

Transport rates can be reduced by increasing the viscosity of the oil phase, by
polymerization of the interfacial adsorbed surfactant molecules, by gelation of the oil
or water phases and by the presence of the right concentration of surfactants
(Schmidts et al., 2009). For example, the water permeation through the oil layer has
been reported to decrease if the oil-soluble surfactant Span 80 was present in high
concentrations (10 to 50wt% of the oil phase) in virtue of the high viscosity in the oil
phase (Wen and Papadopoulos, 2000). Complex formation between proteins and
polysaccharides in the w2-phase decreases the release rate (Muschiolik, 2007).
Addition of gelled gelatin to the w1-phase slowed down the movement of the w1-
phase to the w2-phase (Muschiolik et al., 2006). In the study of Muschiolik et al.
(2006) about multiple emulsions, an increase in PGPR concentration had a reducing
effect on the release of components from the w1-phase. Limited release was obtained
with 4%(w/v) PGPR. This concentration could be reduced to 2%(w/v) by addition of
0.5%(w/v) sodium caseinate in the w1-phase (Muschiolik, 2007).

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Chapter 1 Literature review

1.1.4.3 Gravitational instability of a w/o/w-emulsion

Regarding gravitational instability, according to the law of Stokes, creaming depends


on the density difference between the multiple droplets and the external water, the
multiple droplet size and the viscosity of the w2-phase. The density of the oil phase
can be changed by altering the type of oil or by adding fat crystals (McClements,
2007). However, sub-micron fat crystals can stabilize a w/o-emulsion, but regarding
an o/w or a w/o/w-emulsion, the control of the concentration of these fat crystals is
crucial. A scraped surface heat exchanger can be applied to selectively bring the
crystals at the w1/o-interface and not at the o/w2-interface. If they would be located at
the latter position, aggregation and coalescence might occur. In order to render fat
crystals amphiphilic or adsorbable at the interface, surfactants (monoglycerides,
lecithin) are needed. On addition of PGPR, the crystals are displaced and the interface
consists of mainly PGPR and some crystals, which increases the permeability of the
interface and allows rapid swelling when an osmotic gradient is applied. Hence, the
presence of crystals become superfluous (Frasch-Melnik et al., 2010b).
Monoglycerides and diglycerides will form the membrane around the fat globule and
can increase the density depending on the adsorbed amount, which can decrease the
rate of creaming (Goff, 1997).
The viscosity of the w2-phase can be increased by the use of polymeric compounds
(Lutz and Aserin; 2008).
Creaming can be monitored by measurement of the separation of the serum and cream
layer (Figure 1.6). At time zero, the multiple droplets are homogeneously distributed
in the sample and characterized by a concentration Co. After a while, the emulsion
separates into three distinguishable layers: a serum layer with a lower droplet
concentration than Co, an emulsion layer with a multiple droplet concentration equal
to Co and a cream layer with a larger multiple droplet concentration than Co. Besides
visual observation of the boundary of the layers, also light scattering measurement
can be applied, although in a concentrated emulsion (>10% droplets) this does not
change greatly with increasing droplet concentration. Alternatively, creaming can be
studied by the physical sectioning method, which is a destructive method that requires
freezing of the emulsions. As such the droplet concentration in each frozen section is
measured. The sample can also be divided into pieces by collecting successive
aliquots of the emulsion stored in a burette or separation funnel. When the creaming is

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Chapter 1 Literature review

monitored as a function of time, e.g. by video imaging or by making photographs over


time, the creaming rate can be determined (McClements, 2007).

Figure 1.6: Schematic representation of creaming as a function of time in a (w/o)/w emulsion


(McClements, 2007).

1.1.5 Desired characteristics of a whippable w/o/w-cream

A whipping cream should be quiescently or perikinetically stable prior to whipping


and unstable when sheared during whipping. The resistance against shear induced
destabilization is defined as orthokinetic stability (Goff, 1997; Davies et al., 2001).
In view of sensory resemblance to whippable dairy cream, the size of multiple
droplets of a w/o/w-emulsion should be similar to the oil globules of the dairy cream
(Frash-Melnik et al., 2010).

1.2 Traditional dairy whipping cream

Dairy cream, an oil-in-water emulsion can be defined as the part of milk that is rich in
fat, separated from raw milk by centrifugation at speeds of 4700-6500 rpm, resulting
in cream and skimmed milk (Hoffmann, 2002). Besides a heat treatment, the cream
can be homogenized at a low pressure, which prevents it from creaming and thus

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Chapter 1 Literature review

destabilization. Non-homogenized fat globules are surrounded by a milk fat globule


membrane (MFGM), which prevents them from coalescing (Mulder and Walstra,
1974). The MFGM consists of approximately 25 to 60% of proteins (e.g.
butyrophillin and xanthin oxidase), lipids (e.g. phospholipids) and other compounds,
which results in a thickness between 10 and 50nm (Hui et al., 2007). A
homogenization process disintegrates the MFGM and the fat globules will be
surrounded by mainly caseins.
In the absence of stabilizing additives, the process of whipping of cream is usually
performed in situ by the consumer with a domestic electric mixer. After whipping, the
cream already collapses after 24 to 48h (Allen et al., 2008; Leal-Calderon et al.,
2007).
With regard to the legal aspect of defining cream as a whipping cream, the minimum
fat content matters and differs across countries (Table 1.3).

Table 1.3: Different national agreements of the definition of whipping cream.


Country Fat % Law
Belgium Min 40% Regulation trade of cream, Royal Decree May 23rd, 1934.
The Netherlands Min 30% Commodities Act Decree of dairy products, Article 16, 1994
United Kingdom Min 35% Food Labeling Regulations, Cheese and Cream Regulations, No. 52, 1996

1.3 Changes during whipping of cream

Whipping and the introduction of air destabilize the oil-in-water emulsion, because
the coalescence of fat globules is favored by agitation (Leal-Calderon et al., 2007).
The resulting foam is an emulsion in which the dispersed phase is a gas, from which
the air-water interface is stabilized by fat globules (Schmitt et al., 2005). However,
also in absence of air, cream can be whipped.

1.3.1 Three stages during whipping of cream

Three stages can be distinguished during whipping of cream. In the first stage, most of
the air is incorporated and the foam is protein-stabilized. In the second stage, the
bubbles are coated by a layer of fat globules. The high packing density of fat globule-

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Chapter 1 Literature review

coated gas bubbles inhibits further incorporation of gas. A simultaneous disruption


and coalescence of air bubbles take place. However, the result is a reduction of bubble
size (mostly 10-100 µm in diameter) and distribution (van Aken, 2001; Mulder and
Walstra, 1974) until maximum foam strength is achieved (Stanley et al., 1996).
During air bubble coalescence, the adsorbed fat globules are pushed together at the
bubble surface, flocculate and partially coalesce in clumps at the air-water interface
(Allen et al., 2008b) (Figure 1.7 and Figure 1.8).

Figure 1.7: A floccule and a clump of fat globules. Note that the
identity of the original globule in the clump is still retained
(Mulder and Walstra, 1974).

In the third stage, on prolonged whipping, the formed aggregates of fat globules
become so large that they are expelled into the continuous phase and hence the gas
bubbles are quickly destabilized, which results in a bimodal bubble size distribution
(Jakubczyk and Niranjan, 2006). The consequence is a rapid loss of air and a
phenomenon called churning, which is the formation of butter grains present in a
phased inversed emulsion (van Aken, 2001). The rate and efficiency of beating
determine the balance between whipping and churning. If beaten too slowly, the
cream may churn before a satisfactory foam has been formed. Vigorous beating leads
to high overrun and a foam with small air bubbles (Mulder and Walstra, 1974).

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Chapter 1 Literature review

1.3.2 Foam stabilization by partial coalescence

As discussed in section 1.2.1, partial coalescence of fat takes place in the second stage
of whipping. It results in a rigid network in which bubbles are linked to one another
and liquid is entrapped (Mulder and Walstra, 1974). It prevents the full coalescence
into bigger fat globules that are not capable of structure-building (Mulder and
Walstra, 1974). The fat crystals break and penetrate the interfacial layer around the fat
globules in the emulsion, allowing globules to clump irreversibly together into a
network, whilst the identity of the original globule is retained (Allen et al., 2008a)
(Figure 1.8).

Figure 1.8: (Left) Rigid network due to partial


coalescence of fat globules (Goff, 2011). (Right)
Fat droplets flocculate and partially coalesce into
clumps (A) at the protein-covered air bubble. If
the air bubble bursts, a partially coalesced fat
clump remains (C). If the air bubble remains
stable, fat clumps from the bulk may partially
coalesce with the adsorbed fat clump (B) (Hotrum
et al., 2005a).

Hereby, the presence of partial crystalline fat, which is of a needle or platelet shape in
milk fat, is a crucial factor. The shape of the fat crystals is related to the cooling rate.
A slow cooling rate, e.g. 0.1°C/min, creates a small number of irregularly shaped
crystals, which are characterized by poor coverage of droplet interfaces and are
beneficial for partial coalescence. Rapid cooling, e.g. 10°C/min, promotes the
formation of a lot of fine crystals which are able to form a rigid dense coverage of the
interface and to stabilize the emulsion against coalescence (Frasch-Melnik et al.,
2010a).

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Chapter 1 Literature review

However, almost completely solid fats, e.g. 80%, are not able to practice a spreading
function at the air-water interface, nor can form clumps and hence are not efficient at
stabilizing foam (Dalgleish, 2006; Mulder and Walstra, 1974). Davies et al. (2000)
concluded that partial coalescence of fat droplets is maximized when the solid fat
content is approximately 10-50% (Figure 1.9). Besides the amount of crystalline fat,
also the fat volume fraction is of importance. The rate of partial coalescence is
proportional to the squared fat volume fraction (Fredrick et al., 2010).

Figure 1.9 (Left) rate of partial coalescence as a function of solid fat content (McClements, 2007).
(Right) Correlation between whipping time (emulsion stability) and solid fat content of a 30% fat
emulsion as a function of storage time (ageing time) at 10°C. Black dots refer to the liquid fat
content and white dots refer to whipping time (Darling, 1982).

The fat globule-stabilized air bubble layer (Figure 1.10) will remain intact as long as
the crystals don’t melt. A partial liquid part of fat globules is needed to make adhesion
and partial wetting of the air bubble surface possible. A too high liquid fraction of fat
is characterized by too rapid churning during whipping. Moreover, the excessive
spreading of the liquid fat over the air bubbles prevents the formation of sufficiently
small air bubbles (Mulder and Walstra, 1974).

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Chapter 1 Literature review

Figure 1.10: Scanning electron micrograph of an air bubble


which is stabilized by fat droplets in whipped cream. The scale
bar represents 20 µm (Dalgleish, 2006).

1.3.2.1 Determination of partial coalescence

There are different methods to determine partial coalescence. Firstly, partial


coalescence can be measured by a dye solution absorbance method. It comes down to
the determination of the change in absorbance of a lipophilic dye solution due to the
dilution by free fat. During partial coalescence aggregates of fat globules, which are
considered as free fat, are formed. The Palanuwech method uses the dye Oil Red O
(0.0015wt% in oil), which absorbs maximally at 520nm. Pure oil phase is taken as
blank. The dye solution is poured onto the surface of the emulsion under
investigation, gently mixing and allowing the colored oil to float at the surface under
gentle centrifugation. The free fat in the emulsion will be dissolved in the colored oil,
but the emulsified fat remains in its droplets. After transfer of the diluted-dye solution
fraction from the surface, its absorbance is measured. Out of the change in
absorbance, the free fat fraction in the emulsion can be calculated (Palanuwech et al.,
2003).
Secondly, turbidity can be measured to investigate the aggregates of fat globules
(Kiokas et al., 2004).
Thirdly, partial coalescence can be investigated by measuring the size of clumped fat
droplets. In a study of Kiokas et al. (2004) this was done with pfg-NMR and
supplemented with scanning electron microscopy imaging.

1.3.2.2 Distinction between partial coalescence and complete coalescence

Regarding completely coalesced fat globules, the identity of original fat globules is no
longer retained. Partial coalescing results in an increase of volume fraction and hence

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Chapter 1 Literature review

viscosity, whereas the viscosity doesn’t increase when two droplets coalesce
completely. Another difference lies in the fact that an applied velocity can increase
the rate of partial coalescence without affecting the rate of complete coalescence
(Fredrick et al., 2010).

1.3.3 Alternatives to partial coalescence

Like stated above, stabilization of the foam structure can be done by partial
coalescence of semi-crystalline fat globules, though this is not the only mechanism
(Allen et al., 2008a). An alternative foam-stabilizing method concerns fat globule
aggregation induced by acidification in the presence of a small molecule weight
lipophilic surfactant LACTEM (lactic acid esters of monoglycerides)(0.25wt%). In
protein-stabilized emulsions, a reduction of pH towards the protein’s IEP, diminishes
the electrostatic stabilization and enhances protein-protein interactions. This results in
a protein-stabilized fat globule network, in which the fat droplets are not partially
coalesced but occur as distinct entities, which is similar to the production of yoghurt.
The addition of an emulsifier partially displaces the adsorbed casein at the oil-water
interface, which induces the formation of strong interdroplet crystal–crystal
interactions and fat droplet aggregates upon whipping. The advantage of this method
is that the amount of solid fat enabling whipping can be reduced. Still, its presence is
essential in terms of achieving a similar rigidity as traditional whipped cream (Allen
et al., 2008a).
Márquez and Wagner (2010) concluded that an unwhipped w/o/w emulsion based on
liquid oil, PGPR and 0.12wt% CaCl2 or calcium lactate in the primary emulsion and
0.2wt% xanthan gum in soybean milk as an external water phase, offers an alternative
for whipped dairy cream because of its creamy texture obtained directly after
preparation, which is similar to whipped dairy cream. This is explained by the
swelling of the internal water droplets in the presence of soluble calcium salts in the
w1-phase and the increase of consistency due to the osmotic gradient and by the
interaction of released calcium with soybean proteins at the o/w2-interface, which
results in the flocculation of w/o droplets.

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Chapter 1 Literature review

1.4 Mimicing whipping cream

Recombined cream is an emulsion of butterfat or a higher melting fat stabilized by


skimmed milk products (Hotrum et al., 2005a). If a reconstituted cream contains other
non-dairy sources of fat, it is denoted as a filled cream (Hui et al., 2007).
In order to mimic a whipping cream, partial coalescence can be influenced by the type
and amount of emulsifier in the formulation. For example, proteins in a certain
concentration can reduce the susceptibility of the emulsion to partial coalescence by
formation of a thick layer around the fat globules, which increases the repulsive forces
and the resistance to penetration of fat globules by fat crystals (McClements, 2007).
Segall and Goff (1999) reported a too high stability against whipping for WPH or
SMP-stabilized emulsions. Hydrolysis of whey proteins (WPH) exposes previously
buried hydrophobic sites, which increases its surface activity in comparison to WPI.
SMP consists of large casein micelles which result in a thick layer of surface
coverage, which makes it more resistant to shear. A similar change in surface activity
can be noticed by denaturation of whey proteins. Consequently, an increased fat
membrane integrity and hence, increased stability against partial coalescence can be
observed (Goff, 1997). WPI and sodium caseinate-stabilized emulsions resulted in a
larger susceptibility to partial coalescence than SMP-stabilized emulsions (Goff,
1997; Leser and Michel, 1999). However if more than 0.5% WPI was present, a too
strong film around the fat globules was formed and partial coalescence was impaired
(Leser and Michel, 1999).
Van Lent et al. (2008) investigated the differences between recombined creams made
of different protein sources: skimmed milk powder (SMP) and cream residue powder
(CRP) (Table 1.4). In SMP-creams casein micelles act as surface active material,
whereas in CRP-creams small molecular weight surfactants and whey proteins
stabilize the interface of the o/w emulsion. Moreover, more stabilization by CRP-
surface material per unit weight emulsifier is achieved than casein micelles do in
SMP-cream. With regard to fresh cream, smaller fat droplets and a larger whipping
time were observed than for SMP-creams and the freeze thaw serum leakage was
smaller than for SMP- and CRP-creams. It was concluded that CRP-creams mimicked
fresh cream better than SMP-creams.

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Chapter 1 Literature review

Table 1.4: Differences in characteristics between recombined creams made of skimmed milk
powder or cream residue powder (Van Lent et al., 2008)
Characteristics SMP-cream CRP-cream
Fat droplet diameter >
Free protein content <
Whipping time <
Freeze-thaw serum leakage >
Air phase fraction <
Apparent viscosity >
Creaming rate >

Scott et al. (2003) proved that the separation temperature in obtaining the emulsifying
components, skim milk or sweet buttermilk and butter derived aqueous phase, which
are generated after churning cream that is obtained by separation at 49 or 55°C,
mattered. Mimicked creams manufactured from components at a 55°C separation
temperature were more stable than those coming from a 49°C separation temperature,
perhaps because of the more efficient separation and inactivation of agglutinins that
might cause cold agglutination. Regarding the sensory analysis, the flavor of creams
formulated with sweet butter milk or butter derived aqueous phase were said to be
rich and creamy (Scott et al., 2003).

Besides the type and concentration of protein in the emulsion, the presence of small
molecular weight surfactants influences the susceptibility to partial coalescence.
Examples of small molecule surfactants are monoglycerides, diglycerides, and
polysorbates. Also phospholipids such as soy lecithin, exceeding a minimum
concentration, can render an emulsion more susceptible to partial coalescence upon
shearing action. Small molecule surfactants act as protein displacers, due to their
better surface active properties than proteins, which results in preferential migration to
the fat-water interface (Dalgleish, 2006). Consequently, the fat globules are no longer
covered by a thick stabilizing layer, break easier during whipping and more extensive
partial coalescence takes place (Segall and Goff, 2002). In conclusion, the fat globule
membrane should not be too strong because the aim of whipping is to destabilize the
emulsion (Van Lent et al., 2008; Mulder and Walstra, 1974), which can be obtained
by addition of small molecule surfactants, which are potential destabilizers of the
emulsion.

Small molecule surfactants might also act on the morphology of the fat crystals, e.g.
mono-olein (unsaturated MAG), which doesn’t show much protein displacement, but
it results in a cream that is more susceptible to partial coalescence (Fredrick et al.,

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Chapter 1 Literature review

2010). Hotrum et al. (2005b) noticed shorter whipping times upon addition of small
molecule surfactants. In comparison to non-isolated whey products, caseins are more
readily displaced by surfactants, whereby water soluble surfactants are more effective
in doing this than oil-soluble surfactants (Cornec et al., 1998).

Instead of the application of small molecule surfactants, Goff (1997) promoted partial
coalescence by preparing an emulsion stabilized by milk proteins in such a
concentration that a weak yet sufficient interfacial layer stabilized the emulsion
during storage, which could be destabilized during a whipping process (Goff, 1997).
Additionally, additives that act on the properties of the continuous phase can be
added. Carrageenan in a concentration of 0.02% can prevent creaming by increasing
the viscosity. It reduces the drip volume in whipped creams without affecting the
whipping properties (Kováčová et al., 2010). Xanthan gum, an anionic
polysaccharide, increases the viscosity of the emulsion. In a concentration of 0.1% it
doesn’t affect the overrun (Zhao et al., 2009).
Concerning the stability of mimicked creams, Parkinson and Dickinson (2007)
suggested that the change of a 3wt% beta-lactoglobulin stabilized emulsion (45vol%
n-tetredecane, o/w-emulsion) into a 2.97wt% beta-lactoglobulin stabilized emulsion to
which 0.03wt% sodium caseinate was added, can improve the shelf-life of imitation
cream. The researchers observed an enhancement of long-term (up to 1.5 years)
stability, which was measured by looking at the phase separation.

1.5 Quality characteristics of whipped cream

Quality parameters of whipped cream are the increase in volume or overrun, the
whipping time or whipping rate, the texture of the whipped cream, its physical
stability (i.e. the extent of drainage and coarsening), and the chemical and
microbiological stability (De Meulenaer et al., 2009; Templeton and Sommer, 1932;
Lampert, 1975). An overview of the changes of some quality parameters during
whipping of cream is given in Figure 1.11.

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Chapter 1 Literature review

Figure 1.11: Changes of some quality parameters during whipping of cream. The parameter of
firmness is related to the time needed to lower a weight into the product. Leakage is the amount
of liquid drained from a certain volume in a certain time. Between the broken lines the product
is acceptable. Curves are approximate (Mulder and Walstra, 1974).

1.5.1 Overrun

Overrun depends on the effectiveness of the introduction of gas during the first stage
of whipping. According to Graf and Müller (1965) a well whipped cream should have
an overrun of approximately 50-60%. Overrun can be determined after a fixed time
period or the cream can be whipped until maximum overrun. Maximum overrun
corresponds with maximum stability and stiffness of the foam, and all air bubbles at
this point are encapsulated by coalesced fat droplets which adsorbed at the air-serum
interface (Jakubczyk and Niranjan, 2006).
The amount of emulsifier can affect the overrun, e.g. in a study of Zhao et al. (2008)
the highest overrun was obtained with 0.7% sodium caseinate in a recombined cream
that consisted of hydrogenated palm kernel oil, stabilizers, proteins, sucrose esters and
sugar slurry. As soon as the concentration exceeded 0.9% sodium caseinate, the foam
of the cream collapsed and hence the overrun decreased. Adding stabilizers, e.g.
0.05wt% λ-carrageenan or 0.1wt% locust bean gum (LBG) to a casein based dairy
cream decreases air incorporation capacity of the cream in comparison to creams
without these stabilizers. Despite this disadvantage, they also increase the serum
phase viscosity and interact with proteins in the cream, leading to a decrease of the
serum drainage and whipping time (Camacho et al., 1998).

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Chapter 1 Literature review

1.5.2 Whipping time

The whipping time depends on the rapidity with which the partially coalesced
network of fat globules can be built up. Shorter whipping times are achieved when the
cream is whipped at higher rate (rotational speed of the whisks) (Hotrum et al.,
2005b). Templeton and Sommer (1932) defined the whipping time as the time needed
until desired stiffness, which was determined by observing the torque (load) on the
drive shaft of the whipping apparatus. In van Aken’s (2001) experiment, the endpoint
of whipping was set at the maximum of a peak in the electrical current, related to a
maximum of stiffness. Whipping time in the study of Van Lent et al. (2008) was
based on visual inspection, until a maximum overrun was reached. Ihara et al. (2010)
defined the endpoint of whipping as when a certain cone penetration depth was
reached.
A clear correlation exits between the whipping time (emulsion stability) and the solid
fat content of the emulsion (Darling, 1982) as shown in Figure 1.9.
Just like for overrun, the whipping time can be affected by the type and concentration
of emulsifier and stabilizer in the cream. In a study of van Hotrum et al. (2005b),
shorter whipping times were obtained with 1wt% whey protein isolate stabilized
emulsions than with 1wt% sodium caseinate. The mimicked cream consisted of
40wt% fat. Whey protein isolate (WPI) consists of mainly beta-lactoglobulin, which
forms brittle adsorbed layers at the air-water interface, whereas beta-casein forms
more fluid like layers. Addition of 2.7mM and 5.5mM Tween 20 to the mimicked
cream reduced the whipping time to such an extent that it was similar to the whipping
time of natural cream at the same rotational speed. The lower concentration of Tween
20 resulted in a higher overrun than the higher concentration (Hotrum et al., 2005b).
The lowest whipping time was obtained without locust bean gum or λ-carrageenan in
dairy cream, suggesting that these stabilizing additives cause kinetic hindrance to the
cream foaming, which could be due to not only the increase in the viscosity of the
liquid phase, but also to stabilizer-protein interactions that could partially inhibit the
foaming properties of milk proteins (Camacho et al., 1998).

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Chapter 1 Literature review

1.5.3 Textural analysis

Whipping converts the viscous liquid emulsion into a stiff aerated viscoelastic solid
(Allen et al., 2008b). The viscoelastic behavior can be analyzed by measuring
rheological properties, the storage (G’) and loss modulus (G”), which both increase
when air is introduced (Jakubczyk and Niranjan, 2006). Also the viscosity,
cohesiveness and consistency are texture parameters. A back extrusion test and a flat
base plate rheometer can be used to study the structure of semisolid food and to
quantify the shelf life of whipped creams (Piazza et al., 2009; Allen et al., 2008b).
Differences in firmness and viscosity can exist between creams made of different
proteins. Zhao et al. (2008) found that creams with whey proteins were characterized
by a smaller increase in viscosity during whipping and a lower firmness after
whipping than sodium caseinate-whipped creams. The foam structure of dairy cream
can be better preserved during chilling when higher concentrations (>0.05%) of
additives such as λ-carrageenan or locust bean gum are applied. Higher cream
viscosities can be obtained with λ-carrageenan in comparison to locust bean gum, due
to protein-polysaccharide interactions (Camacho et al., 1998).

1.5.4 Physical stability

Just like emulsions, foams are never thermodynamic stable, the destabilization can
only be kinetically slowed down. Two (physical) destabilization processes can take
place: coarsening and drainage (Indrawati et al., 2008; Butt et al., 2006).

1.5.4.1 Coarsening of foam

During Ostwald ripening, which is a thermodynamically favourable process, diffusion


of gas through liquid films from small to large compartments is taking place, which is
driven by the pressure difference between them (Butt et al., 2006; Dalgleish, 2006).
The resulting phenomenon of coarsening or disproportionation can be slowed down
by having equally sized air bubbles, but this is not feasible with normal kitchen
equipment. Other options are increasing the viscosity of the liquid phase with

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Chapter 1 Literature review

hydrocolloids or strengthening the interfacial layer around the gas bubbles with
surface-active proteins. Although globular proteins favor foam formation and stability
due to interfacial unfolding, the most successful way is to coat air bubbles with semi-
crystalline fat globules, undergoing partial coalescence at the air-water interface
(Dalgleish, 2006; Schmitt et al., 2005; Indrawati et al., 2008).
Coarsening can be studied by measuring the increase in transmitted intensity in a laser
light scattering-CCD camera experiment (Saint-Jalmes et al., 2005). Coarsening can
also be evaluated by microscopical image analysis, facilitated by low T (for
preservation of the original foam structure), scanning electron microscopy (SEM) and
computer assisted quantitative stereology (Smith et al., 1999).

1.5.4.2 Drainage

According to Belitz (2009), no serum separation should occur at 18°C after 1h in


order to have qualitative whipped cream. Due to the pressure difference between the
inside of a bubble and the liquid film, serum drainage or syneresis occurs. The
pressure inside the liquid film is significantly smaller than in the air compartments,
resulting in liquid being sucked into the ‘Plateau border’, which is the channel at the
contact line between liquid films (Figure 1.12).

Figure 1.12: Plateau


border in foam (Butt et
al., 2006)

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Chapter 1 Literature review

Once the liquid reaches a Plateau border, the downward flow, driven by gravity,
becomes substantial (Butt et al., 2006). Liquid flow can be slowed down by increasing
the viscosity of the liquid, e.g. by adding xanthan gum, or by keeping the temperature
low (Mulder and Walstra, 1974; Indrawati et al., 2008).
In Table 1.5 a summary of different drainage analysis methods mentioned in literature
is given.
The serum leakage can also be determined after a freeze-thaw experiment, which is an
example of an accelerated stability test. In Van Lent et al. (2008) cups with whipped
cream were frozen at -18°C for 24h and placed upside-down on an aluminum dish at
16-18°C. After 15 min, the cup was removed and the cream weight was determined.
The serum was poured out and weighted after 150 minutes and the serum leakage
(ftSL) was determined as the mass of serum over the mass of whipped cream, times
100% (Van Lent et al., 2008).

Table 1.5: Overwiew of drainage analysis methods in literature


Reference Drainage analysis method
Allen et al., 2008b Serum drainage stability is the amount liquid drained under gravity from 10 g
sample over 5h period.
Templeton and Sommer, The drainage time is the time between placing the cream in a funnel and the first
1932
drop of serum falling from the end of the glass stem of the glass funnel. The drain
amount is gravimetrically determined.
Van Aken, 2001 Serum loss was measured by placing 30g whipped cream on a sieve and measuring
the volume of serum leaking that passes through the sieve during 2h at 20°C.
Shamsi et al., 2002 40g whipped cream is immersed into a warm water bath at different temperatures
for 6h. Measurement of separated serum in mm.
Van Lent et al., 2008 The drainage stability was determined by placing 50g whipped cream on a sieve
with openings of 1mm. After 1h, at 16-18°C, serum leakage (SL) was determined
as the mass serum over the mass whipped cream, times 100%.

1.6 Factors determining functional properties of whipping cream

1.6.1 Temperature

During whipping of cream, the presence of a certain proportion of fat solidification is


crucial for partial coalescence of fat globules and firmness of the whipped cream,
hence, temperature is important (Figure 1.13).

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Chapter 1 Literature review

Milk fat is a mixture of many different triacylglycerols (about 98%) with individual
melting points and thus it has an extensive melting range of -40°C to 40°C (Smet,
2010). Generally the fat should be partially solid at 5°C, solid enough at ambient
temperature and melt at temperatures below 37°C (Shamsi et al., 2002). Prior to
whipping, it is advised to keep cream, metal bowl and beaters below 7.2 °C (Lampert,
1975).

Figure 1.13: Effect of whipping temperature (in °C) on the


firmness (yield stress, g/cm2) of whipped cream of different
fat contents (%) (Mulder and Walstra, 1974).

1.6.1.1 Tempering

The stability of whipped cream, based on crystallizable oils with a large melting
range, can be enhanced by a process called cycling or tempering. This treatment
implies a temperature increase of the whipped cream to 25-30°C immediately after
whipping and subsequently the cream is cooled down. A clearly stiffened foam,
storable at 4°C for several weeks without any visible structural change (no fat or gas
segregation) can be observed, especially for native dairy creams with 20-40wt% fat
(Leal-Calderon et al., 2007). By contrast, non-tempered whipped cream collapses
already after 48h (Gravier et al., 2006).
In detail, the increase in temperature and a holding time of 5 minutes doesn’t melt all
of the crystals, hence the foam will not be collapsed. Upon cooling, the remaining
crystals serve as catalytic impurities, which results in a controllable nucleation and
increase in crystal size, which can be achieved without deep supercooling (Fredrick et
al., 2010). Drelon et al. (2006) could not relate tempering with polymorphism of fat
nor with an increase of the solid fat content. Though they could report a higher
increase of partial coalescence of whipped tempered creams upon whipping in
comparison to non-tempered whipped creams, which was measured by laser

27
Chapter 1 Literature review

diffraction after application of a heating step to transform the partially coalesced fat
globules into fully coalesced spherical fat globules (Drelon et al., 2006).

1.6.1.2 Heat treatment

The thermal treatment might have an effect on the whipping properties of cream.
Higher temperature pasteurization denatures whey proteins, which become unfolded
and interact with kappa-casein on the fat globules in order to form a complex that
makes the fat globules stable against partial coalescence (Bruhn and Bruhn, 1988;
Smith et al., 1999). Regarding gravitational stability, the complex formation is related
to a decreased creaming rate due to an increase of the density of the fat globules
(Parkinson & Dickinson, 2007).
UHT-treated cream exhibited larger fat globules than HTST-treated creams. The
former was associated with lower overrun and lower foam stability (Smith et al.,
2000).

1.6.2 Fat content

Whipped dairy cream should not contain less than 30% fat in order to form a stable
network capable of enclosing air bubbles. As illustrated in Figure 1.14, an increase in
fat content results in a shorter whipping time, a higher firmness and less serum
leakage (Mulder and Walstra, 1974), but a fat content of more than 38% decreases the
overrun and doesn’t improve the foam firmness any longer (Lampert, 1975). Whipped
creams with less 30% fat are characterized by a long whipping time and an increase of
the rate and extent of serum drainage (Templeton and Sommer, 1932).

1.6.3 Homogenization

During whipping of homogenized dairy cream, the fat globule membrane is


disintegrated twice. The first time happens when the cream is homogenized, resulting
in a newly formed membrane, the second time occurs when the cream is whipped.

28
Chapter 1 Literature review

The newly formed membrane gradually disintegrates and the liquid fat is released and
cements the remaining fat globules.

Figure 1.14: Properties of whipped cream. From left to right: whipping time (minutes), overrun
(%), firmness, leakage of liquid (ml) as a function of fat content for conventional whipping cream
( ____ ) and for a cream with surfactants ( ----). Data are approximate (Mulder and Walstra,
1974).

Apart from the beneficial effect on the color (more white) (Hui et al., 2007) and the
creaming rate of cream by reduction of the fat globule size, homogenization impairs
whipping properties, particularly for a low fat cream: whipping time is much longer
and the foam is less firm. The clumping tendency of the homogenized fat globules is
probably too low, so more fat globules are needed to form a clump (Mulder and
Walstra, 1974).
Two-stage homogenization is used for the manufacture of recombined creams. The
first stage at higher pressure creates smaller fat globules, increases the rate of fat
globules collision and promotes the formation of heat-stable clusters (Figure 1.15),
which are more pronounced with increasing fat content and will separate much
quicker than non-aggregated fat globules (Mulder and Walstra, 1974). In the second
stage at lower pressure fat globules clusters are broken up, while avoiding the
destruction of the single fat globules.

1.6.4 Miscellaneous factors

Aging affects the rearrangement of the composition of the protein membrane in


homogenized cream (Darling, 1982) and it allows time for fat crystallization (Segall
and Goff, 2002).

29
Chapter 1 Literature review

Figure 1.15: Homogenization clusters in homogenized cream (Mulder and Walstra, 1974)

30
Chapter 2 Materials and methods

Chapter 2

Materials and methods

2.1 Commercial butters

Two types of commercial butters with a fat content of 82% were analyzed with the
Maran Ultra 23 spectrometer: an organic churned butter, Bio Karneboter (Delhaize)
and a cream butter, (Zachte) Ardense Roomboter (Carlsbourg), as illustrated in Figure
2.1. Bio Karneboter contains less than 0.1% salt and on the package of Ardense
Roomboter nothing is mentioned about the salt content. The salt content of salted
butters ranges from 0.5 to more than 3%, so the commercial butters used in this
experiment can be assumed to be non-salted.

Figure 2.1: Analyzed commercial butters: Ardense Roomboter and Bio Karneboter

2.1.1 Preparation of butter samples

Calibration of pfg-NMR requires a pure fat sample and a dispersed phase sample. The
separation of the phases was obtained by melting 125g of butter in an oven at 45-50°C
and by centrifugation at 2800g for 20 minutes, resulting in clarified butter oil at the
top and serum at the bottom of the recipient. Two glass NMR-tubes were filled to the
marked line (8mL) with fat and dispersed phase respectively, by using a syringe with
long needle to reach the separated phases.

31
Chapter 2 Materials and methods

Three glass NMR-tubes of each commercial butter were filled to the marked line
(8mL) with butter samples by using a cheese trier.

2.1.2 Analysis of butter samples

The samples at 5°C were analyzed in a Maran Ultra 23 spectrometer, which was set at
-7°C in order to get +5°C in the probe of the spectrometer. Water droplet size was
obtained with the software Droplet Size application (Resonance Instruments Ltd),
Excel and Matlab (see 2.3.3).

2.2 Water-in-oil emulsions

2.2.1 Materials needed for the preparation of w/o-emulsions

The materials used are a lipophilic emulsifier (polyglycerol polyricinoleate,


Palsgaard® 4150, Palsgaard A/S, Denmark), a hydrophilic emulsifier (sodium
caseinate, kindly provided by Armor Protéines, Saint Brice en Cogles, France), high
oleic sunflower oil (Hozol, Contined B.V., Bennekom, The Netherlands), soft-
palmitine mid fraction (Figure 2.2) (soft PMF, Unigra Sp., Conselice, Italy), a buffer
solution and an anti-microbial agent (sodium azide, Acros Organics, Geel, Belgium).
The buffer solution was made, according to the Henderson-Hasselbalch equation and
an aimed pH of 7, by mixing KH2PO4 (Merck KGaA, Darmstadt, Germany) and
K2HPO4 (Alfa Aesar, Karslruhe, Gemany) solutions of equal molarity (0.1M) in a
volume ratio 1:0.63. This pH avoids flocculation of sodium caseinate in the water
phase (I.E.P. is 4.6). By means of a pH-meter, the pH of the buffer solution at room
temperature was measured and amounted to 6.7. The difference in measured and
intended pH might be explained by the applied calibration liquids. Some emulsions
were made with whey protein isolate (WPI, BiPro, Davisco, Le Sueur, USA) instead
of sodium caseinate.

32
Chapter 2 Materials and methods

2.2.5 Composition of the w/o-emulsions

Different emulsions were obtained by varying the concentration of hydrophilic


emulsifier, lipophilic emulsifier, the composition of the oil phase and the water-to-oil
ratio.

Palm Oil

Stearin Olein
fraction fraction

Hard Soft Super


stearin PMF Olein

Hard Mid
PMF olein

Figure 2.2: Fractionation scheme of palm oil

The composition of different emulsions with 20% water (w/w) is represented in Table
2.1. In Table 2.2 shows the composition of emulsions with water to oil-ratios (w/w)
varying from 20% to 60%. The codes of the emulsions start with a letter,
corresponding to the first letter of the composition of the oil phase, followed by two
numbers, separated by a slash. The first number refers to the percentage (w/v) of
sodium caseinate in the water phase and the second refers to the percentage (w/v) of
PGPR in the oil phase.
The water phase was made by weighing sodium azide (all emulsions) and sodium
caseinate (some emulsions) in a volumetric flask and was filled with buffer solution to
the marked line. The oil phase was made by weighing PGPR in a volumetric flask and
filling with Hozol (at room temperature) or soft PMF (heated to 60°C) or both (at
60°C) to the marked line.

33
Chapter 2 Materials and methods

Table 2.1: Composition of the 20% (w/w) W/o-emulsions (components in %, w/v)


W/o-emulsion H0/1 H0,5/1 P0/1 P0,5/1 P0,5/2 M0/1
Water phase
Sodium azide (%) 0.02 0.02 0.02 0.02 0.02 0.02
Sodium caseinate (%) 0.00 0,50 0.00 0,50 0,50 0.00
Buffer solution (pH 6.7) (%) ad 100 ad 100 ad 100 ad 100 ad 100 ad 100
Oil phase
PGPR (%) 1 1 1 1 2 1
Hozol (%) 99 99 0 0 0 49.5
Soft PMF (%) 0 0 99 99 98 49.5

Table 2.2: Composition of w/o-emulsions with different water to oil ratios. The oil phase consists
of Hozol. The water phase contains sodium azide (0.02%, w/v) and a phosphate buffer (pH 6.7).
Percentage water (%, w/w) in the w/o-emulsion
20 30 40 50 60
Water phase
Sodium caseinate (%,w/v) 0.50 0.75 1.00 1.25 1.50

Oil phase
PGPR (%, w/v) 1.00 1.50 2.00 2.50 3.00

2.2.6 Preparation of the w/o-emulsions

An Ultraturrax (type TV45, IKA) and a Microfluidizer (type M110S, Cobra


Engineering NL) (Figure 2.3) were used to premix and homogenize the emulsion at
60°C, respectively. To comply with the demands from good practicing when using the
Ultraturrax, about 75mL of emulsion was prepared, which comes down to weighing
52g oil phase and 13g water phase in a beaker. The Ultraturrax was set at position 1 to
2 and the emulsion was premixed for 1 minute. The emulsion was homogenized in the
Microfluidizer at an air pressure of 6bar for 1.5minutes. All emulsions were analyzed
in triplicate with a fresh emulsion prepared for each replicate.
When preparing an emulsion containing soft PMF or a mix, special care should be
given to preheat the Microfluidizer with boiling water, in order to prevent obstruction,
as presented in Flow chart 2.1.

34
Chapter 2 Materials and methods

Piston

Figure 2.3: Schematic representation of the Microfluidizer M110S: premixed emulsions (A)
were filled into the reservoir (B) and cycled through the dissipation zone (C), cooled by passing
the heat exchange coil (D) and then collected at the outlet by opening the valve (E) .

To assure that the ratio of water to oil in the very first collected emulsion samples
(after preheating), was 1:4, an oven test was conducted. The first six weighted
collected samples in weighted metal recipients with coverage were kept overnight at
105°C in an oven, by this removing the water phase. The difference in weight before
and after placing the samples in the oven, made it possible to calculate the w/o-ratio.
The outcome of the oven test suggested to start collecting the sample after preheating
and executing the first four steps of the Flow chart 2.1, followed by repeating twice
the third and fourth step. In other words, just after preheating, the first three collected
samples should be rejected. The next emulsions of the same and different composition
were collected by operating as illustrated in the Flow chart 2.1. The collected samples
in glass NMR-tubes (18mm outer diameter) were kept in the refrigerator for 24h and
covered with parafilm. After 24h all transport processes occurring are under
equilibrium conditions (Hindmarsch et al., 2005). Some hours before analysis, the
samples were kept at 5°C in a water bath (Julabo F12) filled with a mixture of glycol
and water.

35
Chapter 2 Materials and methods

Flow chart 2.1: A procedure for handling the Microfluidizer M110S


Preheat with boiling deionised water -> circulate -> empty by opening the valve until the liquid level in the reservoir is ~1 cm,
then let the piston move to the right, stop circulation and
suck the remaining liquid out of the reservoir with a syringe

STEP 1
Fill the reservoir with 20mL oil phase -> empty without circulation, empty by opening the valve until the liquid level in the reservoir is ~1 cm,
then let the piston move to the right, stop circulation and
suck the remaining liquid out of the reservoir with a syringe

STEP 2
Fill the reservoir with 20mL premixed emulsion -> circulate for 1.5 min -> empty by opening the valve until the liquid level in the reservoir is ~1 cm,
then let the piston move to the right, stop circulation and
suck the remaining liquid out of the reservoir with a syringe

STEP 3
Fill the reservoir with 10mL premixed emulsion -> empty without circulation, by opening the valve until the liquid level in the reservoir is ~1 cm,
then let the piston move to the right, stop circulation and
suck the remaining liquid out of the reservoir with a syringe

STEP 4
Fill the reservoir with 20mL premixed emulsion -> circulation for 1.5 min -> empty, by opening the valve until the liquid level in the reservoir is ~1 cm,
then let the piston move to the right, stop circulation and
suck the remaining liquid out of the reservoir with a syringe

Repeat twice STEP 3 and 4

To collect the first sample: execute STEP 3


then STEP 4* :
Fill the reservoir with 20mL premixed emulsion -> circulate for 1.5 min -> collect the sample from the second stroke of the piston onwards
(reject the liquid from the first stroke of the piston)

To collect next samples with the same composition, execute STEP 2, 3 and 4*:

To collect samples with a different composition, execute STEP 1*:


Fill the reservoir with 10mL premixed emulsion-> empty by opening the valve until the liquid level in the reservoir is ~1 cm,
then let the piston move to the right, stop circulation and suck the remaining liquid out of the reservoir with a syringe
then execute STEP 2, 3 and 4*

2.4 Quantitative particle size analysis of water in w/o-emulsions

2.4.1 Pulsed field gradient-Nuclear Magnetic Resonance

In order to characterize a w/o-emulsion, its particle size distribution was determined


by pulsed field gradient-Nuclear Magnetic Resonance (pfg-NMR). Pfg-NMR
measurements were conducted with a Maran Ultra 23 spectrometer. In the next
paragraphs, essential highlights of this method are given, clarifying its application to
determine the water droplet size distribution through its ability to measure restricted
molecular self-diffusion, as described by Packer and Rees (1972), Lönnqvist et al.
(1991), Lönnqvist et al. (1997), Johns (2009), Voda and van Duynhoven (2009), van

36
Chapter 2 Materials and methods

Duynhoven et al. (2002), Balinov et al. (1994) and Calliauw (2009). Reference is
made to the latter study for more detailed information about pfg-NMR.
Besides information with regard to characterization of the emulsion, one should notice
the relationship between water droplet size and sensorial properties and microbial
activity. The microbial activity is bigger in larger droplets, because more nutrients are
present. Further elaboration of this aspect is beyond the scope of this thesis.

2.3.2 The pfg-NMR experiment

NMR-spectrometry detects the emission of electromagnetic radiation by nuclei. The


nucleus of an atom absorbs energy of an applied magnetic field. Here, the nucleus of
the proton is studied for determination of the water droplet size distribution.
Pfg-NMR consists of a 90° and 180° radio frequency pulse and two pulsed field
gradients, as illustrated in Figure 2.4. Applying a 90° radio frequency pulse, which is
a rotating magnetic field, supplies energy to be absorbed, with a continuous spectrum
of frequencies. The pulse results in the fact that protons resonate at their Larmor
frequency, which isn’t the same for each nucleus, due to the difference in chemical
environment. The obtained NMR-signal will decrease as signals of protons dephase.
The 180° radio frequency pulse reverses the direction of the rotation of the protons,
which is called rephasing, which will be followed by dephasing. Together, the
rephasing and dephasing signal are called the spin echo signal. If protons resonate at
the same frequency before and after the 180° radio frequency pulse, the NMR-signal
will be maximum. Diffusion of protons attenuates the echo signal, because their
frequency changes due to the different perceived magnetic field. The application of
pulsed field gradients is needed to influence the spin echo signal, due to the diffusion
of protons, more pronounced. The gradients enlarge the differences of perception of
magnetic field and fasten up the dephasing. The stronger the gradients, the greater the
reduction in echo intensity. If there wouldn’t be any diffusion, after the second pulsed
field gradient, protons rephase at time 2τ, resulting in a maximum echo signal,
identical to the signal without gradient pulses. Hereby, τ represents the time between
the 90° and the 180° radio frequency pulse. Pulsed field gradients (Figure 2.4) are
characterized by a duration δ (s) and a strength g, which was fixed at 1.73953
Tesla/meter.

37
Chapter 2 Materials and methods

Figure 2.4: Schematic representation of the pfg-NMR diffusion experiment (Hindmarsh et al.,
2005).

In the RINMR-software the δ-value was varied in 17 or 18 steps, which was done by
using the DSD.RIS.tif script and DSD_Lien.RIS.tif script. The latter script is an
adapted version of the DSD.RIS.tif script, from which the Droplist is altered. In the
Droplist of DSD_Lien.RIS.tif originally 19 δ-values (µs) were contained, but only for
18 values an output was given, because at δ=10000 µs, the duration of the gradient
pulse was too long with regard to the time interval of the two radio frequency pulses.
Hence, the δ-value of 10000 µs was dropped out of this Droplist. Running the script
with 18 δ-values takes about 10 minutes.

Droplist of DSD.RIS.tif script with 17 δ-values in µs: 400; 600; 800; 1000; 1250;
1500; 1750; 2000; 2250; 2500; 2750; 3000; 3250; 3500; 3750; 4000; 4500

Droplist of DSD_Lien.RIS.tif script with 18 δ-values in µs: 500; 750; 1000; 1250;
1500; 1750; 2000; 2250; 2500; 2750; 3000; 3250; 3500; 4000; 4500; 5000; 6000;
8000

Samples in glass NMR-tubes with outer diameter 18mm were filled until the marked
line (8mL) and analyzed at an aimed temperature of 5°C in the Maran Ultra 23
spectrometer. In the performed experiments the time between two gradient pulses (∆)
(Figure 2.4) amounted of 0.2s. As discussed in 2.3.3, two other parameters are needed
to calculate the water droplet size: the magnetogyric or gyromagnetic ratio (γ), which
for H-atoms is equal to 267518000 T-1s-1, and the bulk diffusion coefficient of water
in the dispersed fluid (D) at 5°C, which differs among different composed water

38
Chapter 2 Materials and methods

phases of the w/o-emulsion and was experimentally determined in the calibration


procedure.

2.3.2.1 Calibration procedure

Before analysis, a calibration procedure is needed, which is included in the mentioned


scripts in the RINMR software. First, a pure fat sample (without emulsifier) is used in
order to suppress NMR-signals coming from the fat phase. Secondly, pure deionized
water is used to be able to calculate the magnetic field gradient strength (g) from the
known bulk diffusion coefficient of deionized water (Dw=1.3253E-09 m2/s at 5°C)
and different values of δ. This is based on a pfg-NMR diffusion experiment that
acquires a set of data for each δ-value. Thirdly, a dispersed phase sample is used to
determine the bulk diffusion coefficient of water in this environment at 5°C, which
may be smaller than the diffusivity of pure water due to the presence of dissolved
molecules, such as emulsifiers and salts. Hereby D is calculated from the obtained
gradient strength (g) and a pfg-NMR diffusion experiment that results in a set of data
for each δ-value.

2.4.3 Restricted diffusion in w/o-emulsions

With regard to w/o-emulsions, the water molecules inside the dispersed phase are
characterized by restricted diffusion. This means that the mean displacement of the
liquid molecules inside the emulsion droplets is of the same order of magnitude or
larger than the diameter of the droplet. There is a limit to the amount of diffusion and
hence to the echo intensity reduction. If the movement of a molecule over relatively
short times is observed, the diffusion will be unrestricted. Observations over a longer
time, will result in a restricted diffusion because the molecule cannot move further
than the droplet diameter. By measuring the attenuation ratio of the NMR-signal at
different times, it is possible to identify when the diffusion becomes restricted and
estimate the droplet size distribution.
A function of the ratio (E) of the intensity of the echo signal I (with gradient pulses)
to I0 (without gradient pulses) has been described by Murday and Cotts (1968). The

39
Chapter 2 Materials and methods

function contains the following parameters: δ (duration of the gradient pulse), γ (the
magnetogyric or gyromagnetic ratio), ∆ (time between two gradient pulses), g
(gradient strength), D (bulk diffusion coefficient of water in the dispersed fluid) and
additional parameters λm (the mth-square root of a Bessel function) and R (the radius
of the water droplet):

 2 2 ∞ 1 
(−2γ g ) ∑ * 
 m = 1 (λ 2m R 2 − 2)λ 2m 
E(R, δ ) = exp  
 2δ 2 + exp(−λ 2m D(∆ − δ )) − 2 exp(−λ 2m D∆) − 2 exp(−λ 2m Dδ ) + exp(−λ 2m D(∆ + δ )) 

− 
 λ 2 D ( λ 2 D) 2 
 m m

Equation 2.1

The echo attenuation is smaller as the droplet size decreases (Figure 2.5). The use of
the bulk diffusion coefficient D of the dispersed phase is justified by the fact that only
two or three layers adjacent to the emulsion droplet interface have altered properties
as compared to the bulk.
Echo attenuation ratio Ep (-)

1,0 0.25µm

0,9 0.75µm

1.25µm
0,8
1.75µm
0,7
2.25µm
0,6 2.75µm

0,5 3.25µm

0,4 3.75µm

4.25µm
0,3
4.75µm
0,2
sample1
H0.75/1.50
0,1 free H2O
diffusion
0,0
0 0,001 0,002 0,003 0,004 0,005 0,006 0,007 0,008 0,009

δ (s)
Figure 2.5: Calculated decay of the echo intensity of w/o-emulsions characterized by an R-value,
δ (0s to 8ms) and D=1.30E-09 m2/s, and obtained from Equation 2.1 assuming γ=267518000T-1s-1,
g=1.73953T/m and =0.2s. Experimental decay of the echo intensity versus magnetic gradient
pulse width of pure deionized water (Dw=1.3253E-09 m2/s) and of a w/o-emulsion (H0.75/1.5,
sample 1) (D=1.289E-09 m2/s), both measured at 5°C (γ, g and  are identical as for pure
deionized water).

Depending on the applied method of data processing, different equations are used in
order to get the water droplet size distribution, based on the measured echo intensities.

40
Chapter 2 Materials and methods

The three methods that are used, are the data processing method by the Droplet Size
application (Resonance Instruments Ltd), by Excel or by Matlab.

2.3.3.1 Data processing by the Droplet Size application

This method gives directly the volume-weighted arithmetic mean radius R43 (denoted
in the software as R33), the number-weighted arithmetic mean radius R10 (denoted in
the software as R00), the median radius Rmed of the number-weighted distribution
(denoted in the software as R0) and the distribution width σ. Assuming a lognormal
number-weighted distribution, Pn(R) is introduced, which is the probability
distribution of each droplet radius:

Pn(R) = (Rσ√(2π))-1exp[-(lnR-lnRmed)2/(2σ2)] Equation 2.2

where R is the radius (µm), σ is the distribution width and Rmed is the median radius
considering a lognormal number-weighted distribution, which equals the number-
weighted geometric mean radius. In addition, the distribution width σ corresponds to
the natural logarithm of the geometric standard deviation σg of the particle radius
distribution:

σ = lnσg Equation 2.3

In order to determine the droplet size distribution, the measured echo attenuation
ratios (I/I0), which are denoted as Ep(δ), are compared to the calculated echo intensity
ratios (Equation 2.4). This is done for each magnetic gradient pulse width δ, and by
means of a least-squares analysis the best estimates for Rmed and σ are obtained.

Ep(δ) = ( ∫(0-∞)R3Pn(R)E(R,δ) dR) /( ∫(0-∞) R3Pn(R) dR ) Equation 2.4

where Ep is the total attenuation of the NMR-signal of the population of possible


spherical droplet radii and E(R,δ) can be calculated by using Equation 2.5.

41
Chapter 2 Materials and methods

 2γ 2 g 2 ∞ 
( − R4 
) ∑ *
 D m = 1 λ 4 (λ 2 − 2) 
m m
E(R, δ ) = exp  
 2  2 2 2 2  
 R  − λ m D∆ − λ D(∆ − δ ) − λ Dδ − λ D ( ∆ + δ ) 
2δ − ( 2 )2 − 2 exp( ) + 2 exp( m ) − 2 exp( m ) + 2 exp( m ) 
 λ m D  R 2 R 2 R 2 R2 

Equation 2.5

2.3.3.2 Data processing by Excel

This method gives the volume-weighted geometric mean diameter D33 and uses the
exported data from the Droplet Size Application and consists of 6 steps.
Firstly, echo attenuation ratios are calculated by implementing a certain droplet
radius, a δ-value, a set of λm values as well as values for D, γ, ∆ and g in Equation 2.1.
This is repeated for all δ-values that were used in the analysis, while keeping the
droplet radius constant.
Secondly, the echo attenuation ratios are calculated for another droplet radius for the
different δ-values, obtaining attenuation ratios as function of the droplet radius and δ.
This yields curves as shown in Figure 2.5, in which the relative positioning of the
measured and the calculated data can be observed. In the Excel file, the set of radii is
0.25; 0.75; 1.25; 1.75; 2.25; 2.75; 3.25; 3.75; 4.25 and 4.75 µm. Choosing a different
set of radii is possible, but time consuming. In Appendix A, a part from a data sheet in
Excel that represents the calculated echo attenuation ratios as a function of the radius
and δ of the emulsion H0.75/1.50 (sample 1) is represented.
Thirdly, in prospect of expressing the water droplet size in diameter and since the
lognormal volume-weighted probability distribution as a function of droplet radius is
equal to the lognormal volume-weighted probability distribution as a function of
droplet diameter, the latter will be used in the next equations. The relative frequencies
(P(ɸ)) for each droplet diameter ɸ are calculated and normalized
(Pnorm(ɸ)=Pnorm(Rx2)) by dividing it by the sum of the relative frequencies by using
Equation 2.6.

42
Chapter 2 Materials and methods

Pv (R) (φ σ 2π ) -1 exp[-(ln(φ ) - ln(R 33 x2) 2 /(2σ 2 )]


Pv, norm (R) = Pv, norm (φ ) = =
∑ (Pv (R)) ∑ (Pv (φ ))
Equation 2.6

Hereby, Pv represents the lognormal volume-weighted distribution and the following


parameters are brought in: the distribution width (σ), whose starting value is directly
obtained from the Droplet Size application, and the volume-weighted geometric mean
diameter D33 (or R33x2), whose initial guess is calculated by putting the number-
weighted geometric mean radius (Rmed) in Equation 2.7 and converting it to diameter.
The radius Rmed is directly obtained from the output of the Droplet Size application.
Equation 2.6 also needs an arbitrary diameter (ɸ).

R33= Rmed exp(3σ2) Equation 2.7

Fourthly, the echo attenuation ratios (Ep(δ)), that will be compared to the ones
measured, are obtained by multiplying the matrix consisting of rows with E values
corresponding to different δ-values and columns with E values corresponding to
different droplet radii (Appendix A) by the vector consisting of normalized relative
frequencies for the different droplet diameters (or radii). This operation boils down to
executing Equation 2.8, which is a modified version of Equation 2.4. The main
modification is the replacement of a number-weighted distribution in Equation 2.4 by
a volume-weighted distribution in Equation 2.8.

Ep(δ) = Σ [Pv, norm(ɸ) E(R,δ)] Equation 2.8

Fifthly, the sum of the squared differences between the calculated and the measured
echo attenuation ratios is calculated.
Sixthly, through ‘solver’ of Excel, the best fitting volume-weighted geometric mean
diameter D33 (or R33x2) and the distribution width (σ) are determined, so that the sum
of squared differences between the calculated and experimental echo attenuation ratio
is the smallest. The volume-weighted arithmetic mean diameter D43 is obtained by
using Equation 2.9. Also here, the distribution width (σ) is converted to the geometric
standard deviation (σg) by using Equation 2.3.

43
Chapter 2 Materials and methods

R43= R33 exp(σ2/2) Equation 2.9

Figure 2.6 illustrates relative normalized frequencies of a lognormal volume-weighted


distribution of the emulsion H0.75/1.50 (sample 1) as a function of particle diameter
class, which are obtained by calculation in Excel and by usage of R43 from the output
in the Droplet Size application, which is first converted to R33 by Equation 2.9 and
then included into Equation 2.6. A discrepancy between the lognormal volume-
weighted distribution by Excel and the Droplet Size application can be observed,
which might be due to the difference in Equation 2.1 and Equation 2.5.

Figure 2.7 illustrates the experimental and the best fitting calculated echo attenuation
ratios versus magnetic gradient pulse width (δ). To get a better fit, the product of
calculated normalized relative frequencies and calculated echo attenuation ratios is
multiplied by a factor (the multiplication factor MF (Equation 2.10)) which is also
included as a freely adjustable parameter in the solver.

Ep(δ) = Σ (MF)[Pv, norm(ɸ) E(R,δ)] Equation 2.10

The highest measured echo intensity at the smallest value for δ is considered as Io (the
echo intensity without gradient). This results in the fact that the ratio (E) of the echo
intensity I relative to Io, associated with the smallest δ-value, is allocated a value of
unity. As it is not possible to have an experimental point corresponding to a δ-value of
0µs, the measured relative echo decay data (Ei) are slightly overestimated, which can
be compensated by multiplication of the calculated data by a multiplication factor,
which is generally slightly larger than unity.
A major drawback of the Excel approach is that it includes only a limited number of
particle size classes, which are quite broad (especially in the low size region). This
will surely be problematic for samples with a rather narrow particle size distribution.

44
Chapter 2 Materials and methods

0.8
0.7
0.6
Excel
0.5
Relative DSA
norm alized 0.4
frequency 0.3
0.2
0.1
0
µm

µm

µm

µm

µm

µm

µm

µm

µm

µm
-1

-2

-3

-4

-5

-6

-7

-8

-9

0
-1
0

8
9
Particle diam eter class

Figure 2.6: Relative normalized frequencies of the lognormal volume-weighted distribution of the
emulsion H0.75/1.50 (sample 1) measured at 5°C as a function of particle diameter class,
obtained by calculation in Excel according to section 2.3.3.2 and by inclusion of the output (σ and
the R43) of the Droplet Size application (DSA) in Equation 2.6 after conversion of R43 to R33 via
Equation 2.9 (γ=267518T-1s-1, g=1.73953T/m, =0.2s, D=1.289E-09 m2/s).

1
0.9 Ep calculated
0.8 Ep measured

0.7
Ep (-)

0.6
0.5
0.4
0.3
0.2
0.1
0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009
δ (s)
Figure 2.7: Measured and calculated (using R33=3.0277µm, σ=0.0782 and MF=1.0448) echo
attenuation ratio versus magnetic gradient pulse width (δ) of the emulsion H0.75/1.50 (sample 1)
at 5°C (γ=267518T-1s-1, g=1.73953T/m, =0.2s, D=1.289E-09 m2/s).

45
Chapter 2 Materials and methods

2.3.3.3 Data processing by Matlab

Also this method uses the exported data from the Droplet Size Application. The
difference between applying the Droplet Size application or Excel, lies in the fact that
this method is less cumbersome in terms of time consumption to result in an outcome.
Data processing by Matlab is based on the ‘inverse’ approach. Instead of calculating
the (lognormal) probability for a certain droplet size, which is done in Excel and the
Droplet Size application, a set of radii is calculated from the cumulative probability
(Figure 2.8), which ranges from 0.01 up to 0.99 with a fixed interval width of 0.01
units so that each radius has the same probability. In contrast, as stated in 2.3.3.2,
Excel uses only ten droplet diameters to calculate the lognormal probabilities (starting
from 0.5 µm in steps of 1 µm up to 9.5 µm diameter). This limitation results in the
fact that in Figure 2.8 only a few diameters out of ten are represented. For the sake of
clarity of Figure 2.8, only interval widths of 0.1 units are represented and to allow
comparison with Excel, the probability as a function of diameter instead of radius is
given.
In order to calculate the water droplet size, three files are used: Simulation_data.m,
Fitlien.m and Minim_Lien.m (Appendix B), where the latter actually refers to
Fitlien.m. The values for the free diffusion coefficient of water in the dispersed phase
and the gyromagnetic constant (γ), the reference to the Excel file, which contains the
values of the magnetic gradient pulse width (δ), the magnetic field gradient (g), the
measured intensities I and I0, are used in Simulation_data.m.
Firstly, the echo intensities I(δ,R) (where I refers to the echo intensity and R refers to
the radius that corresponds to the cumulative probability of a lognormal distribution),
are calculated by multiplying Equation 2.1 by I0. As initial guess for the parameter I0,
the experimental echo intensity obtained at the smallest δ is used. The radius is
calculated (and not arbitrarily chosen as in Excel or the Droplet size application) by
means of the inverse approach (Figure 2.8), based on the natural logarithm of the
geometric mean radius R33 of the lognormal volume-weighted particle radius
distribution (µ) and the natural logarithm of the geometric standard deviation σg of the
particle radius distribution (σ), according to Equations 2.11 and 2.12.

µ = ln(R33) = ln[R432/√(stdev2+ R432)] Equation 2.11


σ = ln(σg) = √[ln(stdev2/ R432 +1)] Equation 2.12

46
Chapter 2 Materials and methods

Thereby, stdev and R43 represent the arithmetic standard deviation and arithmetic
mean radius of the volume-weighted particle radius distribution.

Figure 2.8: Cumulative distribution of the diameter in micrometer (assuming a median diameter
of 1.62µm and stdev of 0.2µm) with graphical representation of the two approaches: the
horizontal arrows illustrate the inverse approach (used in Matlab) and the vertical arrows
illustrate the direct approach (used in Excel).

Secondly, the echo intensities (Raccum(δ)) are obtained by the transformation of the
I(δ,R).
Thirdly, the file Fitlien.m calculates the best fitting volume-weighted arithmetic mean
radius (R43) (which is denoted as ‘mean’ in the Matlab file) and the arithmetic
standard deviation of the particle radius distribution (which is denoted as ‘stdev’ in
the Matlab file), so that the squared difference between the experimental echo
intensities I(δ) and calculated echo intensities (Raccum(δ)) is the smallest. Figure 2.9
is a graphical representation of the experimental and best fitting calculated echo
intensities versus magnetic gradient pulse width (δ). A logarithmic transformation of
the least squared difference results in the so-called maximum likelihood (MLH).
Matlab examines the possible combinations of means and standard deviation and
ceases its action when the lowest MLH is found.
The best fitting arithmetic standard deviation of the particle radius distribution is
converted to σg by using Equation 2.12.

47
Chapter 2 Materials and methods

8000
I(small delta)
Raccum(small delta)
7000

6000
Echo intensity

5000

4000

3000

2000

1000
0 1 2 3 4 5 6 7 8 9
Small delta (s) -3
x 10

Figure 2.9: Experimental (I) and best fitting calculated (Raccum) echo intensity (assuming
R33=1.5596µm, σg= 1.0860 and I0= 7,72E+03) versus magnetic gradient pulse width (δ) of the
emulsion H0.75/1.50 (sample 1), measured at 5°C. (γ=267518T-1s-1, g=1.73953T/m, =0.2s,
D=1.289E-09 m2/s).

2.4.4 Statistical methods

It is assumed that water droplet sizes in w/o-emulsions are characterized by a


lognormal distribution. Whether a confidence interval of the mean of three volume-
weighted geometric mean diameters of three samples can be constructed, depends on
the distribution of the volume-weighted mean diameters. A small scale experiment
(Kolmogorov Smirnov-test, S-Plus) of replica’s with the same composition revealed
no rejection of the hypothesis of a normal distribution of the volume-weighted
geometric mean diameters on the 5% significance level. Hence, the construction of a
95% confidence interval of the mean of three volume-weighted mean diameters of
three samples is possible. The average of three samples for an emulsion, the 95%
confidence interval of the mean and the standard deviation of three samples are
calculated with Excel (Microsoft Office 2003).

48
Chapter 2 Materials and methods

Furthermore, parametric tests, which rely on the normal distribution, can be applied.
In order to compare the values of D33 between the three data processing methods or
compositions of emulsions, two-sample paired and two-sample independent sample t-
tests (Excel) are used, respectively. A significance level of 5% is applied. If more than
two groups need to be compared simultaneously, a Bonferroni correction (S-Plus) was
applied.
R-squared values are calculated in Excel and indicate the variability in a measured
data set that is accounted for by the calculated data set. For the Droplet Size
application and Excel, the calculated and measured echo attenuation ratios are
compared, whereas for Matlab, the echo intensities are compared. Whenever multiple
outcomes for the same sample were obtained, the outcome characterized by the
highest R-squared value was used in the statistical analysis.

2.4.5 Important issues during pfg-NMR analysis

2.3.5.1 Temperature of the emulsion during pgf-NMR analysis

Measurements should be done at a stabilized temperature, because diffusion data are


highly temperature sensitive. The Stokes-Einstein equation shows the connection
between temperature and diffusion coefficient:

D=kT/6πηR

where k is the Boltzmann constant, T is temperature, η is the viscosity and R is the


radius of a spherical particle. The diffusion coefficient increases at higher
temperatures because of the temperature effect on the viscosity. Samples are analyzed
at 5°C for having a maximum microstructural stability of the sample. At this
temperature, interdroplet water diffusion is minimized and only intradroplet diffusion
results in the attenuation of the NMR-signal (Van Lent et al., 2008a; Price, 1997;
Voda & van Duynhoven, 2009). Fourel et al. (1994) noted that as temperature
decreases, the standard deviation is approximately constant, but there is a large
decrease in mean diameter of the distribution, because then the condition of restricted

49
Chapter 2 Materials and methods

diffusion is better obtained. At higher temperatures, the continuous phase becomes


more permeable for the water and interdroplet diffusion becomes more interfering.
In conclusion, as long as the temperature is around 5°C and the calibration step is
performed at the same temperature, then the exact temperature is of less importance
than the stability of the temperature due to the adaption of the output to the
calibration.

2.3.5.2 Assumption of a lognormal size distribution

Systematic deviations between results of the fit and the experimental data can be due
to random errors or to the not completely appropriate assumed size distribution,
namely the lognormal size distribution. A way to cope with the fact that the
assumption of log normality cannot hold, is by modification of the equations. In a
study of Fourel et al. (1994) an unrestricted diffusion term was added to the equation
of Ep (Equation 2.4) to get better results in the case of no perfect lognormal
distribution of droplet size or in the case of existence of possible unrestricted
diffusion due to poor emulsification.

2.3.5.3 Advantages of pfg-NMR analysis for determination of water droplet size

In comparison of other methods, pfg-NMR has the advantage that the same samples
can be tested repeatedly, and dilution and centrifugation are not necessary. This non-
invasive technique is fast (about 10 min) and able to test opaque emulsions in which
the continuous phase is not conducting. However, there is a lack of a reference for
validation (Peña and Hirasaki, 2006).

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Chapter 2 Materials and methods

2.5 Qualitative particle size analysis of water in w/o-emulsions

2.4.1 Fluorescence microscopy

Water droplets of w/o-emulsions are visualized with the water-soluble fluorescent


agent eosinY (MP Biomedicals Inc., Illkirch, France) and a fluorescence microscope
(Leitz Diaplan Incident light fluorescence 3-λ Ploemabak illuminator, Wild Leitz,
Germany) (Figure 2.10) equipped with Cell-software (Olympus Soft Imaging
Solutions GmbH, Germany).

Figure 2.10: Incident light


fluorescence 3-λ Ploemabak
illuminator. 1: lamp housing; 2:
lamp housing mount; 3: BG38
red-absorption filter; 4:
excitation light blocking slide; 5:
field diaphragm; 6: filter block
interchange control; 7: stop for
twin wavelength study; 8: 3-
lambda Ploemabak; 9:
localization of the camera.

In this fluorescence microscope, light from a mercury lamp is filtered by an excitation


filter in the filter block, which reflects the light under an angle of 90° through the
objective lens on the sample. The excitation filter selects the incoming light or
excitation wavelength range. EosinY absorbs the light and emits fluorescent light. The
latter passes through the same objective and straight through an emission filter of the
same filter block towards the camera or binocular tubes. The emission filter selects
the emission wavelength range. The choice of filter block depends on the maximum
excitation and emission wavelength of the applied fluorescent agent. The applied filter
block (I2/3) passes excitation wavelengths between 450 to 490nm (BP450-490 or
band pass filter) and emission wavelengths above 520nm (LP520 or long-pass filter).
Depending on the settings, the microscope can be used as a fluorescence or polarized

51
Chapter 2 Materials and methods

light microscope. The fluorescence mode requires opening of filters nr.4 in Figure
2.10 and centration of the mercury or metal halide light by adjustment of the lamp
housing. Filter nr.3 (BG38) is a short-pass filter that passes excitation wavelengths
around 500nm, hence this resembles the excitation filtration of filter block I2/3.

2.4.2 Fluorescence

The fluorescence process comprises three steps (Figure 2.11). Firstly, the fluorescent
molecule is excited by absorption of a photon, which comes from the light source in
the fluorescence microscope or fluorimeter, by which the molecule moves from the
ground state to one of the vibrational levels of the exited state in 10-15 to 10-16s.
Secondly, vibrational relaxation (or internal conversion) occurs towards a lower
energy vibrational excited state without emission of light. This step takes
approximately 10-12s. Thirdly, by emission of a photon, the fluorescent molecule
returns to one of the levels of the ground state, which results in different emitted
photon energies. As a consequence, light is emitted over a range of wavelengths in a
time span of 10-9s.

Figure 2.11: The fluorescence process. S0, S1 and S2


stand for ground singlet state, first excited singlet
state and second excited singlet state, respectively.
Wavy arrows denote that the timescale is larger than
for straight arrows.

The emitted light is characterized by a smaller energy and hence in accordance to the
law of Planck (E = hʋ = hc/λ), a longer wavelength than the light that leads to

52
Chapter 2 Materials and methods

excitation of the molecule. The difference in energy or wavelength between the


absorbed and emitted photon is called the Stokes shift. A large Stokes shift is
beneficial with regard to the sensitivity of the detector, because in this way by the
usage of filters, the excitation light that reaches the detector can be limited.

2.4.3 EosinY

EosinY or tetrabromo fluorescein is a red dye. Besides the usage as a coloring agent
in histological research and as a pharmaceutical product that dehydrates varicella-
blisters, eosinY can be used as a fluorescent agent (Figure 2.12). The letter Y is an
abbreviation of ‘yellowish’ due to the slightly yellowish shade. In contrast, eosin B
has a blue shade and is a dibromodinitro fluorescein derivate.

Figure 2.12: Chemical structure of eosinY (tetrabromofluorescein)

Although the aqueous solubility is 400g/L, the dissolution rate is quite low. Therefore,
a magnetic stirrer was used.
Repeating the analysis of the same sample in the fluorimeter or illumination by UV
and visible light results in repetitions of the excitation and emission cycle until the
fluorescent agent is destructed or photo bleached. Illumination decreases both
absorption and fluorescence. In a study of Kola (2010), within one day, fluorescence
of eosinY-solutions, which were stored in colorless glass in a light room, was found to
be destroyed for 98% and completely after 2 days. Even when the eosinY-solutions
were stored in brown colored flasks in a light room, 92% of the fluorescence intensity
is gone after 3 days. When the aqueous solutions were kept in brown flasks in the
shade, only 15% reduction of the fluorescence of eosinY was observed after 3 days

53
Chapter 2 Materials and methods

(Kola, 2010). Hence, the investigated eosinY-solutions in this study are covered with
aluminum foil.
Above pH 5.58, the fluorescence intensity is constant and higher than below this pH.
Regarding temperature, Kola (2010) demonstrated that the fluorescence intensity is
stable within the studied temperature range of 10°C to 41°C.
Stockert et al. (1994) found that the addition of sodium azide to eosinY-solutions
might create new absorption (520nm) and emission (478nm) peaks. Seema and
Babulal (2009) reported on the interaction between eosinY and some surfactants other
than sodium caseinate. Therefore, the spectrofluorimetric behavior of eosinY in the
presence of sodium caseinate was investigated.

2.4.4 Fluorimetric analysis of w/o-emulsions

2.4.4.1 Fluorimeter

A fluorimeter (Varian fluorescence spectrophotometer, Cary Eclipse) was used to


perform preliminary investigation of aqueous eosinY-solutions in the presence of
different agents. As such, the influence of addition of a phosphate buffer, sodium
azide and sodium caseinate to the aqueous solution on the maximum excitation and
emission wavelength of eosinY was examined.

2.4.4.2 Determination of a suitable concentration of eosinY

A suitable concentration of eosinY in distilled water that is associated with high


fluorescence intensity was looked for. This concentration is used in the water phase of
the w/o-emulsion (50:50, w/w) for fluorescence microscopic investigation. The
software tool ‘Concentration’ of Cary Eclipse was applied. The limitation that the
fluorimeter becomes saturated at very high fluorescence intensities must be taken into
account and forces the quest for a suitable concentration of eosinY to be done at sub
maximum excitation and emission wavelengths. On the basis of Figure 2.13, an
eosinY concentration of 10µg/mL was chosen for the water phase in emulsions.
Linear regression on the first nine data points results in y=537277x-11 and R2=0.93.

54
Chapter 2 Materials and methods

1000

Fluorescence intensity (a.u.)


100

10

1
1.0E-08 1.0E-07 1.0E-06 1.0E-05 1.0E-04 1.0E-03 1.0E-02 1.0E-01 1.0E+00

0.1
Eosin Y concentration (g/100mL)

Figure 2.13: Fluorescence intensity as a function of the logarithm of eosinY concentration in


water. Excitation wavelength=514nm. Emission wavelength= 564nm.

2.4.4.3 Determination of maximum excitation and emission wavelength

By application of the 3D Mode and selection of the Excitation or Emission-button in


the software tool ‘Scan’ of Cary Eclipse, the maximum excitation or emission
wavelength can be determined, respectively. During the scan, the fluorescence
intensity, which is related to a predetermined range of excitation wavelengths for a
predetermined range of emission wavelengths, is recorded. At an emission
wavelength of 480nm the intensity is measured for excitation wavelengths from 480
to 600nm. The next scan measures the intensity at an emission wavelength of 482nm.
Scanning is proceeded until the emission wavelength of 600nm. The highest recorded
intensity corresponds to the scan at the maximum emission wavelength. An example
of an excitation spectrum scan is represented in Figure 2.14, in which excitation and
emission range from 480 to 600nm in steps of 2nm. Fluorescence intensity is
indicated by different colored regions, which is defined as the y-axis of the spectrum.
High fluorescence intensity (or red colored regions) gives an idea about the range of
the maximum excitation and emission wavelength and can be read from the x-axis
and z-axis, respectively. The z-axis represents the number of steps between the lowest
and highest analyzed emission wavelength. In this example, the maximum unit on the
z-axis is 60, since the difference between 600 and 480nm amounts of 120nm and the
size of the steps is 2nm. Conversion of units on the z-axis to the emission wavelength
in nanometer is done by Equation 2.13. Analogously, in an emission spectrum, the

55
Chapter 2 Materials and methods

excitation wavelength can be calculated from the units on the z-axis. Hereby, the x-
axis and y-axis represent the emission wavelength and fluorescence intensity,
respectively.

Emission wavelength (nm) = [(Z-axis units)(size of the steps to go from the first to the
last selected emission wavelength) + lowest analyzed emission wavelength (nm)]

Equation 2.13

60.00 1000
55.00 962.92
888.75
50.00 800
814.58
Intensity (a.u.)

51 8.05 , 13 2.512

45.00
740.41
40.00
600 666.24
35.00 592.07
Z Axis

30.00 517.91
400 443.74
25.00
20.00 369.57
15.00 200 295.40
221.23
10.00
147.07
5.00 0 72.90
480 500 520 540 560 580 600-1.27
480.00 490.00 500.00 510.00 520.00 530.00 W avelength
540.00 550.00 (nm)
560.00 570.00 580.00 590.00 600.00
Wavelength (nm)

Figure 2.14: Contour plot of the scan for excitation and emission wavelength ranging from 480 to
600nm in 60 steps of 2nm. X-axis: excitation wavelength (nm). Z-axis: number of steps that are
used to go from 480 to 600nm emission wavelength. Red color denotes high fluorescence
intensity, green color denotes low fluorescence intensity. Eosin concentration in distilled water is
5µg/mL.

Concerning the determination of the maximum excitation and emission wavelength, a


concentration of eosinY in water of 5µg/mL was used and can be performed in two
ways. In the case that the maximum excitation wavelength must be determined, the
first method consists of narrowing down the emission wavelength range which lies
symmetrically around the maximum emission wavelength. However for determination
of the maximum excitation and emission wavelength, Sometimes a lower
concentration of eosinY must be analyzed to avoid approaching the maximum
recordable fluorescence intensity. The second method comprises the measurement of
the fluorescence intensity by shifting the narrowed range of emission wavelengths to
sub maximum regions. Hence, the maximum recordable fluorescence intensity is not
reached. The eosinY-concentration is left unchanged. In the case that the maximum

56
Chapter 2 Materials and methods

emission wavelength must be determined, the same reasoning applies, but now the
excitation wavelength range must be narrowed down.

An example of such an excitation spectrum is given in Figure 2.15, for which the
maximum excitation wavelength matches 517nm. As the emission wavelength was
located at sub maximum regions, this is an example of the second method. Eleven
different cultured lines can be observed because the emission wavelength is elevated
from 554nm to 564nm in steps of 1nm.

Figure 2.15: Graphical representation of the determination of the maximum excitation


wavelength of eosinY at a concentration of 5µg/mL. Excitation wavelength range: 490-540nm.
Emission wavelength range: 554-564nm.

In order to demonstrate that the maximum excitation or emission peak at different


suboptimal emission or excitation wavelengths, respectively, is located at the same
wavelength, the maximum emission was recorded at different ranges of sub maximum
excitation wavelength as depicted in Table 2.3. No deviating trends could be
observed. The average and standard deviation of the nine maximum emission
wavelengths is 547nm and 0.7nm, respectively. Hence, identical outcomes can be
achieved at excitation wavelengths that are 67nm (527nm minus 460nm) lower than
the maximum excitation wavelength.
In order to test the repeatability of the fluorimetric analysis, the same sample was
repeatedly analyzed at the same excitation wavelength range of 480 to 490nm. The
average maximum emission wavelength and standard deviation of ten repetitions was
547.2nm and 0.7nm, respectively.

57
Chapter 2 Materials and methods

Table 2.3: Recorded maximum emission wavelengths at different excitation wavelength ranges of
eosinY (5µg/mL) in aqueous phosphate buffer (pH6.7) with sodium azide (0.02%,w/v) and
sodium caseinate (1.25%,w/v).
Excitation range (nm) Max. emission wavelength (nm)
500-510 547
495-505 548
490-500 548
485-495 547
480-490 547
475-485 546
470-480 546
465-475 547
460-470 547

2.4.5 Confocal laser scanning microscopy

Two major differences between fluorescence microscopy and confocal laser scanning
microscopy (CLSM) are the depth selectivity and the applied light source. Whereas
fluorescence microscopy detects fluorescence of all layers in the specimen, images by
CLSM can be taken of a selected layer or depth of the specimen. This is achieved by
focusing the laser beam, which is the light source, by an objective lens into a certain
volume of a certain layer of the specimen whereby the emitted light from other layers
does not reach the detector due to the presence of a detector-aperture (Figure 2.16).

Figure 2.16: Schematic representation of the principle of confocal laser scanning microscopy

58
Chapter 2 Materials and methods

2.6 Water-in-oil-in-water emulsions

2.9.3 Composition of the w/o/w-emulsions

Depending on the preparation method, 70g or 100g of w/o-emulsion (50:50, w/w) was
prepared. Addition of the external water phase to a w/o-emulsion in a ratio 40:60
(w/w) gave rise to a w/o/w emulsion.
The water phase of the w/o-emulsion contained 1.25% (w/v) sodium caseinate, 0.02%
(w/v) sodium azide, 0.001% (w/v) eosinY and a phosphate buffer (pH6.7). The fat
phase consisted of 2.5% (w/v) PGPR in soft PMF or a mix of soft PMF and Hozol.
Here, eosinY is added to ameliorate the visualization of the internal and external
water phase. Unless stated differently, the external water phase only differs from the
internal water phase in the concentration of sodium caseinate (1%, w/v) and the
absence of eosinY.

2.9.4 Preparation of the w/o/w-emulsions

Four manufacturing methods were attempted, which differed in the utilized apparatus
and the modus of sampling.

2.9.4.1 Method A

This method entails subsequent application of an Ultraturrax TV45 (until visual


satisfaction) and a Microfluidizer M110S (at 6bar for 1.5 minutes) in order to
fabricate 100g of w/o-emulsion (50:50, w/w) with soft PMF as the fat phase. W/o-
emulsions were kept in a water bath of 50°C before mixing of the external water
phase with an Ultraturrax (for 0.5 minutes) and Microfluidizer (at 1bar for 1.5
minutes) in prospect of making a double emulsion. The samples were collected in
glass NMR-tubes with an outer diameter of 18mm, filled for 40mm height (or 8mL),
covered with parafilm and cooled in an ice bath. Prior to NMR-analysis, samples were
vortexed and meanwhile maximum 100µL 10mM MnCl2 (Normapur,
VWRinternational, Fontenay sous Bois, France) was added to 8mL emulsion.

59
Chapter 2 Materials and methods

2.9.4.2 Method B

This method comprises the generation of 70g w/o-emulsion by an Ultraturrax TV45.


Fourty grams of w/o-emulsion were blended with 60g of external water phase with an
Ultraturrax S25-10G (Figure 2.17) at 24000rpm for 2 minutes, unless stated
differently. In a few samples, the concentration of sodium caseinate was 1.25% (w/v)
instead of 1% (w/v) (2.5.2) in the external water. The type of fat phase is mentioned
for each experiment. Afterwards, double emulsions were further mixed with a
continuous Ultraturrax DK25 (Figure 2.17) at 24000rpm for a specified time. The
advantage of the latter type of Ultraturrax is the reduction of foam formation and
hence higher availability of surfactant to stabilize the emulsion instead of air. Unless
stated differently, samples were cooled in an ice bath. For NMR experiments, the
samples were filled in NMR-tubes up to 40mm with an outer diameter of 18mm and
received MnCl2 directly after production or just before NMR-analysis. The latter
required vortexing.

Figure 2.17: Construction of a continuous Ultraturrax


DK25 flow chamber (bottom) and an Ultraturrax S25-
10G (top)

60
Chapter 2 Materials and methods

2.9.4.3 Method C

The third method differs from the previous methods only in the filling of the NMR-
tubes. NMR-tubes with an outer diameter of 18mm, were filled in such a way that the
detection zone of the spectrometer with a length of 25mm (Zhu, 2011) (Figure 2.18)
comprises the complete sample. It is chosen to fill the tube for 15mm, which means
that it needs to be elevated for 27mm in the spectrometer by means of wrapping a
rubber band around the tube. Right after production, 28µL 10mM MnCl2 is added to
3mL of emulsion and vortexed. In this manner, errors owing to improper
homogenization of the creamy layer by vortexing are avoided.

Figure 2.18: The detection zone (red) of a Maran Ultra 23 spectrometer lies between 22mm and
47mm height (Zhu, 2011). Hence, incompletely filled tubes (e.g. considering 15mm of sample)
must be elevated (e.g. over 27mm) to enable the detection of their whole contents.

Regarding the effect of the elevation of a tube in the Maran Ultra 23 spectrometer on
the T2-distribution, Figure 2.19, 2.20 and 2.21 illustrate the location of the detection
window. Elevation for 0cm, 0.75cm, 1.5cm, 3cm, 4cm, 5cm and 6cm of an NMR-tube
in the Maran Ultra 23 spectrometer was realized by means of a rubber band. To a
double emulsion with P1.25/2.5/1, 75µL of 10mM MnCl2 was added to a 4cm height
filled NMR tube (about 8mL) right after production. Twenty four hours later, analysis
was performed on the phase separated sample, that was not vortexed.

61
Chapter 2 Materials and methods

According to Zhu (2011), the detection window of the Maran spectrometer is situated
between 2.2 and 4.7cm above the bottom. As long as the sample is located in the
detection zone of the spectrometer and the signal is maximized by optimization of the
receiver gain, the total area under the curve stays constant. Upon elevation, the area
under the curve that is related to the fat phase and internal water decreases, whereas
the area under the curve of external water increases. The fat globules with internal
water are enriched at the top of a phase separated sample, whereas the external water
content is higher at the bottom of a tube.

Figure 2.19: Schematic representation of the experiment concerning the elevation of the NMR-
tube in the Maran Ultra 23 spectrometer. P1.25/2.5/1 with 75µL of 10mMMnCl2 in a 4cm filled
(about 8mL) NMR-tube of 1.8cm diameter, added directly after production. (P/A 1-3/03/11)

The fact that at an elevation of 5cm a signal could still be received, might be due to
the inaccurate elevation by means of the rubber band or a broader detection window
than 2.5cm. In order to assess the detection zone a small experiment was performed.
An NMR-tube of 18mm outer diameter was gradually filled with water from 0 to
12mL with a pipet. For each water volume the area under the curve in the T2-
distribution was plotted versus the water volume in the tube (Figure 2.22). By linear

62
Chapter 2 Materials and methods

regression it was found that the area under the curve linearly increases with a water
volume of 3.7 to 9.2mL, which can be converted to a detection window of 20.5 to
50.5mm (5.51mm/mL). Based on this experiment, the width of the detection window
is 3.0cm, which can explain the recorded signal at 5cm elevation in Figure 2.21.
However, close to the detection window margins, more inaccuracy can be noticed
(Zhu, 2011), hence the detection zone of 2.2cm to 4.7cm can still be applied.

1800

1600

1400

0cm elavation
1200
Signal amplitude

0.75cm elavation
1000 1.5cm
3cm elavation
800 4cm
5cm elevation
600
6cm elevation
400

200

0
1 10 100 1000 10000
Time (ms)

Figure 2.20: T2-distribution of samples at different heights in the Maran Ultra 23 spectrometer.
Black arrows denote the direction of change upon elevation. P1.25/2.5/1, 75µL of 10mM MnCl2
after production. (P/A 1-3/03/11).

16000

14000
Area under the curve

12000

10000 Fat phase


External water
8000
Internal water
6000 Total AUC

4000

2000

0
0 1 2 3 4 5 6
Elevation of the NMR-tube (cm)

Figure 2.21: Area under the curve for different relaxation modes and elevation of the NMR-tube.
P1.25/2.5/1 with 75µL of 10mM MnCl2 directly added after production. (P/A 1-4/03/11)

63
Chapter 2 Materials and methods

18000
Area under the curve 16000
14000
12000
10000
8000
6000
4000
2000
0
0 2 4 6 8 10 12
Water volume (mL)

Figure 2.22: Area under the curve in the T2-distribution versus water volume in an NMR-tube of
18mm outer diameter. Receiver gain of the spectrometer was kept constant (RG=1.64).

2.9.4.4 Method D

Method D is a combination of Method B and C. This method is similar as Method B


regarding the preparation of a w/o-emulsion, but besides an Ultraturrax TV45 (until
visual satisfaction), also a Microfluidizer (at 6bar for 1.5 minutes) is utilized in order
to make a w/o-emulsion. The w/o/w emulsion is made exactly like in Method B. The
filling of the NMR-tube is done as in Method C.

2.10 Quantitative analysis of the enclosed water volume and yield of water-in-oil-in-
water emulsions

2.10.1 The CPMG-experiment

The enclosed water volume was estimated by transverse relaxation time constant (T2)
distribution measurements via CPMG-experiments (Carr Purcell Meiboom Gill) in the
presence of MnCl2 with a Maran Ultra 23 spectrometer. The temperature of the
samples was +5°C and the Set Temperature of the spectrometer was -7°C. One radio

64
Chapter 2 Materials and methods

frequency pulse of 90° is applied and N radio frequency pulses of 180°, which results
in N spin echoes or CPMG spin-echo trains (Figure 2.23).

Figure 2.23: The CPMG-experiment

The basis of an NMR experiment is the change of the total magnetic moment or
magnetization of a sample, which can be represented in a xyz-space. Without pulses,
the magnetization vector is aligned with and precesses about the static field B0 or z-
axis. Application of a 90° pulse (radio frequency pulse or B1) puts the magnetization
vector (M0) into the xy-plane (Figure 2.24). All spins experience the same applied
pulse and are in phase along the y-axis.

Figure 2.24: Change of the magnization by a 90°pulse, without 180°pulses. The magnetization
vector aligns back with the static field B0 over time scales of T2.

Without subsequent 180° pulse, the magnetization vector is completely restored along
the z-axis. More into detail, over time scales of T2, the spins lose phase coherence in
the xy-plane due to differences in magnetic field in the sample (spin spin interactions)
or inhomogeneity in the magnetic field. Because the dephasing is measured in the xy-
plane, which is perpendicular to the z-axis, this is called a transverse magnetization
experiment. Alternatively, the loss of coherence can also be observed along the z-axis,

65
Chapter 2 Materials and methods

which happens over time scales of T1 and is denoted as the recovery of the
longitudinal magnetization.
Application of a 180° pulse flips the magnetization vector around the y-axis (Figure
2.25). The so-called spin isochromats are bundles of spins that experience the same
magnetic field. The sum of the spin isochromats is equal to M0 or magnetization
vector at equilibrium, which is equal to the magnetization vector M when aligned with
the static field B0. As each spin isochromat continues to precess with its frequency, all
isochromats will rephase. This rephasing removes the influence of inhomogeneity of
the magnetic field on the spins, hence the change of magnitude of the echo will be due
to the spin spin interactions only.
Application of multiple 180° pulses, results in multiple echos, from which the
magnitude further decreases as more pulses are applied (Figure 2.23). The amplitudes
of the decaying spin echoes as a function of time yield an exponentially decaying
curve, which is characterized by a time constant T2.

Figure 2.25: A 90°x pulse puts M0 into the Y-direction (a). Spin isochromats fan out with time
(b). A 180°y pulse rotates the spins around the Y-axis (c). Rephasing of the spins results in an
echo (d). The difference of magnitude of M in (d) and (a) is a direct measure of T2.

After running a script (Appendix C), this (experimental) relaxation time curve (Figure
2.23) is recorded by the software RINMR. Unless stated differently, the receiver gain
(RG), which is a parameter that maximizes the signal, is optimized automatically prior
to analysis. The calculated NMR is obtained after uploading the experimental data in
the software WinDXP and exporting them to Excel (Appendix D). The x-axis or time-
axis of the relaxation time curve (Figure 2.26) increases from 403.95µs in steps of
400µs to 3276804µs. The y-axis, which represents the relaxation magnitude, differs

66
Chapter 2 Materials and methods

among samples. The decay can be mono or bi-exponential, depending on the different
water phases present and their differentiation with the paramagnetic agent MnCl2. A
stock solution of 10mM MnCl2 in water was prepared. This compound reduces the T2-
relaxation time of water in contact with it and therefore decays faster. In a double
emulsion, MnCl2 will only end up in the external water phase, on the condition that
there is no exchange of water between both water phases through the fat phase. If no
distinction between the internal and external water phase of a double emulsion is
made, e.g. in the absence of MnCl2 (Figure 2.26, red curve), or if only an internal
water phase is present, e.g. in w/o-emulsions, then a mono exponential decay is
acquired. A bi-exponential decay is achieved for w/o/w emulsions after addition of
MnCl2. The fast and slow decaying part of the bi-exponential curve (Figure 2.26,
black curve) are associated with the external and internal water of the double
emulsion, respectively.

14000

12000
Relaxation magnitude

Experimental NMR data


10000 + MnCl2
8000
Calculated NMR data
6000
+ MnCl2

4000 Experimental NMR data


- MnCl2
2000
Calculated NMR data
0 - MnCl2
0 500 1000 1500 2000 2500 3000 3500

Time (ms)

Figure 2.26: CPMG T2-relaxation decay profile of a w/o/w emulsion P1.25/2.5/1. Before (-MnCl2)
and after (+MnCl2) addition of 100µL 10mM MnCl2. to 8mL sample, P/A: 25-26/02).

By means of illustration, the CPMG T2-decay for various emulsions is represented in


Figure 2.27. Fast decays are associated with T2-analysis of the fat phase. Irrespective
of the fat phase, a bi-exponential decay is obtained for double emulsions upon
addition of 75µL 10mM MnCl2 to 8mL sample. A mono-exponential decay can be
observed for w/o/w emulsions with an insufficient amount of MnCl2, which comes
down to an addition of less than 75µL 10mM MnCl2 to 8mL sample.
Contin analysis of the relaxation time curve creates a T2-distribution (Figure 2.28)
time scale goes up from 100µs to100s, where the increase occurs in steps of (t3-

67
Chapter 2 Materials and methods

t2)=(t2-t1)*1.056. A T2-distribution can be constructed directly with the raw data from
the relaxation time curve in the software WinDXP or can be rebuilt by exporting the
data from WinDXP to Excel (Appendix D). Irrespective of the volume of the sample
in the NMR-tube, the total area under the curve is approximately constant.
Figure 2.28 (red curve) reveals that without MnCl2, only one peak associated with
water is observed at a T2 of about 1s. This peak comprises both the external and the
internal water. By addition of MnCl2 the water peak is split into two peaks
corresponding to the external and internal water (Figure 2.28, black curve): the
transverse (T2) relaxation time will be longer for water molecules within the fat
droplets of the double emulsions than when present in the external water phase to
which MnCl2 is added.

16000 W/o (P1.25/2.5)


14000
Relaxation magnitude

12000 Fat phase: soft PMF+


2.5%(w/w) PGPR
10000
W/o/w, insufficient MnCl2
8000
(P1.25/2.5/1)
6000
W/o/w, no MnCl2
4000 (P1.25/2.5/1)
2000
W/o/w, MnCl2
0 (M1.25/2.5/1)
0 200 400 600 800 1000 1200 1400
W/o/w, MnCl2
Time (ms) (P1.25/2.5/1)

100000 W/o (P1.25/2.5)


Relaxation magnitude

10000
Fat phase: soft PMF+
2.5%(w/w) PGPR
1000
W/o/w, insufficient MnCl2
(P1.25/2.5/1)
100
W/o/w, no MnCl2
10 (P1.25/2.5/1)
W/o/w, MnCl2
1 (M1.25/2.5/1)
0 200 400 600 800 1000 1200 1400
W/o/w, MnCl2
Time (ms) (P1.25/2.5/1)

Figure 2.27: CPMG T2-decay profiles for various emulsions on a linear scale (Top) and a
logarithmic scale (Bottom). A w/o/w emulsion with insufficient MnCl2 signifies an addition of less
than 75µL 10mM MnCl2 to 8mL sample. In w/o/w emulsions P1.25/2.5/1 and M1.25/2.5/1
(ratio(soft PMF/Hozol)=3/1), 100µL and 28µL 10mM MnCl2 was added to (vortexed) 8mL and (non-
vortexed) 3mL, respectively.

68
Chapter 2 Materials and methods

Typical T2-relaxation times for water in the absence of MnCl2 amount to 1s, whereas
the relaxation time of the water in contact with MnCl2 can be found at about 100ms.
An additional peak at about 10ms embodies the hydrogen atoms in the liquid part of
the fat phase. Hence, a T2-distribution of a double emulsion is defined as a trimodal
distribution, which is characterized by three modal relaxation times (small, medium
and large) and a signal amplitude of the modes.

700

600
W/o/w
Signal amplitude

500
without
400 MnCl2

300
W/o/w
200 with
MnCl2
100

0
1 10 100 1000 10000
Time (ms)

Figure 2.28: T2-distribution of a w/o/w emulsion (P1.25/2.5/1) with and without addition of 100µL
10mM MnCl2 to 8mL sample (or filled for 40mm) (P/A: 25-26/02).

Figure 2.29 shows the T2-distributions of the emulsions shown in Figure 2.27. A w/o-
emulsion and a w/o/w emulsion in the absence of MnCl2 are characterized by a
similar T2-distribution. Considering the former emulsion, the relative ratio of the peak
associated with fat to the peak associated with water is larger than in the latter
emulsion, which is in accordance to their difference in mass ratio of the mixed fat
phase to water phase. As discussed in section 2.2.3, the mass ratio could be
determined by an oven test. Extraction of the mass ratio from the T2-distribution is
more cumbersome in terms of the requirement of additional information and could be
assessed in two ways. Firstly, the area under the peak that is associated with fat in the
T2-distribution of a w/o-emulsion can be divided by the area under the peak in the T2-
distribution of only fat phase. For example, the data of the w/o-emulsion exhibited in
Figure 2.29 render a mass ratio fat/water of 40%, whereas in theory this would be
50%. The discrepancy might be explained by the difference in solid fat content
between a pure fat sample and a w/o-emulsion at the same temperature. Secondly,
calculation of the fat fraction can be performed on the basis of one T2-distribution,

69
Chapter 2 Materials and methods

whereby the knowledge of the percentage of relaxing protons in the fat and water
phase is required.
Also from Figure 2.29, the T2-distribution of the w/o/w emulsion with insufficient
MnCl2 can be regarded as a transition between the T2-distribution of w/o/w emulsions
in the absence and MnCl2 and w/o/w emulsions with sufficiently added MnCl2.

W/o (P1.25/2.5)
800

700 Fat phase: soft


PMF+
600 2.5%(w/w)PGPR
Signal amplitude

W/o/w,
500
insufficient
MnCl2
400 (P1.25/2.5/1)
W/o/w, no MnCl2
300 (P1.25/2.5/1)

200 W/o/w, MnCl2


(M1.25/2.5/1)
100

0 W/o/w, MnCl2
(P1.25/2.5/1)
1 10 100 1000 10000
Time (ms)

Figure 2.29: T2-distribution for the various emulsions shown in Figure 2.27. A w/o/w emulsion
with insufficient MnCl2 signifies an addition of less than 75µL 10mM MnCl2 to 8mL vortexed
sample. In w/o/w emulsions P1.25/2.5/1 and M1.25/2.5/1 (ratio of soft PMF to Hozol=3/1), 100µL
and 28µL 10mM MnCl2 was added to (vortexed) 8mL and (non-vortexed) 3mL, respectively.

2.10.2 Determination of the enclosed water volume

From the peak areas in the T2-distributions, the enclosed water volume can be
estimated:

Estimated enclosed water volume (%) = [AUC(s) 100%] / [AUC(s) + AUC(f)]


Equation 2.14

where AUC (s) and AUC (f) stand for Area Under the Curve for the peak of the water
that is characterized by a slow and fast T2-relaxation time, respectively. Adding
MnCl2 dilutes the sample. Sabatino et al. (2011) showed that when this extra volume

70
Chapter 2 Materials and methods

was taken into account, no significant effect of the added MnCl2 was observed. The
percentage enclosed water volume is expressed as:

Estimated enclosed water volume (%) = mL of internal water


100mL of total water in the double emulsion

Equation 2.15

where the volume can be replaced by the mass on account of the equal densities of the
two water phases.

2.10.3 Determination of the yield of a double emulsion

The yield (%) of a double emulsion is expressed by the equation:

Yield of a double emulsion (%) = Amount of internal water 100%


Theoretical maximum amount of internal water

Equation 2.16

The amount of water can be either expressed in volume or mass units. Bearing in
mind that the theoretical maximum enclosed water percentage is equal to 20g (w1-
phase) per 80g water (w1 and w2-phase) in w1/o/w2 emulsions that are prepared in a
weight ratio of 20:20:60, then the inclusion of the estimated enclosed water volume
revises Equation 2.16 of the yield of a double emulsion into Equation 2.17:

Estimated enclosed water (%)


Yield of a double emulsion (%) = 100%
Theoretical maximum enclosed water (%)

Estimated enclosed water (%)


= 100%
25%
Equation 2.17

71
Chapter 2 Materials and methods

2.10.4 Statistical methods

Assumptions were checked prior to application of parametric tests, such as the two
sample t-test and ANOVA. If they didn’t hold, non-parametric tests were used, such
as Wilcoxon rank sum test and Kruskal Wallis (S-Plus). Three repetitions of one
sample of each condition were analyzed, 24h after production. Hence, the standard
deviation associated with the three values per sample corresponded more to the
variation in measurement than to the variation in production. If more than two groups
need to be compared simultaneously, a Bonferroni correction (S-Plus) was applied.

2.11 Fat globule analysis by laser diffraction

The size of multiple droplets in a double emulsion was analyzed by a Mastersizer S


(Malvern Instruments) with settings 3PDD and breaking indices 1.596 and 1.33.

2.12 Profilometric analysis of double emulsions

One dimensional pulsed field gradient NMR profilometry is introduced in this thesis
as a novel technique to analyze the extent of creaming of a double emulsion and to
calculate the water content in the creamed layer of a sample. The advantage of this
method lies in its fast and nondestructive behavior. Besides the extent of creaming,
also the creaming rate could be determined by repetitive measurements at fixed time
intervals. Regarding double emulsions, profilometry offers the advantage over the
determination of the creaming rate by creaming accelerated measurement of the
transmission in a LUMiFuge apparatus, in terms of avoidance of possible detrimental
centrifugal forces on the enclosed water percentage. Profilometric analysis was
performed with a Maran Ultra 23 spectrometer, using a script in the RINMR software
(Appendix E). Reference is made to (Zhu, 2011). The first and second pulse gradient
duration amounted of 3562 and 7124µs, respectively. Their gradient strength was
31mT/m.

72
Chapter 2 Materials and methods

2.13 Whipping of a commercial dairy cream and w/o/w-emulsions

2.13.1 Commercial dairy cream

Whipping cream with 40g milk fat per 100mL (UHT treated, stabilizer: carrageenan,
Campina) was purchased.

2.9.2 W/o/w emulsions

2.9.2.1 First attempt to prepare whippable double emulsions

Two hundreds milliliter of double emulsion were made in accordance to method D.


The composition of this emulsion was M1.25/2.5/1 in which M stands in this
experiment for a mix of 62.5% soft PMF and 37.5% Hozol. EosinY (0.001%, w/v)
was added to the internal water phase (w1) to improve visualization of possible phase
separation. Firstly, about 90g of w/o-emulsion was made, from which 40g was
weighed and mixed with 60g of external water phase (w2). Secondly, again 100g of
w/o/w emulsion was prepared to result in a total produced amount of 200g, which was
collected in four Greiner tubes of 50mL and cooled in an ice bath. Afterwards, the
samples were attached in a Rotator SB2 at fixed rotational speed of 20rpm to prevent
creaming, while being kept overnight in a room at 5°C.

2.9.2.2 Second attempt to prepare whippable double emulsions

The second attempt differs from the first attempt in terms of the manner of prevention
of creaming. Each 30 minutes, the sample was gently twisted. Headspace was
provided to enable mixing while twisting. After production until whipping, the sample
was placed in a refrigerator at 5°C for 6.5h.

73
Chapter 2 Materials and methods

2.9.2.3 Third attempt to prepare whippable double emulsions

In the third endeavor, creaming is prevented by addition of 0.2%(w/w) xanthan gum


to the external water phase (w2) and the double emulsion was prepared analogously as
in the first attempt. Prior to whipping, storage was done at 5°C for 16h.

2.9.2.4 Fourth attempt to prepare whippable double emulsions

Based on the knowledge that small molecular weight surfactant are efficient in
displacing proteins from the interfacial film, which is desired in an intended
destabilization such as whipping, sodium caseinate in the external water phase was
replaced by 1%(w/v) cream residue powder (CRP), which contains phospholipids. In
addition, 0.2%(w/w) xanthan gum was added. The sample was stored at 5°C for 7h.

2.9.3 Determination of the whipping time

A Kenwood mixer (Major KM250) equipped with a standard wire whisk was used
from which the bowl and whisker were kept in the fridge prior to whipping. The
temperature in the fridge was 5°C. The volume of whipping cream was 200mL and
the applied speed level of the mixer was 3. The whipping time was chronometered as
the time between switching on the mixer and visual satisfaction.

2.9.4 Determination of the overrun of a whipped emulsion

A plastic transparent recipient of 200mL was weighted empty (m1) and filled with
unwhipped (m2) and whipped cream (m3). To avoid overestimation of the overrun, the
recipient should be filled completely without including artificial larger bubbles, which
can be removed by tapping gently (van Aken, 2001; Van Lent et al., 2008).
The overrun was determined from the equation:

74
Chapter 2 Materials and methods

(net mass of unwhipped cream) - (net mass whipped cream)


Overrun (%) = 100%
(net mass of unwhipped cream)

(m 2 - m1 ) - (m 3 - m1 )
= 100% Equation 2.18
(m 2 - m1 )

2.9.5 Physical destabilization (drainage) of whipped emulsions

The mass of a sieve with pore size 1mm and an aluminum dish was measured.
Approximately 50g of whipped emulsions was brought on the sieve that rested on the
aluminum dish. After 1h at room temperature (16-18°C) the serum that had leaked
from the whipped emulsion on the sieve into the aluminum dish was determined:

Serum leakage (%) = net mass of serum in the aluminium dish 100%
mass of whipped cream brought on the sieve

Equation 2.19

75
Chapter 3 Results and discussions

Chapter 3

Results and discussion

3.1 Optimization of the water droplet size analysis

3.1.1 Temperature experiment by using a thermocouple

A small temperature test was performed with the aim to find an appropriate Set
Temperature of the Maran spectrometer. The latter can be defined as the temperature
that doesn’t give rise to temperature changes of a representative sample inside this
tube during the time period that the NMR-tube has to stay inside the Maran
spectrometer in order to complete the analyses of all magnetic gradient pulse width
values (δ). After programming a certain Set Temperature, some time was required
until the Set Temperature was reached and stabilized. Before placing the emulsion in
the spectrometer, it was kept in a water bath (Julabo) with Set Temperature of 5.3°C.
The temperature inside the emulsion, while kept in the water bath and covered with
parafilm, amounted to 4.2 to 4.3°C (Table 3.1), which was measured with a
thermocouple (Agilent 3497 OA). Regarding the accuracy of the temperature
measurements with the thermocouple, a digital calibrated thermometer displayed
about 0.65°C higher than the thermocouple. As such, the data represented in Table 3.1
and Figure 3.1 are corrected for this. Afterwards, the NMR-tube with sample covered
by a parafilm was transferred to the Maran spectrometer. The (transfer) time between
the removal of the tubes from the water bath and the beginning of the pfg-NMR
measurement (time zero) was shorter than 45s (Table 3.1), which resulted in an
average temperature of the emulsion at time zero of 4.8°C. The thermocouple probe
was positioned in such a way that it was immobile and measured the temperature
inside the emulsion without touching the glass of the NMR-tube.
Figure 3.1 represents the follow-up of the temperature of an emulsion (H1.25/2.50),
which is placed in the Maran Ultra 23 spectrometer as a function of time during a pfg-

76
Chapter 3 Results and discussions

NMR measurement of approximately 9.5 minutes. Based on this figure, the Set
Temperature of -7°C and -8°C is recommended for analysis of w/o-emulsions because
it results in a stable probe temperature. Consequently, all emulsions were analyzed
accordingly, except the emulsions that were analyzed at an earlier date than the
conducted temperature experiment, for which the Set Temperature is mentioned. Once
the samples are brought in the spectrometer, attention must be paid to the fact that
temperature control in the probe is not excellent, meaning that the samples should be
analyzed right away and not after a set time delay.

A first remark about this temperature measurement concerns the dependency of the
optimal Set Temperature on the composition of the emulsion. The thermal heat
capacity of a more fluid like emulsion, containing Hozol, is bigger than the thermal
heat capacity of a solid like emulsion, containing soft PMF or a mix of Hozol and soft
PMF. Hence, more energy is needed to increase the temperature of a sample with
Hozol. A more representative sample is a more sensitive sample towards temperature
change, such as a sample made of soft PMF or a mix of soft PMF and Hozol.
A second remark has to do with the definition of an optimal Set Temperature. During
measurement of the sample in the Maran spectrometer, the objective is to find an
approximately constant temperature. Whether this results in a sample temperature of
exactly 5°C is of less importance, since this will be corrected for by performing a
calibration at the same Set Temperature as the sample.
A third remark concerns the heat capacity of the applied thermometers. The digital
thermometer responds quickly to temperature changes and the temperature was
recorded in less than 1min. The non digital thermometer is characterized by a larger
heat capacity and was therefore, prior to usage, cooled down to 5°C in order to
compensate the slow temperature adaptation.

77
Chapter 3 Results and discussions

Table 3.1: Temperature of the emulsion (H1.25/2.5) in the water bath, transfer time, temperature
of the emulsion at time zero and average temperature of the emulsion during a pfg-NMR
analysis. Transfer time is the time between removal from the water bath and time zero. All
parameters are given for different Set Temperatures of the Maran Ultra 23 spectrometer.

T(emulsion) (°C) in Transfer T(emulsion) (°C) at Average T(°C) during pfg-


Set T (°C) water bath time (s) time zero NMR analysis
-5°C 4.2 40 5.0 5.61
-6°C 4.2 33 4.6 5.28
-7°C(A) 4.3 36 4.8 4.99
-7°C(B) 4.3 36 5.1 4.92 4.98
-7°C(C) 4.3 44 4.7 4.83
-7°C(D) 4.3 36 5.0 4.92
-7°C(E) 4.3 30 4.9 4.89
-8°C(A) 4.3 30 4.5 4.59 4.62
-8°C(B) 4.3 39 4.6 4.56
-9°C 4.3 39 4.5 4.28

7
Emulsion temperature (°C)

6.5
Set T=-5°C
6
Set T=-6°C
5.5 Set T=-7°C
Set T=-8°C
5
Set T=-9°C
4.5

4
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5
Time (min)

Figure 3.1: Follow-up of the temperature of the emulsion (H1.25/2.5) during NMR-analysis with
different Set Temperatures of the Maran Ultra 23 spectrometer. For the Set Temperature -7°C
and Set Temperature -8°C, averages of five and two measurements are shown respectively,
whose standard deviation is given as error bars.

3.1.2 Repeatability experiment

Aiming for a correct manner of rounding up of values for D43, a repeatability


experiment was conducted on three samples of emulsion P0.5/1. According to the
definition of repeatability by the ISO (1993), the following conditions must be
fulfilled: the same measurement procedure, the same observer, the same measuring
instrument, used under the same conditions, the same location and repetition over a
short period of time. IUPAC states that the measure of repeatability is the standard
deviation qualified with the term ‘repeatability standard deviation’. Repeatability may

78
Chapter 3 Results and discussions

also be defined as the value below which the absolute difference between two single
test results obtained under the above conditions, may be expected to lie with a
specified probability.
In the experiment the sequence of conducted measurements is as follows:
sample 1- sample 2- sample 3- sample 1- sample 2- sample 3- sample 1- sample 2-
sample 3.
This sequence was chosen because the experiment had to be conducted over a short
period of time, but not too short. If, for example, sample 1 would have been
conducted three times in a row, the elapsed time (about 10 minutes) between two
measurements would be too short. Moreover, this would mean that a sample stays
continuously for about 30-35 minutes inside the spectrometer, which is characterized
by a poor temperature control. With the proposed sequence of conducting
measurements, the elapsed time between the first and second measurement of the
sample 1 and of sample 2 was 36 minutes. For sample 3, this took 34 minutes, which
was identical to the time duration between the second and third measurement of this
sample 3. The time duration between the second and third measurement was 33
minutes for sample 1 and 2. Thus, approximately, the repetition period was 34
minutes.
In Table 3.2, the repeating measurements are presented in the same order as they were
conducted. The volume-weighted arithmetic mean diameter (D43) and the geometric
standard deviation (σg) of the samples P0.5/1 were calculated by using different data
processing methods.
In Table 3.3 the statistical analysis of the repeatability experiment with the parameters
received from the Droplet Size application and Matlab are presented. The standard
deviation of the three measurements of the volume-weighted arithmetic mean
diameters of sample 1, 2 and 3 are 0.012, 0.004 and 0.003µm, respectively. Based on
these values, it is suggested that, when measuring the same sample again after 34
minutes under the same conditions, more than one digit after the comma of the
volume-weighted arithmetic mean diameter will be less reliable than the first digit
after the comma. In other words, it is expected that the difference between two tests of
the same sample, with a time interval of 34 minutes and under the same conditions,
may be of the magnitude of two digits after the comma. Also from Table 3.3 it can be
seen that the replicate samples are significantly different from each other if they are
measured over a time scale of 90minutes.

79
Chapter 3 Results and discussions

In conclusion, in order to offer sufficient information and implement the outcome of


this repeatability experiment, all D43-values will be rounded up to two digits after the
comma.

Table 3.2: Calculated volume-weighted arithmetic mean diameter (D43) and geometric standard
deviation (σg) of the water droplets in the P0.5/1 emulsion by using Droplet Size application and
Matlab. No results could be obtained with Excel. Three samples were made and each sample was
analyzed three times.
Droplet Size application Matlab
P0.5/1 D43 (µm) σg D43 (µm) σg
Sample 1 -first time 2.66 1.002 1.81 1.000
Sample 2 -first time 2.58 1.002 1.83 1.000
Sample 3 -first time 2.60 1.009 1.81 1.014

Sample 1 -second time 2.64 1.001 1.75 1.190


Sample 2 -second time 2.58 1.002 1.82 1.009
Sample 3 -second time 2.62 1.002 1.82 1.000

Sample 1 -third time 2.64 1.003 1.79 1.034


Sample 2 -third time 2.60 1.008 1.83 1.000
Sample 3 -third time 2.62 1.004 1.81 1.001

Table 3.3: Statistical analysis of the repeatability experiment indicating the average, standard
deviation of the three repetitions of each sample and 95% confidence interval.
Droplet Size application Matlab
Sample 1
Average D43 (µm) 2.64 1.78
Standard deviation (µm) 0.012 0.034
95% Confidence interval (µm) [2.618 ; 2.675] [1.698 ; 1.869]

Sample 2
Average D43 (µm) 2.59 1.83
Standard deviation (µm) 0.004 0.005
95% Confidence interval (µm) [2.580 ; 2.599] [1.812 ; 1.839]

Sample 3
Average D43 (µm) 2.61 1.81
Standard deviation (µm) 0.003 0.008
95% Confidence interval (µm) [2.604 ; 2.621] [1.793 ; 1.832]

3.2 Quantitative particle size analysis of water droplets in commercial butters

In prospect of the preparation and analysis of w/o-emulsions, the droplet size of water
droplets in an industrially prepared (commercial) butter was determined. For all

80
Chapter 3 Results and discussions

analyses on butter, the running script, DSD.RIS.tif as described in section 2.3.2, was
used. The diffusion coefficient of water in the dispersed phase at 5°C was equal to
9.698E-10m/s2 for Bio Karneboter and 9.122E-10m/s2 for Ardense Roomboter. For
comparison, the diffusion coefficient of pure water at 5°C is equal to 1.3253E-09
m2/s.
By means of illustration, the experimental data of the first sample of Bio karneboter
and the best fitting calculated data, which were obtained by the three data processing
methods, are graphically represented in Figure 3.2a,b,c. In Figure 3.3 the averaged
echo intensity and standard deviation of three samples versus magnetic gradient pulse
width (δ) is graphically represented for both butters. As seen from this graph, a faster
echo decay as a function of magnetic gradient pulse width (δ) is observed for Bio
Karneboter than for Ardense Roomboter, hence larger water droplets will be found in
the former butter.
In Table 3.4 the volume-weighted arithmetic mean diameter (D43) and the geometric
standard deviation (σg) of the water droplets in the commercial butters are obtained by
using different data processing methods (Droplet Size application, Excel and Matlab),
as described in section 2.3.3.
In Table 3.5, a statistical analysis of the parameters D43 and σg, obtained by different
data processing methods, is presented: the average and standard deviation of three
samples for each commercial butter and the 95% confidence interval of the average
are mentioned.
The mean D43 of Bio Karneboter is significantly larger than the mean D43 of Ardense
Roomboter and this is valid for data coming from the Droplet Size application
(p=0.0086), Excel (p=0.0081) and Matlab (p=0.0079). This is in accordance to the
study of Van Lent et al. (2008). In the latter study a Bruker instrument was used.
Also, based on the samples of Bio Karneboter, the mean D43 obtained by the Droplet
Size application was significantly larger than the mean D43 obtained by Excel
(p=0.001) and by Matlab (p=0.001). Similarly, based on the samples of Ardense
Roomboter, the mean D43 obtained by the Droplet Size application was significantly
larger than the mean D43 obtained by Excel (p=0) and by Matlab (p=0).

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Chapter 3 Results and discussions

1.1
Ep measured
1.0
Ep calculated
0.9
Ep (-)

0.8

0.7

0.6

0.5
0 0.0005 0.001 0.0015 0.002 0.0025 0.003 0.0035 0.004 0.0045
δ (s)

Figure 3.2a: Measured and calculated echo attenuation ratio versus magnetic gradient pulse
width by Droplet Size application, for Bio Karneboter (sample 1).

1.1
Ep measured
1.0
Ep calculated
0.9
Ep (-)

0.8

0.7

0.6

0.5
0 0.0005 0.001 0.0015 0.002 0.0025 0.003 0.0035 0.004 0.0045
δ (s)

Figure 3.2b: Measured and calculated echo attenuation ratio versus magnetic gradient pulse
width by Excel, for Bio Karneboter (sample 1).

3,200
3,000 I (measured)
2,800 Raccum (calculated)
Echo intensity

2,600
2,400
2,200
2,000
1,800
1,600
1,400
0 0.0005 0.001 0.0015 0.002 0.0025 0.003 0.0035 0.004 0.0045
δ (s)

Figure 3.2c: Experimental (I) and best fitting calculated (Raccum) echo intensity versus magnetic
gradient pulse width by Matlab, for Bio Karneboter (sample 1).

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Chapter 3 Results and discussions

Table 3.4: Volume-weighted arithmetic mean diameter (D43) and geometric standard deviation (σg) of commercial butter samples calculated by different data
processing methods (Droplet Size application, Excel and Matlab)
Droplet Size application Excel Matlab
OUTPUT OUTPUT OUTPUT OUTPUT OUTPUT OUTPUT OUTPUT
Rmed (µm) σ (µm) D43 (µm) σg σ (µm) MF D33 (µm) D43(µm) σg R43 (µm) Stdev (µm) σg
Bio Karneboter
sample 1 0.9942 0.4159 3.64 1.516 0.3221 1.0138 2.59 2.72 1.380 1.3738 0.5024 1.425
sample 2 1.0839 0.3578 3.40 1.430 0.2809 1.0079 2.47 2.57 1.324 1.2943 0.3960 1.349
sample 3 0.9811 0.4060 3.50 1.501 0.3138 1.0096 2.51 2.63 1.369 1.3247 0.4695 1.411

Ardense roomboter
sample 1 0.7107 0.4610 3.00 1.586 0.3640 1.0046 2.17 2.31 1.439 1.1482 0.4711 1.484
sample 2 0.5234 0.5512 3.04 1.735 0.4407 1.0035 2.09 2.30 1.554 1.1583 0.5875 1.614
sample 3 0.5968 0.5155 3.02 1.674 0.4062 1.0225 2.13 2.31 1.501 1.1552 0.5403 1.560

Key to symbols:
Rmed: median radius or number weighted geometric mean radius of the log-normal distribution (µm)
R43: volume-weighted arithmetic mean radius (µm)
D43: volume-weighted arithmetic mean diameter (µm)
D33: volume-weighted geometric mean diameter (µm)
σ: distribution width or neperian logarithm of the geometric standard deviation of the particle radius or diameter distribution (µm)
σg : geometric standard deviation of the particle radius or diameter distribution (dimensionless)
Stdev: arithmetic standard deviation of the volume-weighted particle radius distribution (µm)
MF: multiplication factor that is entered in the solver of Excel together with R33 and Sigma
OUTPUT: data obtained directly from the output of the data processing method

83
Chapter 3 Results and discussions

3500
Ardense Roomboter
Bio Karneboter

3000
Echo intensity

2500

2000

1500

1000
0 0.001 0.002 0.003 0.004 0.005

δ (s)
Figure 3.3: Plot of the average echo intensity of three samples of Ardense Roomboter and Bio
Karneboter versus magnetic gradient pulse width. For each average echo intensity, the standard
deviation of three samples is represented by an error bar.

Table 3.5: Statistical analysis of D43 and σg, obtained by different data processing methods
(Droplet Size application, Excel and Matlab), obtained from three samples with the same
composition.
Droplet Size application Excel Matlab
Bio Karneboter
Average of D43 3.51 2.64 2.66
Standard deviation 0.121 0.078 0.080
95% Confidence interval [3.214 ; 3.813] [2.447 ; 2.835] [2.463 ; 2.861]

Ardense Roomboter
Average of D43 3.02 2.31 2.31
Standard deviation 0.020 0.008 0.010
95% Confidence interval [2.970 ; 3.070] [2.289 ; 2.328] [2.282 ; 2.334]
Droplet Size application Excel Matlab
Bio Karneboter
Average of σg 1.482 1.355 1.395
Standard deviation 0.0457 0.0292 0.0406
95% Confidence interval [1.369 ; 1.596] [1.283 ; 1.428] [1.294 ; 1.496]

Ardense Roomboter
Average of σg 1.665 1.493 1.552
Standard deviation 0.0753 0.0578 0.0654
95% Confidence interval [1.478 ; 1.852] [1.350 ; 1.637] [1.390 ; 1.715]

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Chapter 3 Results and discussions

3.4 Quantitative particle size analysis of water droplets in w/o-emulsions

3.3.1 Analysis of emulsions with composition H0/1, H0.5/1, M0/1 and P0/1

Water in oil-emulsions H0/1, M0/1 and P0/1 were prepared and analyzed with the
running script DSD.RIS.tif of the Maran Ultra 23 spectrometer. The diffusion
coefficient of the dispersed phase at 5°C for these emulsions was 1.457E-09 m2/s,
whereas for H0.5/1, which was analyzed with the script DSD_Lien.RIS.tif, this was
1.298E-09 m2/s. To the water phase of emulsion H0.5/1, 0.5% (w/v) eosinY was
added in order to enable better visualization of serum separation, which occurred after
approximately 2 weeks.
The experimental numerical data of the echo intensities of the analyzed w/o-
emulsions as a function of magnetic gradient pulse width (δ) can be found in
Appendix F.
In Table 3.6 the volume-weighted arithmetic mean diameter (D43) and the geometric
standard deviation (σg) of the water droplets in different w/o-emulsions were obtained
by using different data processing methods. In the column of determined parameters
in Excel, the error message ‘No results, cannot divide by zero’ denotes that the sum of
relative frequencies was zero, resulting in the error message (#DEEL/0!) for the
normalized relative frequencies. In this case, D33, which is converted to D43, and the
distribution width (Sigma) were not able to be calculated. In the same column of
Table 3.6, the sentence ‘No change of filled in values’ refers to the fact that when
filling in the values of D33 and Sigma from the column of Droplet Size application in
Table 3.6, together with the multiplication factor (for better fit, as explained in section
2.3.3.2.) into the solver of the Excel file, the parameters D33 and Sigma remained
identical to the filled in values. Furthermore, flat bars denote that no results could be
obtained and empty cells refer to lack of measurements. Normally, the first measured
echo attenuation intensity point is a good estimate for I0 (or the intensity of the echo
attenuation without pulsed field gradient), however, when the first measured point is
not the largest measured point, then the largest point was taken as the best estimate for
I0.
The impairment of Excel to produce values for D43 and Sigma is explained by the fact
that in Excel, the relative frequencies are calculated for a chosen range of diameters
with a relatively broad bin width, namely 1µm. This approach has the disadvantage

85
Chapter 3 Results and discussions

that, for samples characterized by a small distribution width, the measured


distribution might be located in between two adjacent values of the chosen set of
diameters, resulting in a zero percentage relative frequency for the chosen set of
diameters. The inverse approach for determination of the calculated echo attenuation
ratios is an approach that calculates the droplet diameter for a certain relative
frequency. This is the approach which is used in Matlab.

Table 3.6: The volume-weighted arithmetic mean diameter (D43) and the geometric standard
deviation (σg) of the water droplet distribution in different w/o-emulsions by using different data
processing methods. R-squared values are given whenever multiple outcomes of one sample by a
data processing method was collected (see section 2.3.4).
Droplet Size application Excel Matlab
D43 (µm) σg MF D43 (µm) σg D43 (µm) σg R2
H0/1
Produced 8/9/10
sample 1 - - - - - 3.94 1.000 0.9848
sample 1 Io(2.1) 3.94 1.000 0.9848
sample 1 * 5.50 1.097 1.410 4.00 1.004 4.00 1.000 0.9955
sample 2 4.90 1.001 no results, cannot divide by 0 3.72 1.000 0.9367
sample 2 * Io(3.1) 3.81 1.000 0.9731
sample 3 5.04 1.004 no change of filled in values - - -
sample 3 Io(2.1) 3.82 1.000 0.9439
sample 3* 3.92 1.000 0.9831

M0/1
Produced 9/9/10
sample 1 4.38 1.002 no change of filled in values - - -
sample 1 * 3.36 1.000
sample 2 - - - - - 3.23 1.000
sample 2 ** 4.32 1.002 no change of filled in values - - -
sample 3 - - - - - 3.22 1.000 0.9949
sample 3 ** 4.30 1.000 no results, cannot divide by 0 3.22 1.000 0.9969

P0/1
Produced 9/9/10
sample 1 4.76 2.065 1.012 3.35 1.821 3.44 1.895
sample 2 3.90 1.861 1.010 2.86 1.670 2.91 1.735
sample 3 4.60 2.309 1.012 3.23 2.013 3.30 2.094

H0.5/1
Produced 22/9/10
sample 1 4.28 1.116 1.044 3.06 1.081 - - -
sample 1 *** 3.26 1.000
sample 2 - - - - - 3.26 1.000 0.9967
sample 2 *** 4.26 1.001 no results, cannot divide by 0 3.26 1.000 0.9967
sample 3 4.26 1.101 1.037 3.05 1.079 3.26 1.000

*: output is calculated without including the first measured data point (echo intensity)
**: without including the second measured data point
***: without the last measured data point. MF: multiplication factor

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Chapter 3 Results and discussions

As illustrated in Table 3.6, data processing by Matlab of sample 1 of the w/o-


emulsion with code H0/1 and production date 8/9/10 has three values for the volume-
weighted arithmetic mean diameter and geometric standard deviation. This is
explained by the fact that three sets of data were obtained: the complete set, the set
from which one data point was removed and the set with a different I0. The complete
set didn’t give any results by the Droplet Size application. An error message
appeared: “Invalid floating point operation”. This was due to the fact that the
measured data were too much deviating from the calculated data and hence no good
fit could be achieved. By deleting a deviating data point, values for parameters were
obtained. For some samples the geometric standard deviation is equal to 1, which
would mean that all droplets are exactly the same. Figure 3.4 illustrates the effect of
the echo intensity decay as a function of gradient pulse width δ for a fixed volume-
weighted mean radius (R33) and a variable geometric standard deviation σg. Only the
data points at large δ are slightly influenced by σg. Hence, the estimation of the
distribution width is less accurate as compared to the distribution mean.

1.2

0.8 Sigma g=1.005


Echo intensity

Sigma g=1.01
0.6 Sigma g=1.015
Sigma g=1.05
0.4 Sigma g=1.1

0.2

0
0 1 2 3 4 5 6 7 8 9

δ (ms)

Figure 3.4: Echo intensity decay as a function of gradient pulse width for variable geometric
standard deviations. R33 is fixed at 1.5µm. Ds=1.31E-09m2/s.

Figure 3.5 shows a constant relationship between higher volume-weighted arithmetic


mean diameters obtained from the Droplet Size application and lower values for D43,
calculated by Matlab or Excel. W/o-emulsions made with Hozol as the fat phase
render significantly higher mean D43 than in emulsions prepared with a mix of soft

87
Chapter 3 Results and discussions

PMF and Hozol (p=0.044). This can be explained by the contraction of soft PMF
during solidification at refrigerator temperature, which would coincide with expulsion
of water from the emulsified water droplets and separation of free water. Since the
latter could not be observed, the findings might be explained by an increased
permeabililty for water of the continuous phase.

6.00

5.00

4.00
D43 (µm)

Droplet Size application


3.00 Excel
Matlab
2.00

1.00

0.00
H0/1 (8/9/10) M0/1 (9/9/10) P0/1 (9/9/10) H0.5/1 (22/9/10)
Emulsion composition

Figure 3.5: Plot of the average volume-weighted arithmetic mean diameter (D43) of three samples
for each emulsion composition, calculated by different data processing methods (Droplet Size
application, Excel and Matlab). For each average D43, the standard deviation of three samples is
represented by an error bar.

3.3.2 Analysis of emulsions with composition H0/1, H0.5/1, M0/1, P0/1, P0.5/1 and
P0.5/2

After mastering the Microfluidizer procedure better, emulsions with composition


P0.5/1 and P0.5/2 were made and samples H0/1, H0.5/1, M0/1 and P0/1 were remade.
These emulsions were analyzed with the script DSD_Lien.RIS.tif. The diffusion
coefficient of water in the dispersed phase at 5°C without sodium caseinate was
1.288E-09 m2/s. The diffusion coefficient at 5°C for emulsions containing 0.5% (w/v)
sodium caseinate was 1.247E-09 m2/s (H0.5/1) and 1.295E-09 m2/s (P0.5/1 and
P0.5/2). As illustration, Figure 3.6 gives a graphical representation of the
experimental intensity data of the echo attenuation in function of the magnetic
gradient pulse width (δ), as well as the best fit by Matlab for sample 1 of emulsion
H0.5/1 (Prod. date 21/10/10). The Set Temperature of the Maran Ultra 23
spectrometer was set at -14°C for analysis of P0.5/1 and P0.5/2. The analyzed
samples are presented in Table 3.7.

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Chapter 3 Results and discussions

2500
Measured data
Fitted line

2000
Intensity of the echo attenuation

1500

1000

500

0
0 1 2 3 4 5 6 7 8 9
Small delta (s) -3
x 10

Figure 3.6: Graphical representation of the experimental data of intensity of the echo
attenuation in function of magnetic gradient pulse width (δ) together with the best fit obtained
by Matlab for sample 1 H0.5/1 (Prod. date 21/10/10).

3.2.2.1 Statistical analysis of emulsions H0/1, H0.5/1, P0/1, M0/1, P0.5/1 and P0.5/2

In Table 3.7, the values of the samples that are associated with the highest R2 are
preferentially included in the statistical processing of the data. Concerning the
differences in data processing methods, a closer look to Table 3.7 results in the
conclusion that data processing by Excel often leads to no outcome, as discussed in
section 3.3.1. The statistical analysis of Table 3.7 consisted of the investigation of the
differences of the diameters of samples with the same composition between the
different processing methods. Also, the comparison of the emulsions H0/1, P0/1 and
M0/1 gives an idea about the influence of the fat type on the mean water droplet size
and the comparison between [H0/1 and H0.5/1], [P0/1 and P0.5/1] and the
comparison between [P0.5/1 and P0.5/2] renders information about the effect of the
concentration of the hydrophilic and hydrophobic emulsifier, respectively on the
mean water droplet size.

89
Chapter 3 Results and discussions

Table 3.7: The volume-weighted arithmetic mean diameters (D43) and the geometric standard
deviation (σg) of the water droplets in different w/o-emulsions by using different data processing
methods. MF=multiplication factor. Flat bars denote that no results could be obtained. R-
squared values are given whenever multiple outcomes of one sample by a data processing method
was collected.
Droplet Size application Excel Matlab
D43 (µm) σg R2 MF D43 (µm) σg R 2
D43 (µm) σg R2
H0/1
Produced 19/10/10
Sample 1 2.62 1.232 0.9935 no results, cannot divide by 0 3.72 1.181 0.9893
Sample 1 *** 2.32 1.271 0.9939 no change of filled in values 3.75 1.211 0.9950
Sample 2 4.56 1.107 0.9993 1.035 3.13 0.932 0.9987 3.47 1.043 0.9927
Sample 2 *** 4.58 1.134 0.9994 1.034 3.14 1.000 0.9987 3.48 1.071 0.9998
Sample 3 5.08 1.242 0.9961 1.020 3.75 1.175 0.9973 3.72 1.190 0.9973
Sample 3 *** 5.14 1.271 0.9962 1.022 3.76 1.188 0.9971 3.82 1.212 0.9971

M0/1
Produced 19/10/10
Sample 1 3.72 1.029 0.9893 no change of filled in values 2.87 1.000 0.9911
Sample 1 *** 3.80 1.333 0.9950 1.009 2.89 1.264 2.90 1.283 0.9961
Sample 2 3.68 1.101 0.9918 1.008 2.96 1.092 2.83 1.064 0.9935
Sample 2 *** 3.72 1.319 0.9951 no change of filled in values 2.86 1.271 0.9963
Sample 3 3.66 1.006 0.9917 no change of filled in values 2.83 1.000 0.9916
Sample 3 *** 3.70 1.287 0.9951 1.007 2.85 1.227 2.84 1.241 0.9963

P0/1
Produced 21/10/10
Sample 1 3.24 1.002 0.9719 no change of filled in values - -
Sample 1 *** 3.10 1.429 0.9955 1.006 2.41 1.346 2.41 1.386
Sample 2 3.34 1.002 0.9527 no change of filled in values 2.59 1.000 0.9416
Sample 2 *** 3.58 1.904 0.9896 1.009 2.62 1.714 2.71 1.792 0.9910
Sample 3 3.30 1.249 0.9545 0.981 2.56 1.296 0.9639 2.56 1.224 0.9577
Sample 3 *** 3.84 2.127 0.9937 1.014 2.70 1.845 0.9942 2.85 1.973 0.9948

H0.5/1
Produced 21/10/10
Sample 1 5.18 1.276 0.993 3.84 1.221 3.83 1.220
Sample 2 5.14 1.278 0.985 3.85 1.236 3.80 1.222
Sample 3 5.06 1.275 0.982 3.81 1.237 3.80 1.219

P0.5/1 ■
Produced 30/9/10
Sample 1 2.65 1.002 no change of filled in values 1.79 1.071
Sample 2 2.59 1.004 no change of filled in values 1.83 1.003
Sample 3 2.61 1.005 no change of filled in values 1.81 1.005

P0.5/2
Produced 30/9/10
Sample 1 2.68 1.004 0.9410 no change of filled in values 2.08 1.000 0.9559
Sample 1 *** 2.48 1.018 0.9907 no change of filled in values 1.95 1.118 0.9913
Sample 2 2.80 1.004 0.8626 no change of filled in values 2.17 1.000 0.8615
Sample 2 *** 2.54 1.121 0.9921 1.128 2.47 1.066 1.99 1.000 0.9918
Sample 3 - - no change of filled in values 2.11 1.000 0.8289
Sample 3 *** 2.46 1.002 no change of filled in values 1.91 1.000 0.9835
■: each sample is the average of three repetitions. ***: without the last measured data point

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Chapter 3 Results and discussions

3.3.2.2 Difference between different data processing methods

For each emulsion composition, three samples were produced. Thus, 3 values
calculated with the Droplet Size application are compared with 3 values obtained with
Excel and 3 values from Matlab. This is graphically illustrated in Figure 3.7, in which
it can be observed that Matlab calculates a significantly lower mean volume-weighted
arithmetic mean diameter than the Droplet Size application for the same emulsion (all
p<0.02, except for H0/1). Possibly due to the small Sigma of sample 1 of H0/1, a
deviating value for D43 is obtained. A significantly larger mean volume-weighted
arithmetic mean diameter is recorded for the Droplet Size application than for Excel
for the samples with composition M0/1, P0/1 and H0.5/1 (all p<0.02). Insufficient
data from Excel were available for H0/1, P0.5/1 and P0.5/2. A significantly larger
mean σg by the Droplet Size application in comparison to Excel and Matlab can be
detected for the sample with composition H0.5/1 (p=0.0082 and p=0). For the other
emulsion compositions, concerning the comparison of the replicates per sample
composition, Matlab results in slightly but not significantly lower mean geometric
standard deviation as compared to the Droplet Size application.

6.00

5.00

4.00
D43 (µm)

Droplet Size application


3.00 Excel
Matlab
2.00

1.00

0.00
H0/1 M0/1 P0/1 H0.5/1 P0.5/1 P0.5/2
(19/10/10) (19/10/10) (21/10/10) (21/10/10) (30/9/10) (30/9/10)
Emulsion composition

Figure 3.7: Graphical representation of the difference in mean volume-weighted arithmetic mean
diameter between three data processing methods. Error bars denote the standard deviation of
three samples.

3.3.2.3 Influence of the type of fat on the mean water droplet size

The influence of the type of fat on the mean volume-weighted arithmetic mean
diameter was investigated by comparison of the outcome from Matlab of samples

91
Chapter 3 Results and discussions

H0/1, M0/1 and P0/1. A significantly larger mean D43 (µm) was detected for H0/1
versus M0/1 (p=0.009) and for H0/1 versus P0/1 (p=0.005). As discussed in section
3.3.1, the solidification of soft PMF at refrigerator temperature might contribute to the
difference in mean water droplet size among emulsions with different fat composition.

3.3.2.4 Influence of the emulsifier on the mean water droplet size

To establish the effect of the concentration of the hydrophilic emulsifier on the mean
D43, the outcome by Matlab of samples H0/1 and H0.5/1 were compared. It could not
be stated that the addition of sodium caseinate had a significant effect on the mean D43
(p=0.2). This is not in accordance to the expectations; it is expected that adding
sodium caseinate would decrease the water droplet size, because more interfacial
surface can be stabilized by emulsifier. This statement holds when the applied air
pressure (6bar) of the Microfluidizer is capable of reducing the droplet size even
more. However, it is in accordance to what Calliauw (2009) observed with pfg-NMR
analysis, in contrast to the observations with light microscopy.
The same test was performed on the outcome by Matlab of samples P0/1 and P0.5/1.
A significantly larger mean D43 of P0/1 in comparison to P0.5/1 was found (p=0.011).
In other words, regarding emulsions based on soft PMF without hydrophilic
surfactant, the addition of sodium caseinate to 1% PGPR has a beneficial effect on the
reduction of the mean water droplet size.
The comparison of the mean D43 of P0.5/1 and P0.5/2, which tests the influence of the
concentration of the hydrophobic emulsifier on the mean D43, results in the conclusion
that, if the data are obtained by Matlab, the mean D43 is larger for the emulsion
samples containing soft PMF with 2% PGPR than for the samples containing soft
PMF with 1% PGPR (p=0.0065). If the data are obtained from the Droplet Size
application, then the opposite is reported (p=0.0086). Although the latter observation
is expected, the beneficial effect of PGPR to the water droplet size cannot be
statistically proven, because the data from the Droplet Size application and Matlab are
contradictory.

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Chapter 3 Results and discussions

3.3.3 Elevation of the water fraction of emulsions based on Hozol

Anticipating the creation of a double emulsion in which the fat globules are filled with
a maximal water fraction in order to reduce the caloric content of a whipping cream,
the water percentage of emulsions, which contain Hozol as the fat phase, was
increased from 20% to 30%, 40%, 50% and 60wt%. The emulsifiers sodium caseinate
and PGPR were increased proportionally to the water percentage in the emulsion as
illustrated in Table 3.8.

Table 3.8: Composition of the emulsions with different water percentage


W/o-emulsion H 0.50/1 H 0.75/1.5 H 1.0/2.0 H 1.25/2.5 H 1.5/3.0
Water percentage (wt%) 20 30 40 50 60

Oil percentage (wt%) 80 70 60 50 40


Water phase
Sodium azide (wt%) 0.02 0.02 0.02 0.02 0.02
Sodium caseinate (wt%) 0.50 0.75 1.00 1.25 1.50
Buffer solution (pH6.7) ad 100% ad 100% ad 100% ad 100% ad 100%
Oil phase
PGPR (wt%) 1.0 1.5 2.0 2.5 3.0
Hozol (%) 99.0 98.5 98.0 97.5 97.0

By means of a dilution test with water, a quick assessment of the type of emulsion
was acquired. If water was miscible with the emulsion, then this was an o/w-
emulsion, whereas immiscibility referred to a w/o-emulsion. As such, all prepared
emulsions were classified as w/o-emulsions, except H1.5/3.
Comparing the first echo intensity of the various emulsions, values of 5529, 7718,
10610, 14457 and 185 were found for 20, 30, 40, 50 and 60wt% water in emulsions.
As shown in Figure 3.8, the very small value for the 60% emulsion is due to a nearly
completely decayed echo intensity within the first time period, which is indicative of
free water diffusion as occurs in emulsions with an aqueous continuous phase.

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Chapter 3 Results and discussions

16000
14000 H0.50/1.00 measured
H0.75/1.50 measured
12000
Echo intensity
H1.00/2.00 measured
10000
H1.25/2.50 measured
8000
H1.50/3.00 measured
6000
4000
2000
0
0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009

δ (s)

Figure 3.8: Measured echo intensity as a function of magnetic gradient pulse width (δ) for
various emulsions. The connection line represents the calculated echo intensity obtained from the
Droplet Size application.

The volume-weighted arithmetic mean diameters of the emulsions are presented in


Table 3.9 and this reveals that also H1.5/3 gives a water droplet size outcome by data
processing, but the calculated data fit the measured data less, which is expressed in a
smaller R2. The script that was used was DSD_Lien.RIS.tif. The water diffusion
coefficient in the serum at 5°C of H0.5/1, H0.75/1.5, H1/2, H1.25/2.5 and H1.5/3
amounted to 1.299E-09, 1.289E-09, 1.282E-09, 1.283E-09 and 1.268E-09 m2/s,
respectively. At a certain temperature, an increase of sodium caseinate reduces the
diffusion coefficient as water molecules become less mobile.
Figure 3.9 shows the average volume-weighted arithmetic mean diameter as a
function of mass fraction of water in the w/o-emulsion, as determined by the Droplet
size application, Excel and Matlab. In there, Matlab renders consistently smaller
volume-weighted mean diameters than the Droplet Size application. The mean D43,
obtained by Excel for emulsions with 40 and 50% water fraction, are similar to the
mean D43 obtained by the Droplet Size application, whereas the mean droplet size
data obtained by Excel for the emulsion with 20% water fraction resemble the data in
Matlab. The latter confirms the observations in sections 3.3.1 and 3.3.2.2. In Excel,
the emulsions with 40 and 50% water fraction are characterized by a small
distribution width (│σ│< 0.020µm), whereas │σ│ for the emulsion with 20% water
fraction and for all other prepared emulsions resulting in a equivalent water droplet
size distribution as the data obtained by Matlab, is higher than 0.10µm. Hence, the
accuracy of the data in Excel that are based on deviating calculated distribution
widths might be doubtful.

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Chapter 3 Results and discussions

Table 3.9: Volume-weighted arithmetic mean diameter (D43) and geometric standard deviation
(σg) obtained by different data processing methods, for emulsions with different mass fraction of
water. R-squared values are given whenever multiple outcomes of one sample by a data
processing method was collected. MF= multiplication factor. Flat bars denote that no results
could be obtained.
Droplet Size application Excel Matlab
D43 (µm) σg R 2
MF D43 (µm) σg R 2
D43 (µm) σg R2
H0.5/1
Prod. 04/11/10
sample 1 4.90 1.248 0.9985 1.028 3.60 1.183 0.9983 3.66 1.202 0.9990
sample 1 *** 4.94 1.277 0.9990 1.030 3.61 1.194 0.9982 3.68 1.222 0.9993
sample 2 4.84 1.266 0.9982 1.024 4.57 1.194 0.9983 3.62 1.219 0.9989
sample 2 *** 4.88 1.292 0.9985 1.026 3.57 1.204 0.9982 3.64 1.237 0.9990
sample 3 4.82 1.231 0.9968 1.022 3.55 1.175 0.9974 3.61 1.186 0.9974
sample 3 *** 4.84 1.242 0.9963 1.022 3.55 1.173 0.9969 3.61 1.188 0.9971

H0.75/1.5
Prod. 03/11/10
sample 1 4.22 1.129 0.9992 1.045 3.04 1.085 3.21 1.086
sample1 *** 4.24 1.168 0.9996
sample 2 4.00 1.004 0.9996 no change of filled in values - -
sample 2 **** 4.00 1.001 0.9996 no results, cannot divide by 0 3.08 1.000
sample 3 3.96 1.001 no results, cannot divide by 0 - -
sample 3 *** - - - - - 3.04 1.000

H1/2
Prod. 28/10/10
sample 1 3.98 1.119 0.9982 1.243 3.76 1.000 3.72 1.079 0.9670
sample 1 *** 4.00 1.142 0.9978 no results, cannot divide by 0 3.05 1.092 0.9970
sample 2 **** 3.88 1.001 no results, cannot divide by 0 - -
sample 3 - - - - - 3.01 1.000 0.9984
sample 3 *** 3.94 1.064 0.9990 1.197 3.82 1.001 3.71 1.000 0.9757
sample 3 **** 3.94 1.016 0.9989 no change of filled in values 3.03 1.000 0.9980
sample 4 3.94 1.007 0.9988 no change of filled in values - -
sample 4 *** 3.94 1.000 0.9984 no results, cannot divide by 0 - -
sample 4 * 3.94 1.000 0.9984 no results, cannot divide by 0 3.04 1.000

H1.25/2.5
Prod. 04/11/10
sample 1 3.88 1.043 0.9992 no results, cannot divide by 0 2.98 1.000 0.9988
sample 1 *** 3.88 1.110 0.9993 1.208 3.82 1.006 2.98 1.039 0.9986
sample 2 - - - - - 2.97 1.000
sample 2 *** 3.84 1.000 no results, cannot divide by 0 - -
sample 3 3.92 1.075 0.9992 no results, cannot divide by 0 3.01 1.000 0.9989
sample 3 *** 3.92 1.128 0.9993 1.197 3.70 0.975 3.01 1.071 0.9988

H1.5/3
Prod. 11/11/10
sample 1 3.52 1.001 0.9516 no change of filled in values 2.72 1.000 0.9496
sample 1 *** 3.52 1.002 0.9314 no change of filled in values 2.71 1.000 0.9298
sample 2 3.50 1.001 0.7929 no change of filled in values - -
sample 2 *** 5.12 2.524 0.7693 1.061 2.23 1.883 3.55 2.248
sample 3 - - - - - 2.74 1.000 0.9057
sample 3 *** 3.50 1.005 no change of filled in values 2.70 1.000 0.8706
* or *** signifies that the first or last measured data point is omitted from the calculations,
respectively. **** refers to the exclusion of the last two data points in the calculation.

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Chapter 3 Results and discussions

Emulsion H0.5/1 with 20% water fraction is characterized by a significantly larger


mean volume-weighted arithmetic mean diameter, obtained in Matlab and the Droplet
Size application, than emulsion H0.75/1 (p<0.01), H1/2 (p=0) and H1.25/2.5 (p=0)
with 30, 40 and 50% water fraction, respectively.
Concerning H0.75/1.5, H1/2 and H1.25/2.5, no significant difference of the mean
volume-weighted arithmetic mean diameter can be detected (p=0.054). If the
surfactant concentration would be insufficient, then an increase of the water fraction,
and hence increase of water/oil interface, would result in an increase of water droplet
size, as larger droplets have a lower interfacial area in a w/o-emulsion with constant
water fraction. Since this can not be detected in the prepared emulsions for increasing
water fractions, the applied surfactant concentration is not inadequate at water mass
fractions up to 50%.

6.00

5.00

4.00 Droplet Size


D43 (µm)

application
3.00
Excel
2.00
Matlab
1.00

0.00
10 20 30 40 50 60
Water fraction in w/o emulsion (w/w)

Figure 3.9: Average volume-weighted arithmetic mean diameter and standard deviation (error
bars) of 3 samples per emulsion composition as a function of water fraction (w/w) in the w/o-
emulsion and determined by different data processing methods. The average D43 of emulsion
H1.25/2.5 (50wt% water) was calculated on 4 samples.

3.3.4 Effect of the decrease of the driving air pressure of the Microfluidizer M110S on
the water droplet size in a w/o-emulsion

The aim of this experiment was to find out whether a decrease of air pressure results
in a larger water droplet size. Emulsions with composition H1.25/2.5 (50% water
mass fraction) were homogenized in a Microfluidizer at two air pressures: 4bar or

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Chapter 3 Results and discussions

6bar. Due to pressure fluctuations above 6bar in the Microfluidizer, no higher


pressures were investigated.
The corresponding volume-weighted arithmetic mean diameters were determined by
the Droplet Size application and Matlab as represented in Table 3.10. Data processing
by Excel was omitted due to deficiency of results. The script that was used was
DSD_Lien.RIS.tif. The water diffusion coefficient in the serum at 5°C amounted to
1.283E-09m2/s.
Statistical analysis revealed that the mean volume-weighted arithmetic mean
diameter, obtained by the Droplet Size application, of a H1.25/2.5 emulsion is
significantly larger when the emulsion is homogenized at an air pressure of 4bar than
at 6bar (p=0.034). Increasing the air pressure of a Microfluidizer M110S with 1bar
results in a decrease of D43 of 0.03µm for both data processing methods. Based on this
experiment, an increase of air pressure of 2bar is not very cost-effective.

Table 3.10: Volume-weighted arithmetic mean diameter and geometric standard deviation of the
w/o-emulsion H1.25/2.5 (Production 11/11/10), produced at different air pressures. Data obtained
by the Droplet Size application and Matlab. Flat bars denote that no results could be obtained.
R-squared values are given whenever multiple outcomes of one sample by a data processing
method was collected.
Droplet Size application Matlab
D43 (µm) σg R2 D43 (µm) σg R2
Air pressure= 4 bar
sample 1 3.92 1.001 0.9988 3.02 1.000 0.9986
sample 1 *** 3.92 1.125 0.9995 3.01 1.067 0.9991
sample 2 3.84 1.000 - -
sample 2 *** - - - -
sample 3 - - - -
sample 3 *** 3.86 1.002 2.98 1.000
Air pressure= 6 bar
sample 1 - - - -
sample 1**** 3.76 1.000 2.89 1.000
sample 2 - - - -
sample 2*** 2.94 1.000 0.9929
sample 2**** 3.82 1.001 2.93 1.000 0.9913
sample 3 - - - -
sample 3*** 3.82 1.000 0.9970 2.96 1.000 0.9934
sample 3**** 3.84 1.000 0.9963 2.95 1.000 0.9939
*** signifies that the last measured data point was not included in the calculations. **** refers to
exclusion of the last two data points in the calculations.

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Chapter 3 Results and discussions

3.3.5 Analysis of emulsions with the hydrophilic surfactant whey protein isolate

In this experiment, w/o-emulsions were made with whey protein isolate (WPI) as the
hydrophilic surfactant in a concentration of 1.25% (w/v) instead of sodium caseinate.
The fat phase was Hozol with 2.5% (w/v) PGPR and the air pressure was set at 6bar
in the Microfluidizer. The mass fraction of water was 50% and contained 0.5% (w/v)
eosinY. During manufacturing, some clumps could be observed. This might be due to
thermal denaturation of the whey protein isolate, since during premixing with an
Ultraturrax device, temperatures run up to 60°C. The volume-weighted arithmetic
mean diameters as obtained by the Droplet Size application and Matlab are collected
in Table 3.11. The script that was used was DSD_Lien.RIS.tif. The water diffusion
coefficient in the serum at 5°C amounted to 1.288E-09 m2/s for sample 1 and 2, and
1.281E-09 m2/s for sample 3. The average and standard deviation of the D43 obtained
by the Droplet Size application was 3.93µm and 0.15µm.

Table 3.11: Volume-weighted arithmetic mean diameter and geometric standard deviation of
three samples (H1.25/2.5) with WPI as hydrophilic surfactant. Data are obtained by the Droplet
Size application and Matlab. R-squared is given when different outcomes for one sample were
collected. Flat bars denote that no results could be obtained.
Droplet Size application Matlab
D43 (µm) σg R2 D43 (µm) σg R2
sample 1 - - - -
sample 1 *** 3.94 1.008 - -
sample 2 - - - -
sample 2 *** 3.78 1.000 - -
sample 3 4.12 1.169 0.9953 3.14 1.118 0.9968
sample 3 *** 4.18 1.268 0.9969 3.17 1.207 0.9979
*** signifies that the last measured data point was not included in the calculations.

There is no significant difference of the mean volume-weighted arithmetic mean


diameter, obtained by the Droplet Size application, between the samples made with
WPI and samples made with sodium caseinate (see section 3.3.3, H1.25/2.5)(p=0.29).
WPI differs from sodium caseinate in its molecular size and hence speed of reaching
the interfacial area. Since emulsions were aged for 24h, the WPI had sufficient time to
reach the water oil interface. Besides the time aspect, also the concentration and type
of the surfactant determines the water droplet size in w/o-emulsions which are made
in identical preparation conditions.

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Chapter 3 Results and discussions

3.4 Qualitative particle size analysis of water droplets in w/o-emulsions by


fluorescence microscopy

3.4.1 Preliminary investigation of eosinY-solutions by fluorimetry

The aim of the fluorimetric investigation of eosinY in solution was to visualize water
droplets in a w/o-emulsion, which contains eosinY in the water phase. As mentioned
in section 3.3.3, the w/o-emulsion with 50% water contained 1.25wt% sodium
caseinate, a phosphate buffer (pH6.7) and 0.02wt% sodium azide. In order to see the
effect of these compounds on the maximum excitation and emission wavelength of
eosinY (5µg/mL), four different solutions were made and kept at room temperature.
The first solution was eosinY in distilled water. The second solution contained eosinY
and phosphate buffer in distilled water. The third solution comprised eosinY,
phosphate buffer and sodium azide in distilled water. The ingredients of the fourth
solution were eosinY, phosphate buffer, sodium azide and sodium caseinate in
distilled water. All four solutions were scanned at excitation and emission
wavelengths from 450 to 600nm. Afterwards, the maximum excitation or emission
wavelength was determined at narrower emission or excitation wavelength ranges,
respectively.

3.4.2 Determination of the maximum excitation and emission wavelength of eosinY


(5µg/mL) in water

For more detailed information about the excitation scan in Figure 3.10a, reference is
made to section 2.4.4.3. In Figures 3.10a and 3.10b the maximum excitation
wavelength lies in the heavily red or white colored area, respectively, which is located
between 510 and 520nm wavelength. Some scattering due to suspended particles in
the eosinY-solution at the bisection can be observed. The maximum excitation
wavelength of eosinY in water was extracted from the emission spectra at 533-543nm
with excitation at 470-540nm and amounted to 517nm.

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Chapter 3 Results and discussions

1000
70.00 701.64
647.57
60.00 800
593.51

50.00 Intensity (a.u.) 539.44


600 485.38
431.31
40.00
Z Axis

377.24
400 323.18
30.00
269.11
20.00 200 215.05
160.98
10.00 106.92
0 52.85
450 500 550 600-1.22
450.00 475.00 500.00 W avelength (nm)
525.00 550.00 575.00 600.00
Wavelength (nm)

Figure 3.10a: EosinY in water (5µg/mL). Contour plot of the scan for excitation and emission
wavelength ranging from 450 to 600nm in 75 steps of 2nm. X-axis: excitation wavelength (nm).
Z-axis: number of steps that are used to go from 450 to 600nm emission wavelength. Red color
denotes high intensity, blue color denotes low intensity.

Figure 3.10b: EosinY in water (5µg/mL). 3D-plot of the scan for excitation and emission
wavelength ranging from 450 to 600nm. X-axis: excitation wavelength (nm). Y-axis: fluorescence
intensity (arbitrary units). Z-axis: number of steps to go from the first to the last selected
emission wavelength.

Information about the maximum emission wavelength can be obtained from the Z-
axis in Figure 3.10a. The values 42 and 48 on the Z-axis border the heavily red area
and correspond to an emission wavelength of 534nm and 546nm by conversion with
Equation 2.13. Figure 3.11 is a mirror image of Figure 3.10 by reflection around the
bisection and it shows that the maximum emission wavelength lies between 533 and
545nm and it confirms that the maximum excitation wavelength can be found
between Z-axis values of 32 (or 514nm) and 38 (or 526nm), as described in section
2.4.4.3. The emission maximum was obtained by selecting a range of excitation

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Chapter 3 Results and discussions

wavelengths from 510 to 520nm (first method, see section 2.4.4.3) and amounted to
538nm.

1000
70.00 735.10
678.44
60.00 800
621.77
Intensity (a.u.)

565.11
50.00
600 508.45
451.79
40.00
Z Axis

395.13
400 338.47
30.00
281.81
20.00 200 225.14
168.48
10.00 111.82
0 55.16
450 500 550 600-1.50
450.00 475.00 500.00 W avelength (nm)
525.00 550.00 575.00 600.00
Wavelength (nm)

Figure 3.11a: EosinY in water (5µg/mL). Contour plot of the scan for emission and excitation
wavelength ranging from 450 to 600nm in 75 steps of 2nm. X-axis: emission wavelength (nm). Z-
axis: number of steps to go from the first to the last selected excitation wavelength. Red color
denotes high intensity, blue color denotes low intensity.

Figure 3.11b: EosinY in water (5µg/mL). 3D-plot of the scan for emission and excitation
wavelength ranging from 450 to 600nm. X-axis: emission wavelength (nm). Y-axis: fluorescence
intensity (arbitrary units). Z-axis: number of steps to go from the first to the last selected
excitation wavelength.

3.4.3 Determination of the maximum excitation and emission wavelength of eosinY


(5µg/mL) in an aqueous phosphate buffer (pH6.7)

Figure 3.12 unravels that the maximum excitation wavelength is situated between 500
and 530nm. The maximum excitation wavelength was acquired from sub maximum

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Chapter 3 Results and discussions

emission wavelengths, namely from 565 to 575nm (second method, see 2.4.4.3) and
was found to be 517nm. Figure 3.12 reveals a maximum emission wavelength range
of 534 to 562nm. Fluorescence spectra obtained at excitation wavelengths ranging
from 457 to 467nm revealed that a maximum emission wavelength can be found at
540nm. Whereas the fluorescence intensity of eosinY in water (Figure 3.11b)
remained below 1000a.u., the addition of phosphate buffer to eosinY increased the
intensity. Moreover, in the latter less scattering can be observed.

1000
70.00 962.91
888.74
60.00 800
814.56
Intensity (a.u.)

740.39
50.00
600 666.21
592.04
40.00
Z Axis

517.87
400 443.69
30.00
369.52
20.00 200 295.34
221.17
10.00 146.99
0 72.82
450 500 550 600-1.36
450.00 475.00 500.00 W avelength (nm)
525.00 550.00 575.00 600.00
Wavelength (nm)

Figure 3.12: EosinY (5µg/mL) in aqueous phosphate buffer (pH6.7) in 75 steps of 2nm. Contour
plot of the scan for emission and excitation wavelength ranging from 450 to 600nm. X-axis:
excitation wavelength (nm). Z-axis: number of steps to go from the first to the last selected
emission wavelength. Red color denotes high intensity, blue color denotes low intensity.

3.4.4 Determination of the maximum excitation and emission wavelength of eosinY


(5µg/mL) in aqueous phosphate buffer (pH6.7) with sodium azide (0.02%, w/v)

The maximum excitation wavelength can be found from 495 to 533nm (Figure 3.13).
The maximum excitation wavelength is 517nm and was measured at an emission
wavelength range of 565 to 575nm. The maximum emission wavelength is located
between 530 and 560nm (Figure 3.13) and amounted to 542nm, which was measured
at an excitation wavelength range of 500 to 570nm. The shoulder at 480-482nm as
seen in Figure 3.10 to 3.12 is also present in Figure 3.13.

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Chapter 3 Results and discussions

1000
70.00 962.91
888.74
60.00 800
814.57

50.00 Intensity (a.u.) 740.39


600 666.22
592.05
40.00
Z Axis

517.87
400 443.70
30.00
369.52
20.00 200 295.35
221.18
10.00 147.00
0 72.83
450 500 550 600-1.34
450.00 475.00 500.00 W avelength (nm)
525.00 550.00 575.00 600.00
Wavelength (nm)

Figure 3.13: EosinY (5µg/mL) in aqueous phosphate buffer (pH6.7) with sodium azide (0.02%).
Contour plot of the scan for emission and excitation wavelength ranging from 450 to 600nm in 75
steps of 2nm. X-axis: excitation wavelength (nm). Z-axis: number of steps to go from the first to
the last selected emission wavelength. Red color denotes high intensity, blue color denotes low
intensity.

3.4.5 Determination of the maximum excitation and emission wavelength of eosinY


(5µg/mL) in aqueous phosphate buffer (pH6.7) with sodium azide (0.02%, w/v) and
sodium caseinate (1.25%,w/v)

The maximum excitation wavelength is located between 510 and 542.5nm (Figure
3.14). Fluorescence spectra at emission wavelengths ranging from 566 to 576nm
revealed a maximum excitation wavelength at 527nm. Figure 3.14 reveals a
maximum emission wavelength range of 533 to 565nm. Fluorescence spectra
obtained at excitation wavelengths ranging from 500 to 510nm revealed a maximum
emission wavelength at 547nm.

This eosinY-solution, in comparison to solutions in the absence of the protein,


demonstrated a red-shift in the excitation and emission spectra. Less pronounced
scattering is expected inside an emulsion, as sodium caseinate will migrate
preferentially to the interface.

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Chapter 3 Results and discussions

1000
70.00 962.94
888.82
60.00 800
814.70

Intensity (a.u.)
740.58
50.00
600 666.46
592.34
40.00
Z Axis

518.22
400 444.10
30.00
369.98
20.00 200 295.86
221.74
10.00 147.62
0 73.50
450 500 550 600-0.62
450.00 475.00 500.00 W avelength (nm)
525.00 550.00 575.00 600.00
Wavelength (nm)

Figure 3.14: EosinY (5µg/mL) in aqueous phosphate buffer (pH6.7) with sodium azide (0.02%)
and sodium caseinate (1.25%). Contour plot of the scan for emission and excitation wavelength
ranging from 450 to 600nm in 75 steps of 2nm. X-axis: excitation wavelength (nm). Z-axis:
number of steps to go from the first to the last selected emission wavelength. Red color denotes
high intensity, blue color denotes low intensity.

3.4.6 Selection of the filter block in the fluorescence microscope

Table 3.12 gives an overview of the obtained maxima of excitation and emission
wavelength for different aqueous solutions. The water phase of the water-in-oil
emulsions consisted of sodium caseinate, phosphate buffer and sodium azide. Hence,
the outcomes of this solution (Figure 3.14) are compared to the available filter blocks
in the fluorescence microscope. Filter block I2/3 is appropriate in terms of passing
through wavelengths which include the maximum emission wavelength of the
eosinY-solution. However, the excitation filter passes wavelengths which are located
in the sub maximum excitation regions. Filter block N2 is appropriate in terms of
passing through wavelengths which are close to the maximum excitation wavelength
of the eosinY-solution. However it fails in picking the wavelengths that correspond to
maximal fluorescence intensity. Consequently, filter block I2/3 was preferred,
although it is not perfect for eosinY-solutions.

Table 3.12: Overview of maximum excitation and emission wavelengths for different aqueous
solutions of eosinY. E=eosinY (0.001%w/v), PE=eosinY and phosphate buffer (pH6.7).
PNE=eosinY, phosphate buffer and NaN3 (0.02%w/v). PNEN= eosinY, phosphate buffer, NaN3
and Na caseinate (1.25% w/v).
Max. excitation λ (nm) Max. emission λ (nm)
E 517 538
PE 517 540
PNE 517 542
PNEN 527 547
I2/3 filter block BP 450-490 LP 520
N2 filter block BP 530-560 LP 580

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Chapter 3 Results and discussions

3.4.7 Fluorescence microscopic images of water droplets in a w/o-emulsion

The analyzed emulsion contained 50wt% water, in which 0.001% (w/v) eosinY,
1.25% (w/v) sodium caseinate, phosphate buffer (pH6.7) and sodium azide (0.02%,
w/v) was included. The oil phase was Hozol with 2.5% (w/v) PGPR. The emulsion
was covered with aluminum foil and diluted ten times with Hozol just before
microscopic analysis.
As mentioned in section 3.4.6, the selected filter block I2/3 is not perfect for eosinY.
Consequently, a weak fluorescence of not well-defined water droplets is observed
(Figure 3.15). Both small and bigger (about 10µm) droplets can be noted.

Figure 3.15: (Top) Light microscopic image. (Bottom) Fluorescent


microscopic image. Ten times diluted H1.25/2.50 (three days old)
with Hozol. Concentration of eosinY in water phase is 0.001%.
Objective x100.

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Chapter 3 Results and discussions

3.4.8 Imaging of water droplets in water in oil emulsions by confocal laser scanning
microscopy

Figure 3.16 is a (negative) confocal laser scanning microscopic (CLSM) image of a


w/o-emulsion with more appropriate absorption (514nm) and excitation filters
(538nm) than the light and fluorescent microscopic image (Figure 3.15). Smaller
water droplets in the size range of 3 to 4µm can be observed than in Figure 3.15. This
might be explained, on the one hand, by the difference in time elapse between
emulsion preparation and shooting of the pictures. This could be investigated by pfg-
NMR analysis of w/o-emulsions as a function of time. On the other hand, CSLM
doesn’t require a cover glass, which might in the case of fluorescence microscopy,
cause water droplets to coalesce under the influence of pressure. van Aken and Zoet
(2000) defined coalescence of water droplets by pressure of cover slips as coalescence
induced by flow, which gradually increases upon increase of dispersed phase. The
CSLM-sample is investigated by an objective which is placed under the objective
glass, whereas for fluorescence microscopy, the objective is located above the
objective glass and hence the latter requires separation by means of a cover glass.
Moreover the required dilution step in fluorescence microscopy induces flow in the
emulsion, which might affect the coalescence rate. All the mentioned reasons point in
the direction of the CSLM-image as a more correct representation of the water
droplets. However, in order to assess the cause of the observed difference in water
droplets between the two microscopic devices, more investigation is needed.

Figure 3.16: Confocal laser scanning microscopic (negative) image of an undiluted w/o-emulsion,
H1.25/2.50 (24h after preparation). Concentration of eosinY in water phase is 0.001%.

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Chapter 3 Results and discussions

3.5 Determination of the enclosed water volume and yield of w/o/w-emulsions

3.5.1 Method optimization

The optimization of the determination of the enclosed water volume of double


emulsions consisted of the creation of a double emulsion and the quest for an
appropriate concentration of MnCl2 enabling the characterization of double
emulsions. Moreover it was investigated whether the usage of T2-analysis to
characterize a double emulsion at 5°C was authorized and the preparation method for
double emulsions was further optimized by investigation of the effect of the duration
of mixing and the cooling conditions on the enclosed water volume or yield.

3.5.1.1 Optimization of the preparation method

The aim was to find a preparation method that resulted in a double emulsion with a
significant fraction of enclosed water. A w1/o-emulsion is emulsified in an external
water phase (w2) which results in a w1/o/w2-emulsion. As such, an oil in water
emulsion is obtained, in which the fat phase encloses water and hence the caloric
content is reduced.
As described in section 2.5.2.1, Method A comprises the application of an Ultraturrax
TV45 and a Microfluidizer for the manufacturing of both w/o-emulsions and w/o/w-
emulsions. Twenty four hours after production, the double emulsions were
characterized by a low (<4%) or no enclosed water volume, as determined by T2-
relaxation measurements at 5°C after addition of volumes up to 100µL MnCl2
(10mM) to a sample of 8mL. These findings can be explained by either the absence of
internal water or the permeability of the fat phase. The latter is investigated in section
3.5.1.3. Absence of internal water might be due to the presence of an osmotic gradient
between the water phases in a w/o/w-emulsion or due to destruction of the double
emulsion during preparation. To exclude the possibility of lack of internal water due
to osmotic pressure gradients induced by MnCl2 addition, the osmolarity of
components of the water phase was calculated by multiplication of the molarity (C)
by the amount of dissociated particles per mole (i). The osmotic pressure was
determined by the formula of van ‘t Hoff: Π = iCRT, where (iC) stands for the

107
Chapter 3 Results and discussions

osmolarity (osm/m3), R and T signify the gas constant (8.314J/mol/K) and the
absolute temperature (K).
From Table 3.13, it is clear that there is a small osmotic pressure difference of 1.3kPa
between the water phases. Hence, this physical property plays a minor role in the lack
of internal water. In fact, a minor transport of water from the w1 to the w2-phase
would restore the osmotic equilibrium.

Table 3.13: Calculation of the osmotic pressure in kPa at 278K (storage temperature)
Concentration Molar mass Molarity Osmolarity Osmotic pressure
W1-phase (g/100mL) (g/mole) (mM) (osm/m3) (kPa)
eosinY 0.0010 647.90 0.015 <0.050
Sodium azide 0.0200 64.99 3.10 6.20
Sodium caseinate 1.2500 3E+05 0.042 <0.050
KH2PO4 0.8491 136.09 61.3 123
K2HPO4 0.6731 174.14 38.6 116
244.6 565.4
W2-phase
Sodium azide 0.0200 64.99 3.10 6.20
Sodium caseinate 1.0000 3E+05 0.033 <0.050
KH2PO4 0.8491 136.09 61.3 123
K2HPO4 0.6731 174.14 38.6 116
max. MnCl2 0.0025 125.84 0.200 0.600
245.2 566.7

The inexistence of internal water might be due to the destruction of the double
emulsion by the Microfluidizer at the applied homogenization pressure and time
duration. Samples were collected before and after the second application of the
Microfluidizer step. The emulsions that were collected before the Microfluidizer step
demonstrated separation of an upper creamy layer and a lower water phase in less
than 24h. As judged by the ratio of the thickness of the layers (Figure 3.17) and by T2-
relaxation measurements (Figure 3.18), internal water was present before
microfluidization of the double emulsion. Figure 3.17 shows a lower serum layer and
an upper cream layer. It was clear that the ratio of the volume of the separated phases
of a non-microfluidized sample was not equal to the ratio of a sample with unmixed
pure oil phase and water phase (20:80), but rather similar to the ratio of the volumes
in a sample with unmixed w/o-emulsion and external water (40:60). Hence, the visual
creaming aspect already provided a strong indication that (at least part of) the water
remained encapsulated within the oil phase.

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Chapter 3 Results and discussions

Cream layer

Serum layer

Figure 3.17: Photograph of unmixed sample of oil and external water (20:80), an unmixed
sample of w/o-emulsion (P1.25/2.5) and external water (40:60) and a separated non-
microfluidized w/o/w-emulsion (P1.25/2.5/1).

In Figure 3.18, the T2-distribution of a microfluidized and non-microfluidized sample


is depicted. In the former T2-distribution, the peak associated with internal water is
absent. Hence, the microfluidized emulsion, which was homogeneously distributed
throughout the length of the NMR-tube, can be regarded as a o/w emulsion.
Consequently, in order to result in a w/o/w-emulsion, the mixing of a w/o-emulsion
with the external water phase should be performed without the usage of a
Microfluidizer M110S.

800
700
600
Signal amplitude

500
Before
400 microfluidization
300 After
microfluidization
200
100
0
1000 10000 100000 1000000 10000000
Time (µs)

Figure 3.18: T2-distribution of a sample before and after microfluidization (P1.25/2.5/1) with
addition of 100µL of 10mM MnCl2 to 8mL vortexed sample.

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Chapter 3 Results and discussions

3.5.1.2 Finding an appropriate concentration of MnCl2

Figure 3.19 schematically represents the effect of the addition of no, insufficient,
sufficient and too much MnCl2 to a double emulsion on the T2-distribution. In the
absence of MnCl2, a small fraction of low T2 and a large fraction of high T2 was
observed, which is ascribed to fat and water, respectively. It was seen that 25 to 50µL
of 10mM MnCl2 in a sample of 8mL was insufficient to fully separate the signals that
are related to the internal (not influenced by MnCl2) and external water (affected by
MnCl2) in the T2-distribution. Higher volumes than 100µL of 10mM MnCl2 per 8mL
sample fused the signals that are related to the fat phase and the external water.
Hence, either 75µL or 100µL of 10mM MnCl2 per 8mL sample was found an
appropriate amount of MnCl2. This comes down to a concentration of 94 to 125µM
MnCl2 in the double emulsion (i.e. 156 to 208µM MnCl2 in the external water phase).

Figure 3.19: Schematic representation of the effect of MnCl2 addition on the T2-distribution of a
multiple emulsion.

3.5.1.3 Investigation of the permeability of the oil phase by a temperature experiment

Sabatino et al. (2011) reported that a reliable characterization of a vesicular dispersion


by T2-analysis is possible when there is no permeability of the bilayer. Since the

110
Chapter 3 Results and discussions

bilayer in a liposome acts as a physical barrier similar to the fat phase in a double
emulsion, the permeability of the latter was evaluated by performing a temperature
experiment. Impermeability of the fat phase at the applied temperature, authorizes the
usage of T2-analysis to characterize a double emulsion.
Three 8mL samples, which were made in accordance to Method B (see section
2.5.2.2), with composition P1.25/2.5/1 and 0µL (sample 0), 75µL (sample 1) and
100µL (sample 2) of 10mM MnCl2, added prior to analysis were heated successively
from 5°C to 25°C and 45°C in a warm water bath. Afterwards, the samples were
cooled for 1.5h at 5°C. At each mentioned temperature, T2-relaxation measurements
were performed. In Figure 3.20, the effect of temperature on the T2-distribution in
Sample 1 is given. Whereas at 5°C a clear separation of the two water peaks is visible,
this doesn’t hold anymore at 25°C and 45°C. Comparing sample 0 (Figure 3.21) and
sample 1 (Figure 3.20) at 5°C, it is clearly observed that the addition of MnCl2
separates the water signal into two peaks: a peak characterized by medium relaxation
(or medium relaxation time mode), which corresponds to the external water and a
peak characterized by slow relaxation (or large relaxation time mode), which is
associated with the internal water phase. The signal of the fat phase (fast relaxation
time mode) is located at shorter times. Evaluation of the permeability and the effect of
temperature on the T2-distribution is done by analysis of the area under the curve, the
signal amplitude and the relaxation time at different temperatures.

5°C 75µL MnCl2

1400
1200
Signal amplitude

1000
800
600
400
200
0
-200
1 10 100 1000 10000
Time (ms)

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Chapter 3 Results and discussions

25°C 75µL MnCl2

1000
800
Signal amplitude

600
400
200
0
-200
1 10 100 1000 10000
Time (ms)

45°C 75µL MnCl2

1000
800
Signal amplitude

600
400
200
0
-200
1 10 100 1000 10000
Time (ms)

Back to 5°C 75µL MnCl2

1200
1000
Signal amplitude

800
600
400
200
0
-200
1 10 100 1000 10000
Time (ms)

Figure 3.20: Effect of temperature (5°C, 25°C, 45°C and back to 5°C) on the T2-dsitribution of
Sample 1(P1.25/2.5/1, 1st repetition) upon addition of 75µL of 10mM MnCl2 to 8mL of vortexed
double emulsion.

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Chapter 3 Results and discussions

5°C Without MnCl2

1600
1400
Signal amplitude

1200
1000
800
600
400
200
0
-200
1000 10000 100000 1000000 10000000 100000000
Time (µs)

Figure 3.21: T2-distribution of sample 0 (P1.25/2.5/1 without MnCl2 addition)

3.5.1.3.1 Analysis of the area under the curve at different temperatures

Figure 3.22 depicts the difference in area under the curve of Sample 1 and 2 at 5°C
before and after heating. Since Sample 1 was measured once only at 5°C, Sample 2
was statistically analyzed. The mean area under the curve that is related to the
external water (medium relaxation) significantly decreases after thermal treatment
(p=0.0383). Considering Sample 2, the mean area under the curve that is associated
with fat (fast relaxation) and internal water (slow relaxation) significantly increases
after a heat-cool experiment (for both p=0.0383). Consequently, the mean enclosed
water volume of three repetitions of Sample 2 at 5°C significantly increases
(p=0.0361) after heat application with a factor 1.84 from 16.2% ± 0.6 to 29.8% ± 0.3.
Sample 1 confirms the change of the area under the curve and enclosed water fraction
after heat application.

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Chapter 3 Results and discussions

5°C (sample 1)
16000
14355 13902
14000 Back to 5°C (sample 1)
13598 13853
Area under the curve

12000 5°C (sample 2)

10000
Back to 5°C (sample 2)
8000
Total AUC 5°C (sample2)
6000
Total AUC back to 5°C (sample1)
4000

2000 Total AUC 5°C (sample 1)

0 Total AUC back to 5°C (sample 2)


Fast relaxation Medium relaxation Slow relaxation

Figure 3.22: Area under the curve as a function of relaxation time at 5°C before and after heat
application. Error bars denote the standard deviation of three repetitions of Sample 1 and 2,
except Sample 1 at 5°C, which was measured once only. The total area under the curve is
represented by a flat horizontal line and amounts to 13853 and 13598 for Sample 1 at 5°C and
back to 5°C, respectively, whereas for Sample 2 this is equal to 13902 and 14355, respectively.

Regarding the change of the area under the curve related to fat, this might point out
the possibility of transition of the type of polymorphs of the fat crystals in soft PMF.
The latter is provoked in a process called tempering. One polymorph differs from the
other in melting point, stability, density, melting enthalpy and nucleation rate. In
general, tempering results in more stable polymorphs that are characterized by a
higher melting point, higher density, higher melting enthalpy and lower nucleation
rate (Dewettinck and Fredrick, 2010). This comes down to a more solid like fat phase,
characterized by the selection of a certain type of polymorphs upon a heat-cool
experiment. However, since polymorphs are very small (ångström scale), the
observed change in area under the curve related to fat might be due to slow
crystallization, whereby after cooling to 5°C, a larger fraction of the fat phase remains
liquid.

Figure 3.23 and Table 3.14 indicate that at 25°C the area under the curve for fast
relaxing hydrogens (fat phase) can be recorded in Sample 1, whereas in Sample 2 the
signal related to fat is absent. At 45°C, at which there is less fat crystallization, the
signal of the fat phase in Sample 2, expressed in units of area under the curve, is not
significantly lower in comparison to the initial 5°C (p=0.1). Cooling to 5°C
significantly increases the mean area under the curve for fat in comparison to 45°C, in
virtue of the liquid-solid phase transition (both p=0.0383 for Sample 1 and 2). On the

114
Chapter 3 Results and discussions

one hand, upon temperature rise, a larger signal for more mobile protons might be
expected since the liquid fat fraction increases. On the other hand, at 45°C soft PMF
is completely liquid, but the incubation time (about 30 minutes) might have been too
limited and hence, a fraction of soft PMF might have been still solid. The liquid
fraction is expected to relax slower than the solid fraction. Consequently, the area
under the curve of the fat signal at 45°C might be smaller than at 5°C, due to a shift of
the more liquid fraction of soft PMF towards the signal of the external water.
However, this would coincide with an increase of the area under the curve of the latter
signal, which cannot be evaluated from Table 3.14.

1800
1600
1400
1200
1000
Oil peak (sample1)
AUC

800
Oil peak (sample2)
600
400
200
0
-200 5 25 45 5
Temperature (°C)

Figure 3.23: Effect of temperature on the area under the peak associated with the fastest
relaxation time for two samples. Each sample at each temperature is analyzed three times, except
Sample 1 at 5°C, which is analyzed once only. Error bars denote the standard deviation of three
repetitions.

Due to lack of separation of signals in the T2-distribution at higher temperatures,


nothing can be stated about the enclosed water volume upon temperature increase. A
similar temperature experiment on vesicular dispersions was performed by Sabatino et
al. (2011) and this study concluded that upon temperature rise, the real enclosed water
volume doesn’t change, whereas the estimated enclosed water volume might decrease
due to fast exchange of water between the internal and external water phase.

115
Chapter 3 Results and discussions

Table 3.14: Area under the curve for fast relaxation (fat), medium relaxation (external water) and slow
relaxation (internal water) at 5, 25, 45 and back to 5°C. Each sample is analyzed three times per
temperature, except the first sample at 5°C which is analyzed once. Some cells are merged together due
to lack of separation of peaks.
Sample 1
Temperature (°C) AUC (fat) AUC (Ext. water) AUC (Int. water) Total AUC
5 616 10857 2380 13853
25 1573 12876 14449
25 1696 12471 14167
25 1490 12699 14189
45 14035 14035
45 13903 13903
45 13767 13767
5 1609 7321 4668 13598
5 1568 11934 13502
5 1511 11847 13358
Sample 2
5 425 11458 2098 13981
5 551 11137 2221 13909
5 540 11091 2186 13817
25 15971 3057 19028
25 15182 3963 19144
25 15056 4175 19231
45 106 14243 14349
45 63 14200 14263
45 517 10767 2853 14137
5 1711 9019 3898 14628
5 1481 8959 3778 14218
5 1481 8959 3778 14218

3.5.1.3.2 Analysis of the signal amplitude at different temperatures

At all investigated temperatures, the mean maximal signal amplitude of the T2-distribution
is reached at medium relaxation times (Table 3.15) and is significantly smaller at 45°C than
at the initial 5°C (p=0.05). This means that at increasing temperatures, the peaks flatten and
widen in such a way that they merge together. This might point to the possibility of thermal
destruction of the double emulsion or permeability of the fat phase for water or MnCl2 at
higher temperatures. However, upon cooling after heating, the peak of internal water pops
up again, which indicates that the double emulsion is not destructed by the applied
temperature rise, nor that there is physical contact, which is irreversible, of the internal
water phase with the external water phase wherein MnCl2 is dissolved. Hence, even at
higher temperatures, the permeability of soft PMF for MnCl2 does not impose restrictions

116
Chapter 3 Results and discussions

regarding the authorization of the usage of T2-analyses at 5°C for double emulsions based
on soft PMF.
A broader distribution of the signals in the T2-distribution or lower resolution at higher
temperatures might refer to a less homogeneous relaxation behavior of hydrogens in the
same environment upon temperature rise.
Also in Table 3.15, the high values of signal amplitude for the medium relaxation mode
are attributed to completely fused signals associated with the medium and fast relaxation
mode.

Table 3.15: Maximal signal amplitude at different relaxation times in the T2-distribution of two
samples, analyzed thrice. Values in bold denote maximal signal amplitude per temperature and per
repetition. Empty cells refer to missing data.
Sample 1
Fast relaxation mode Medium relaxation mode Slow relaxation mode
st st
T (°C) 1 rep. 2nd rep. 3rd rep. 1 rep. 2nd rep. 3rd rep. 1st rep. 2nd rep. 3rd rep.
5 171 1206 449
25 234 305 217 827 1054 775 470 755 439
45 64 110 0 844 791 570 458 412 0
5 471 180 146 1140 832 716 1044 698 542

Sample 2
5 159 296 285 1304 1663 1628 352 367 380
25 0 0 0 1887 8120 11103 382 1854 2746
45 16 21 176 853 773 1018 246 179 385
5 305 155 155 1230 977 1135 908 617 617

3.5.1.3.3 Analysis of the relaxation time at different temperatures

Upon temperature rise, it is expected to observe an increase of relaxation time, since


hydrogens become more mobile and the relaxation takes longer.
Fast, medium and low relaxation times at maximal signal amplitude in the T2-distribution for
different temperatures are summarized in Table 3.16 and Figure 3.24, in which a significant
increase of relaxation time can be detected for the fast relaxation mode upon temperature
increase from 5°C to 45°C (p=0.0383). In an additional experiment, two NMR-tubes were
filled with 16mL soft PMF and with Hozol. The purpose of this experiment was to found out
whether emulsification of fat globules affects the influence of temperature on the fast
relaxation mode. The bulk fat samples were subjected to 5°C, 25°C and 45°C (Figure 3.25).
Since the receiver gain of the spectrometer is optimized prior to analysis and hence, the

117
Chapter 3 Results and discussions

signal is optimally amplified, useful information can be drawn from the relaxation time
instead of the area under the curve. Heating soft PMF or Hozol results in an increase of the
relaxation time of the signal at the fast relaxation mode. In conclusion, a temperature rise has
a decisive effect on the fast relaxation mode of bulk fat and this can be confirmed in the
analyzed samples with emulsified fat. In Figure 3.25 also bulk water without MnCl2 was
subjected to the same conditions. An increase of the relaxation time is observed when the
temperature rises from 25°C to 45°C.
Regarding signals at medium relaxation times of the double emulsion, significantly slower
mean relaxation time of the hydrogen atoms is recorded on account of higher mobility of
hydrogen atoms as temperature increases from 5°C to 25°C and from 25°C to 45°C (p<0.04).
As a consequence, longer relaxation times are recorded.
Concerning slow relaxing hydrogens (internal water), a significantly larger mean relaxation
time is noted at 45°C than at (initial) 5°C (p=0.0381).
After cooling, two peaks, associated with fast relaxation, are created, which reflects the
presence of hydrogens in different environments. For Sample 1, these are located at 3.9 and
12.1ms. For Sample 2, these can be found at 3.4 and 10.5ms.

Table 3.16: Fast, medium and slow relaxation time modes (ms) at different temperatures (°C) of Sample 1
and 2. Flat bars refer to absence of signal. Empty cells denote no measurements.
Sample 1
Fast relaxation mode Medium relaxation mode Slow relaxation mode
T(°C) 1st rep. 2nd rep. 3rd rep. 1st rep. 2nd rep. 3rd rep. 1st rep. 2nd rep. 3rd rep.
5 10 99 675
25 16 18 16 120 120 120 818 743 818
45 28 38 - 194 194 176 1202 1202 -
5 4 4 4 109 99 99 506 557 557
14 12 10
Sample 2
5 10 12 12 74 67 67 613 557 557
25 - - - 99 120 132 901 901 992
45 33 13 38 160 160 160 1323 1457 1202
5 3 3 3 82 82 82 506 506 506
12 10 10

118
Chapter 3 Results and discussions

Signal amplitude of the mode 1400

1200

1000
5°C (Sample 1)
800
25°C (Sample 1)
600
45°C (Sample 1)
400

200

0
1 10 100 1000 10000
Modal relaxation time (ms)

12000
Signal amplitude of the mode

10000

8000
5°C (Sample 2)
6000 25°C (Sample 2)
45°C (Sample 2)
4000

2000

0
1 10 100 1000 10000
Modal relaxation time (ms)

Figure 3.24: Exposition of the signal amplitude of the different peaks in the T2-distribution of
Sample 1 (Top) and Sample 2 (Bottom) as a function of the relaxation time of three repetitions of
one sample at 5, 25 and 45°C, except Sample 1 at 5°C, which is analyzed once only.

By calculation of the ratio of average modal values of the fast and slow relaxation time for
each sample (Table 3.17), it can be seen that both relaxation times of the signals of internal
and external water shift to larger times, which might contribute to the drop in resolution of the
T2-distribution. Therefore, T2-measurements should be performed at a low temperature to
optimize the resolution of the T2-distribution.

119
Chapter 3 Results and discussions

500
450
400
Signal amplitude

350
300 5°C soft PMF
250 25°C soft PMF
200 45°C soft PMF
150
100
50
0
1 10 100 1000 10000
Time (ms)

1200

1000
Signal amplitude

800
5°C Hozol
600 25°C Hozol
45°C Hozol
400

200

0
1 10 100 1000 10000
Time (ms)

4000
3500
3000
Signal amplitude

2500 5°C water


2000 25°C water
1500 45°C water
1000
500
0
1 10 100 1000 10000
time (ms)

Figure 3.25: T2-distribution of soft PMF, Hozol and water, analyzed at different temperatures.

120
Chapter 3 Results and discussions

Table 3.17: Average relaxation time mode of three repetitions per sample that correspond to signal
amplitude for external (medium relaxation) and internal (slow relaxation) water. Factor denotes the ratio
of increase in average time between two subsequent temperatures. Stand. dev. and Stand. dev. (Factor)
refer to the standard deviation of three measured relaxation time modes and Factors at one temperature,
respectively.
Medium Stand. Factor Stand. dev. Large Stand. dev. Factor Stand. dev.
relaxation time Dev. (ms) (Factor) relaxation time (ms) (Factor)
Sample T(°C) mode (ms) mode (ms)
1 5°C 98.8 0 675.0 0
25°C 119.7 0 1.2 - 793.0 43.3 1.2 -
45°C 188.0 10.4 1.6 0.09 1165.3 63.5 1.5 0.14
1.9 - 1.7 -
2 5°C 69.5 3.9 575.7 32.3
25°C 117.0 16.7 1.7 0.33 931.3 52.5 1.6 0.16
45°C 160.0 0 1.4 0.21 1327.3 127.5 1.4 0.21
2.3 0.13 2.3 0.32

3.5.1.3.4 Variation of the duration of mixing of the double emulsions

By variation of the duration of the Ultraturrax mixing of the double emulsions, a maximum
enclosed water volume or yield was strived after. The theoretically possible enclosed water is
20g on 80g of total water in the w/o/w-emulsion (20:20:60). The duration of mixing with the
Ultraturrax S25N-10G at 24000rpm was set at 2min, while mixing with the Ultraturrax DK25
at 24000rpm was performed for 1, 2, 4, 6 or 8 minutes. It is essential to subject equal amounts
of sample to the different mixing times.
In this experiment, the w/o/w-emulsions were made in accordance to Method B (see section
2.5.2.2) with soft PMF as a fat phase. Before analysis, samples of 8mL were vortexed and
meanwhile 75 or 100µL of 10mM MnCl2 was added. Regarding gravitational stability, the
double emulsions creamed after 24h. The T2-relaxation distribution was measured after 24h
and illustrated in Figure 3.26, from which it is clear that a larger duration of mixing with the
Ultraturrax DK25 resulted in less enclosed water volume or a lower yield. Based on the
experimental data, at least one difference between the different durations of mixing on the
mean enclosed water volume (or the mean yield) can be detected (p=0.009). A significant
difference between mixing the double emulsion for 1 minute or 8 minutes was observed on
the mean enclosed water volume percentage (p=0.0383). Hence, the highest yield is achieved
after mixing with an Ultraturrax DK25 for 1 minute and all subsequent emulsions were
prepared like this.

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Chapter 3 Results and discussions

According to the manual of the device, longer mixing times don’t achieve further reduction of
droplet size, but only an increase of temperature, which might destroy the double emulsion
and cause the drop of the enclosed water volume.

20 80
18
Estimated enclosed water

70
16
60
14
volume (%)

Yield (%)
12 50
10 40
8 30
6
20
4
2 10
0 0
0 1 2 3 4 5 6 7 8 9
Mixing time with Ultraturrax DK25 (min)

Figure 3.26: Effect of mixing time (minutes) of a w1/o/w2-emulsion with an Ultraturrax DK25 (24000rpm)
on the enclosed water volume (%) and yield (%) by T2-relaxation measurements. Error bars denote the
standard deviation of 3 subsequent measurements of the yield of one sample. All samples needed 75µL of
10mM MnCl2, except the sample of 4 minutes to which 100µL of 10mM MnCl2 was added to 8mL of
double emulsion.

3.5.1.3.5 Effect of the reduction of the duration of mixing with an Ultraturrax S25-10G

In this experiment the effect of the reduction of the duration of mixing with an Ultraturrax
S25-10G on the enclosed water volume or yield was evaluated.
Double emulsions were made by Method B, whereby the duration of mixing with an
Ultraturrax S25-10G was changed from 2 minutes to 1 minute. In the case of 1 minute
mixing, the average enclosed water volume and standard deviation, based on three repetitions
of the same sample, amounted to 18.3% and 0.1%, respectively. For the sample that was
mixed for 2 minutes, this amounted to 18.6% and 0.1%, respectively. No significant
difference of mixing one minute longer with the Ultraturrax S25-10G on the mean percentage
of enclosed water volume (p=0.0722) was observed. In order to provide a balance between
sufficient mixing and the prevention of destruction of the double emulsion, all subsequent
emulsions were prepared by mixing for 2 minutes, because this didn’t reveal more destruction
than mixing for 1 minute with an Ultraturrax S25-10G.

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Chapter 3 Results and discussions

3.5.1.3.6 Effect of quick cooling on the percentage of enclosed water volume

In comparison to bulk fat, fat in emulsion globules is shielded from catalytic impurities for
crystallization by emulsification. Hence, emulsified fat requires a higher density of catalytic
impurities or more likely a higher degree of supercooling to induce nucleation than bulk fat.
Quick cooling by means of placing emulsions after production directly in an ice bath, might
result in more supercooling of the fat in the globules as compared to samples cooled in the
fridge and more fine crystals.
Figure 3.27 shows the enclosed water volume and yield for double emulsions samples, made
in accordance to Method B (see section 2.5.2.2), that are cooled in an ice bath or in the fridge.
The oil phase in this experiment is a mixture of soft PMF and Hozol (87.5/12.5) with 2.5%
PGPR. After 24h, samples of both cooling regimes were characterized by a creamy layer.
By statistical analysis, there was at least one difference between the four analyzed samples on
the mean enclosed water (p=0.0143). Although no unanimous difference could be detected
between samples cooled in an ice bath or in the fridge on the mean enclosed water percentage
(or yield) (p=0.24-0.4), all subsequent samples were cooled down in an ice bath after
preparation.

20 80
Estimated enclosed water volume

18 70
16
60
14
50
Yield (%)

12
(%)

10 40
8 30
6
20
4
2 10

0 0
ice bath no ice bath

Figure 3.27: Effect of cooling regime on the enclosed water volume by T2-relaxation measurements or
yield of the double emulsion. Error bars denote the standard deviation of 3 subsequent measurements of
the yield of one sample. Per cooling regime, two samples were analyzed. To the black colored bars, 100µL
of 10mM MnCl2 was added, whereas to the white colored bars 75µL of 10mM MnCl2 was added to 8mL
vortexed sample, prior to analysis.

123
Chapter 3 Results and discussions

3.5.2 Method application

3.5.2.1 Variation of the composition of the fat phase

In prospect of the aim to create whippable double emulsions, the ratio of soft PMF and Hozol
(and hence the solid fat content) was varied and the effect on the enclosed water volume (or
yield) was investigated.
As mentioned in section 1.2.2, partial coalescence of oil globules in whipping cream is
maximized at 10-50% solid fat content. At 5°C, the solid fat content of bulk soft PMF is 80%
(Verhaeghe, 2004). The type of surfactants plays an additional role in terms of influence on
the solid fat content. Although the solid fat content is not an additive property, the addition of
Hozol with 0% solid fat at 5°C, results in mixtures whose solid fat content in bulk fat may be
roughly approximated by:

% solid fat = 0% solid fat (Hozol)(1-x) + 80% solid fat (soft PMF)(x)

where (x) and (1-x) refer to the percentage of soft PMF and Hozol in bulk fat.

However, the maximum solid fat content of bulk fat at a certain temperature is in general
higher and reached at a faster rate than emulsified fat (Campbell et al., 2002). That’s why it
was chosen to analyse mixes of soft PMF and Hozol in various ratios in double emulsions.
Table 3.18 reports the analyzed mixtures of oils in double emulsions.

Table 3.18: Mixtures of soft PMF and Hozol and the associated calculated solid fat content in bulk fat
Code Soft PMF (wt%) Hozol (wt%) SFC (%) in bulk fat
100/0 100.0 0.0 80
87.5/12.5 87.5 12.5 70
75/25 75.0 25.0 60
62.5/37.5 62.5 37.5 50
50/50 50.0 50.0 40
37.5/62.5 37.5 62.5 30
25/75 25.0 75.0 20
12.5/87.5 12.5 87.5 10

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Chapter 3 Results and discussions

3.5.2.1.1 Effect of the fat composition on the enclosed water volume and yield of double
emulsions made by Method B

Figure 3.28 represents the enclosed water volume and yield using a mixture of soft PMF and
Hozol in the fat phase. As will be seen in section 3.5.2.1.3, the accuracy of the values of the
enclosed water and yield that are obtained with method B, should be interpreted with caution.
However they allow relative comparison of values in this experiment.
Based on the samples, a difference was detected between the different mixtures of fat on the
mean enclosed water volume (p=0.0047), but no trend can be observed from Figure 3.28.

30,0 120
Estimated enclosed water

25,0 100

20,0 80
volume (%)

Yield (%)
15,0 60

10,0 40

5,0 20

0,0 0
12.5/87.5 25/75 37.5/62.5 50/50 62.5/37.5 75/25 87.5/12.5 100/0

Mixture soft PMF/Hozol

Figure 3.28: Effect of the ratio of soft PMF and Hozol on the enclosed water volume and yield by T2-
relaxation measurements. Error bars around the average yield denote the standard deviation of 3
measurements of one sample. All samples required 75µL of 10mM MnCl2 per 8mL vortexed double
emulsion, added prior to analysis, except the 100/0 fat- sample, to which 100µL of 10mM MnCl2 per 8mL
vortexed double emulsion was added.

3.5.2.1.2 Effect of variation of the fat phase on the T2-distribution of double emulsion made
by Method B

Double emulsions with a low ratio of soft PMF/Hozol are characterized by a low fat phase
crystallization. Figure 3.29 gives an overview of the obtained values for the area under the
curve that correspond to fast (fat), medium (external water) and slow (internal water)
relaxation. Based on these samples, the ratio of soft PMF/Hozol doesn’t affect the mean area
under the curve that is associated with fast relaxation (fat phase).

125
Chapter 3 Results and discussions

100000 Oil peak

10000
Area under the peak

External
water peak
1000

100 Internal
water peak
10
Total area
1 under the
12.5/87.5 25/75 37.5/62.5 50/50 62.5/37.5 75/25 87.5/12.5 100/0 curve

Ratio soft PMF/Hozol in fat phase


Figure 3.29: Area under the peak as a function of ratio of soft PMF/Hozol in the fat phase of a double
emulsion. Error bars denote the standard deviation of three repetitions. One sample per ratio was made,
except for ratio 87.5/12.5, two samples were made. To all samples 75µL of 10mM MnCl2 was added prior
to analysis, except one sample with ratio 87.5/12.5, which contained 100µL of 10mM MnCl2.

3.5.2.1.3 Effect of the variation of the fat phase on the T2-distribution of double emulsion
made by Method C

An overview of the area under the different peaks for different ratios of soft PMF/Hozol in
double emulsions is given in Figure 3.30.

100000 Fat peak

10000
Area under the peak

External
1000 water peak

100
Internal
water peak
10

1 Total area
under the
12.5/87.5 25/75 37.5/62.5 50/50 62.5/37.5 75/25 87.5/12.5 100/0
peak
Ratio soft PMF/Hozol

Figure 3.30: Area under the peak as a function of ratio of soft PMF/Hozol in the fat phase of a double
emulsion. Error bars denote the standard deviation of three repetitions of one sample. To all samples
28µL of 10mM MnCl2 was added.

Whereas in 3.3.2.5 (Method B) this could not be reported, Figure 3.30 shows that if double
emulsions are made by Method C, emulsions with a low ratio of soft PMF/Hozol are

126
Chapter 3 Results and discussions

characterized by a lower area under the curve for the signal that is associated with fat, than at
higher ratios of soft PMF/Hozol. A trendline drawn through the points that connect the area
under the curve that is related to fat as a function of the ratio soft PMF/Hozol, gives the
equation y=92.9x+30.1 (R2=0.82). This could be explained by the fact that since the
relaxation time is higher for protons in a more liquid fat, a part of the fat signal merges with
the signal of the medium relaxation mode and hence, the area under the curve for fat at lower
ratios of soft PMF/Hozol decreases. However, this would coincide with an increase of the
area under the curve associated at medium relaxation mode, which cannot be observed.

Whereas in Figures 3.24 and 3.25 an increase of relaxation time of the smallest mode in the
T2-distribution of P1.25/2.5/1 and bulk soft PMF, respectively, was observed upon
temperature increase and hence, decrease of solid fat content and Adam-Berret et al. (2011)
reported that pure fat mixes with a low solid fat content at a constant temperature have a high
T2-relaxation time and vice versa, Figure 3.31 shows for the analyzed emulsions that the
smallest mode of the relaxation time distribution (fat phase) is not different among samples
with ratios of soft PMF/Hozol from 37.5/62.5 to 100/0 and hence, different solid fat contents
in the double emulsion. This might be explained by the presence of a fraction of soft PMF in
all analyzed emulsions, by which the relaxation time related to fat remains approximately
constant for the analyzed ratios of soft PMF/Hozol.

100
Relaxation time (ms)

10

0.1
12.5/87.5 25/75 37.5/62.5 50/50 62.5/37.5 75/25 87.5/12.5 100/0
Ratio of soft PMF/Hozol

Figure 3.31: Smallest mode of relaxation time distribution as a function of the ratio of soft PMF/Hozol in
the fat phase of a double emulsion. Error bars denote the standard deviation of three repetitions for one
sample.

127
Chapter 3 Results and discussions

3.5.2.1.4 Effect of the fat composition on the enclosed water volume and yield of double
emulsions made by Method C

Method C (see 2.5.2.3) comprises the filling of an NMR-tube for 15mm height. All tubes are
elevated for 27mm during analysis in the spectrometer. Right after production, 28µL of
10mM MnCl2 is added and vortexed. The same experiment was carried out as described in
section 3.5.2.1.
Figure 3.32 represents the enclosed water volume and yield as a function of the ratio of soft
PMF and Hozol in the fat phase of a double emulsion, for which no linear trend can be
reported.
In comparison to the same experiment, performed with Method B (3.5.2.1.1), Method C
resulted in a significantly lower mean enclosed water volume (p=0). This is explained by the
way of filling of the NMR-tubes. Tubes in Method B are filled until the mark (40mm),
whereas in Method C, samples of 15mm height are analyzed, while elevated. As the detection
zone for tubes in Method B covers mainly the upper part of the sample and, if not completely
homogeneously vortexed, this contains more less dense or oily particles, which are filled with
internal water, less external water is recorded. As a consequence, based on Equation 2.14, a
higher enclosed water volume in Method B than in Method C is achieved. In terms of the
accuracy of the enclosed volume and yield, Method C is more reliable, due to the complete
coverage of the sample in the detection zone of the Maran Ultra 23 spectrometer.
A double emulsion with a soft PMF/Hozol ratio of 67.5/32.5 is characterized by a mean
enclosed water volume of 15.6 ± 0.1% and a mean yield of 62.4 ± 0.4%. This means that this
w1/o/w2-emulsion (20/20/60) actually contains 32.5g w1/o emulsion per 100g double
emulsion (12.5/20/67.5). A commercial whipping cream contains approximately 30 to 40g fat
per 100g cream. Hence, based on the enclosed volume, the volume fraction of the 20%
emulsion might be sufficient to enable efficient whipping.

128
Chapter 3 Results and discussions

18,0 70
Estimated enclosed water volume

16,0
60
14,0
50
12,0

Yield (%)
10,0 40
(%)

8,0 30
6,0
20
4,0
10
2,0

0,0 0
12.5/87.5 25/75 37.5/62.5 50/50 62.5/37.5 75/25 87.5/12.5 100/0
Ratio of soft PMF/Hozol

Figure 3.32: Effect of the ratio of soft PMF and Hozol on the enclosed water volume (or yield) by T2-
relaxation measurements. Error bars around the average yield denote the standard deviation of 3
measurements of one sample.

3.5.2.1.5 Effect of the fat composition on the enclosed water volume and yield of double
emulsions made by Method D

Method D (see 2.5.2.4) is similar to Method C, but comprises the production of the w/o-
emulsion with an Ultraturrax device ánd Microfluidizer.
Two different fat mixes with a soft PMF/Hozol ratio of 62.5/37.5 and 100/0 were investigated
by T2-analysis. In Table 3.19 the average and standard deviation of three repetitions of one
and three samples with a soft PMF/Hozol ratio of 62.5/37.5 and 100/0 are represented,
respectively. Comparison with the values from section 3.5.2.1.4 results in the conclusion that
based on these samples, the mean enclosed water volume is lower in double emulsions made
with Method D than with Method C (p=0.05). Probably, during the second emulsification
step, more water is expelled from fat globules if the internal water droplets are smaller.

Table 3.19: Average enclosed water volume and standard deviation of three repetitions per sample.
Samples were filled for 15mm height, 28µL MnCl2 was added after production and measured in a 27mm
elevated position.
Soft PMF/Hozol Enclosed water volume (%)
100/0 12.8 ± 0.4
12.7 ± 0.2
11.8 ± 0.1
62.5/37.5 10.5 ± 0.3

129
Chapter 3 Results and discussions

3.5.2.2 Effect of the concentration of hydrophilic emulsifier in the external water phase on the
enclosed water volume

As discussed in section 1.1.4.1, Garti and Aserin (1996) reported high concentrations of
external hydrophilic emulsifier reduce the encapsulation efficiency. In this experiment the
effect of the increase of sodium caseinate from 1% (w/v) to 1.25% (w/v) in the external water
phase on the enclosed water volume was investigated. Regarding emulsion P1.25/2.5/1 that
was prepared by Method B, the average and standard deviation of three repetitions of the
same sample, amounted to 18.6% and 0.1%, respectively, which was significantly higher than
17.1% and 0.3%, respectively, for sample P1.25/2.5/1.25 (p=0.038). In conclusion, already by
increasing the external hydrophilic surfactant sodium caseinate with 0.25% (w/v) the
encapsulation efficiency of a double emulsion is negatively influenced.

3.6 Fat globule analysis by a Malvern Mastersizer S

Three repetitions of a particle size analysis of a double emulsion with composition


H1.25/2.5/1 and made in accordance to Method B (section 2.5.2.2), were performed on the
same day of production with a Malvern Mastersizer S. A few drops of double emulsion were
brought into the MS-17 wet sample dispersion unit filled with deionized water. The average
D43 and the standard deviation of the average of three repetitions was 16.94µm and 0.04µm
(Figure 3.33).
Volume-weighted distribution (%)

14

12

10

0
0.1 1 10 100 1000
Diameter (µm)

Figure 3.33: Volume-weighted distribution as a function of diameter of oil globules of H1.25/2.5/1 (P/A
11/11-03).

130
Chapter 3 Results and discussions

3.7 Visualization of double emulsions by light microscopy

An image of a double emulsion with composition H1.25/2.5/1 made by Method B (see section
2.5.2.2) was taken (Figure 3.34). A lot of very small water droplets can be observed in the oil
globules, which coincides with type C of water droplet entrapment as discussed in section
1.6.1.

Figure 3.34: Light microscopic image of H1.25/2.5/1 three days after production (P/A 11-14/03), upon
tenfold dilution with external water phase. Objective 50x.

3.8 Research on the thickness of the separated cream layer of double emulsions

The thickness of the separated cream layer can be determined manually with a ruler and can
be illustrated by one dimensional pulsed field gradient NMR profilometry.

3.8.1 Determination of the thickness of the separated creamy layer with a ruler

Thickness determination by the manual method points out that the oil containing (upper) part
or cream layer of the separated double emulsion by Method B, C and D makes up
approximately 50 to 60% of the total volume. No differences could be observed among

131
Chapter 3 Results and discussions

different fat phases. If these oil globules are monodisperse spherical particles that contact
each other without deformation, the porosity would be approximately 40%. Since for an
emulsion, a distribution of globule sizes exist, the porosity or extraglobular water in the cream
layer might be rather between 5 to 20%.

3.8.2 Profilometric analysis of double emulsions

One dimensional pulsed field gradient NMR profilometry allows to analyze the extent of
creaming of a double emulsion and the calculation of the water content of the cream layer of a
double emulsion. Moreover, the creaming rate can be determined. Concerning the latter, the
analyzed samples already creamed after 24h. Hence, focus was laid on investigation of the
thickness and water content of the creamed layer of the double emulsion.
Regarding the extent of creaming, a setup is applied which embodies a gradual rise of the
NMR-tube, whereby after each ascent the signal amplitude is recorded of each slice of the
sample as a function of frequency. The NMR-tube is filled for 47mm with a double emulsion
(P1.25/2.5/1) and was prepared by Method C. Figure 3.35 demonstrates its profilometric
analysis. A complete data-output is given in Appendix G. Also a sample with soft PMF and
2.5% PGPR is included in the figure, whose maximal signal intensity is 8 times lower than the
maximal intensity of the w/o/w-sample. Protons in fat are characterized by a small T2-
relaxation time and hence, more pronounced relaxation occurs than for water protons. A small
T2-relaxation time is related to a small profilometry signal intensity. Regarding the w/o/w-
sample, one can observe two maximal signal amplitudes. The higher maximal signal intensity
at 80000 units is measured when the detection zone of the spectrometer converges with the
separated serum phase at the bottom of the sample, which occurs after an elevation of the tube
for 2 or 3cm. The lower maximal signal intensity at 55000 units coincides with the detection
of the creamy layer of the separated sample. At an elevation of 1 and 2cm, both maxima are
recorded, indicating that the separation border between the cream layer and water phase is
located within the detection window, which is illustrated for an elevation of 1cm in Figure
3.36. In there, a drawing of a NMR-tube is placed next to the profile. It has to be mentioned
that the frequency axis is in fact a rescaled height axis, since the frequency is proportional to
the height of a sample, which is based on the equation:
ω = ωo+ γ∆xG

132
Chapter 3 Results and discussions

where ω is the Larmor frequency of precession of hydrogen atoms in a magnetic field, ω0 is


the Larmor frequency in a magnetic field B0, γ is the gyromagnetic ratio of hydrogen atoms,
∆x is the position along the axis on which G, the field gradient, is applied (Zhu et al., 2008).
From γ and G, the relationship between ω and ∆x is known. In Figure 3.36, the lower
frequencies are related to the lower part in the detection window, at which the water phase is
situated. The hydrogens in the creamy layer are detected at higher frequencies. The signal
amplitude is proportional to the amount of mobile hydrogens (Zhu et al., 2008), but also
strongly affected by T2. As more mobile hydrogens are present in the separated serum phase
than in the upper creamy part, this explains the higher signal amplitude for serum versus the
creamy part of the sample.
Since both the cream and water phase are symmetrically recorded at a tube elevation of 1 and
2cm, the creamy layer must be located in the middle of the detection window at about 1.5cm
elevation. As the detection zone starts from 2.2 to 4.7cm, the middle of this is found at
3.45cm, or, considering 1.5cm elevation, the middle of the detection zone is positioned about
2.0cm above the bottom of the tube. The ratio of the thickness of the cream layer (47-20mm)
over the total filling height (47mm) is equal to the approximate thickness of the creamy layer,
which amounts to 58.5%. This is in accordance to the measured thickness of the creamy layer
with a ruler in section 3.8.1.
From this, the water content can be evaluated in the cream layer, which will be partly
encapsulated within the oil droplets. Hundred milliliter double emulsion contains 58.5mL
cream and 41.5mL serum phase or also 22mL (or 20g) fat and 80mL (or 80g) total water. The
latter reasoning gives a volume fraction of 21.6mL fat and 78.4mL total water per 100mL
double emulsion. The water content in the cream layer is calculated by subtraction of 41.5mL
serum phase from 78.4mL water, which amounts to 36.9mL water per 58.5mL cream (63%).
This enables the calculation of intraglobular and extraglobular water in the cream layer. A
double emulsion P1.25/2.5/1 contains on average 10.5mL enclosed water on 100mL water
(section 3.5.2.1.5). This results in 8.2mL enclosed water on 78.4mL water or 100mL double
emulsion. Based on the thickness of 58.5% cream layer, 36.9mL (78.4mL-(100-58.5)mL)
water was present in the cream layer. Subtraction of 36.9mL water in the cream layer in
100mL double emulsion and 8.2mL enclosed water in 100mL double emulsion amounts to
28.7mL extraglobular water in the cream layer in 100mL double emulsion.
Alternatively, the water content in the creamy layer can be assessed after performing a
profilometric analysis on a bulk water phase. Two profiles were read in Figure 3.37 for a tube
filled with deionized water and a tube with external water phase w2 (1% (w/v) sodium

133
Chapter 3 Results and discussions

caseinate). The area under the profile of deionized and external water phase amounts to 2643
and 2703 units, respectively. The separated serum layer of the double emulsion is not
deionized water, nor is it the external water phase, since the concentration of sodium caseinate
is lower in the separated sample due to preferential localization of this compound at the water-
oil interface. As all other compounds in the external water phase are similar to the separated
serum phase, the true area under the profile of the water fraction of the double emulsion must
approximate its value. The calculation of the water content of the cream layer is performed
with the signal intensity of the external water phase. At 0cm elevation, at which no signals of
separated serum phase are registered, the average signal intensity of 27 data points in the
middle of the detection zone is 54173 units. The water content in the cream layer is calculated
by dividing the latter by the average signal intensity of external water of 28 data points in the
middle of the detection zone, which amounts to 85954 units. This gives a water content of the
creamy layer of 63%, which is exactly the same value as calculated from the thickness of the
creamy layer.

90000

80000

Soft PMF and


70000 PGPR

ocm elevation
60000
w /o/w
Signal intensity

50000 1cm elevation


w /o/w
40000 2cm elevation
w /o/w
30000 3cm elevation
w /o/w
20000
4cm elevation
w /o/w
10000
5cm elevation
w /o/w
0
-0.04 -0.02 0 0.02
Frequency (MHz)

Figure 3.35: Pfg-NMR 1D profilometry at different elevations of a double emulsion (P1.25/2.5/1) and a
sample of soft PMF with 2.5% PGPR (w/v), determined at 5°C (P/A 2-3/03/11).

134
Chapter 3 Results and discussions

Frequency 1cm elevation w/o/w


(MHz)
0,05

0,04

0,03

0,02

0,01

0
0 10000 20000 30000 40000 50000 60000 70000 80000 90000
-0,01

-0,02

-0,03

-0,04

-0,05

Signal Intensity

Figure 3.36: Frequency as a function of signal intensity of a 1cm elevated w/o/w sample (P1.25/2.5/1) filled
over 47mm in a Maran NMR glass tube. The red bar denotes the detection window from 22 to 47mm.
Black bar denotes the filling degree.

100000
90000
80000
Signal Intensity

70000
60000
50000
40000
30000
20000
10000
0
-0,06 -0,04 -0,02 0 0,02 0,04 0,06
Frequency (Mhz)

External w ater phase (w 2) Deionized w ater

Figure 3.37: Pfg-NMR 1D profilometry of external water phase (W2) and deionized water. NMR-tubes
were filled for 47mm height.

135
Chapter 3 Results and discussions

3.9 Whipping of a commercial dairy cream

3.9.1 Whipping time of a whipped commercial dairy cream

The average whipping time and standard deviation of three repetitions of 200mL cream was
281s and 1.7s.

3.9.2 Overrun of a whipped commercial dairy cream

The average and standard deviation of the net mass of three unwhipped cream samples and
two whipped cream samples in recipients of 200mL was (202.89 ± 0.45)g and (92.09 ±
3.29)g, respectively. Hence, the average overrun is 54.6%.

3.9.3 Physical destabilization (drainage) of a whipped commercial dairy cream

After storage at 16-18°C of 50g of whipped cream on a sieve, no leaked serum could be
detected.

3.10 Whipping of w/o/w-emulsions

3.10.1 First attempt

A double emulsion was prepared by Method D as described in section 2.8.2.1. The applied
ratio of soft PMF/Hozol (62.5/37.5) was chosen after consideration of several aspects that
affect the solid fat content, besides the fact that this is a empirical experiment.
Firstly, the temperature of the refrigerated room in which the emulsion was stored amounts to
5°C, which results in a solid fat content of bulk soft PMF of 80% (Verhaeghe, 2004) or a solid
fat content of the bulk mix of soft PMF/Hozol (62.5/37.5) of 50%.
Secondly, after emulsification the solid fat content drops. In Fredrick et al. (2011) in which
homogenized recombined emulsified milk fat (35wt%) with a fat globule diameter of about
3µm was compared to bulk milk fat, the solid fat content at 5°C after one day differed about

136
Chapter 3 Results and discussions

5%. In this thesis the fat globules are larger and filled with water. Section 3.5.2.1.5
demonstrated that the internal water of this double emulsion amounted to 10.5g/100g, which
comes down to 8.4g on 80g of total water in a w/o/w-emulsion (20/20/60). This means that
100g of a stable double emulsion actually contains 28.4g w1/o-droplets, which is built up of
2/3 (20g) fat and 1/3 (8.4g) enclosed water. The volume of a fat globule with diameter 17µm
(see section 3.6) is 2572µm3, from which two third or 1715 µm3 is occupied by fat. A fat
globule with a diameter of 3µm gives a volume of 14µm3. Hence, there is a volume difference
of a factor 122. This probably renders the difference in solid fat content between bulk fat and
emulsified fat in this experiment lower than the observed difference in Fredrick et al. (2011).
A third aspect in consideration is to mimic dairy cream that is well whippable at a solid fat
content of 10-50% (see section 2.1.1).
Creaming of a recombined cream depends on the fat droplet size (Stokes’s law), the viscosity
of the external water phase and the interfacial film properties. Changing the fat droplet size
by homogenization in a Microfluidizer appeared to be detrimental for the enclosed water
volume. Alteration of the viscosity and interfacial film properties requires the addition of
compounds. Bearing in mind the popularity of clean label foods on the one hand and the
increase of complexity by introduction of additional compounds on the other hand, it was
decided to reduce the gravitational destabilization by rotation of the sample at 20rpm, while
being kept overnight at 5°C. However, this made the emulsion churn. This churned sample
was whipped in order to ameliorate separation of the coalesced cream phase from the serum.
After 1 min and 4 min and 40sec of whipping, the ratio of cream to serum amounted to
34.4/65.6 and 33.5/66.5, respectively, which shows that an increase of the whipping time
reduces the amount of water and/or increases the amount of fat in the fat phase. Allowing for
a fat/water ratio of 20/80 for a completely separated double emulsion, the ratios of this sample
reveal that the coalesced cream phase must still contain water droplets and hence is a w/o-
emulsion.

3.10.2 Second attempt

The second attempt differs in terms of the manner of prevention of creaming. Each 30
minutes, the sample was gently twisted. Headspace was provided to enable mixing while
twisting. After production until whipping, the sample was placed in a refrigerator at 5°C for

137
Chapter 3 Results and discussions

6.5 hours. Nevertheless this sample exhibited a small creamy layer, which made the emulsion
unwhippable.

3.10.3 Third attempt

In the third endeavour, creaming is prevented by addition of 0.2wt% xanthan gum to the
external water phase (w2) and the double emulsion is prepared analogously as in the first
attempt. After about 3 minutes of whipping, churning was observed.

3.10.4 Fourth attempt

Analogously as in section 3.10.3, the external water phase contained 0.2wt% xanthan gum.
Sodium caseinate in the external water phase was replaced by cream residue powder. Already
after 15s of whipping, the emulsion churned. The difference in onset of churning or
perikinetic instability upon whipping between emulsions with sodium caseinate or cream
residue powder in the external phase can be explained by the presence of phospholipids in the
latter external phase that replaces proteins from the interfacial film upon shear action and
hence, perikinetically destabilizes the emulsion faster than in the absence of such small
molecular weight surfactants.

138
General conclusions

General conclusions

In order to create w/o/w-emulsions, the preparation of a w/o-emulsion is required. The size of


the water droplets in the w/o-emulsions was analysed by pfg-NMR. Comparing three data
processing methods (Droplet Size application, Excel and Matlab) it was observed that the
latter two methods resulted in a smaller water droplet size than the former method. Excel
frequently failed to give an output.

From the experiments on w/o-emulsions, it became clear that the type of fat phase had a
significant effect on the mean water droplet size, whereby emulsions with soft PMF or a mix
of soft PMF and Hozol were characterized by a smaller particle size than emulsions based on
Hozol only. Addition of the hydrophilic emulsifier sodium caseinate (0.5%, w/v) to
emulsions based on soft PMF with 1% PGPR had a significant beneficial effect on the
reduction of the mean droplet size.
W/o-emulsions with a mass fraction of water up to 50% could be obtained without alteration
of the type of emulsion, whereby the water droplet size was not significantly different for
water mass fractions of 30, 40 or 50%.
Since there was a small but significant effect on the mean droplet size for emulsions made at a
driving air pressure of 4bar or 6bar in the Microfluidizer, it is advised to prepare w/o-
emulsions at the lower pressure setting.

Fluorimetric experiments indicated that it should be possible to analyse water droplets of w/o-
emulsions with 50% mass fraction of water, in which 0.001% (w/v) eosinY is included,
qualitatively by light microscopy. This requires the use of a filter block that passes the
maximum excitation and emission wavelength range of 517 to 527nm and 542 to 547nm,
respectively.

Considering the optimization of the preparation of w/o/w-emulsions, the use of a


Microfluidizer M110S at 1bar for 1.5min for mixing the w/o-emulsion with the external water
phase destroyed the double emulsion and hence no enclosed water could be detected. The
percentage of enclosed water was be determined by T2-analysis, which was shown to be an
authorized and valuable tool for characterization of w/o/w-emulsions, whereby a

139
General conclusions

concentration of 94 to 125µM MnCl2 in the double emulsion (i.e. 156 to 208µM MnCl2 in the
external water phase) was needed to ensure complete resolution of the internal and external
water phase.
Increasing the duration of mixing of the w1/o-emulsion and the external phase w2 significantly
lowered the enclosed water volume or yield. Moreover, increasing the concentration of
sodium caseinate in the external water phase by 0.25% (w/v) significantly reduced the
encapsulation efficiency of the double emulsion.

The thickness of the separated cream layer in w/o/w-emulsions was determined either
manually with a ruler or spectrometrically by profilometry. The latter method offers the
possibility to calculate the water content in the cream layer. On the other hand, the addition of
0.2wt% xanthan gum to the external water phase makes the w/o/w-emulsion stable against
cream separation.

In comparison to commercial dairy cream, the desired textural change during whipping at 5°C
was not observed in w/o/w-emulsions based on a ratio of soft PMF/Hozol of 62.5/37.5,
whereby churning occured much quicker. No improvement regarding the whippability was
observed by replacement of sodium caseinate by cream residue powder in the external water
phase. In a next step, the solid fat content of w/o/w-emulsions with different fat composition
can be investigated.

In conclusion, w/o-emulsions with up to 50wt% of water could be obtained. This mixture


could be incorporated in an aqueous phase with retention of about 42 to 62%, depending on
the preparation method, of water inside the emulsion droplets if the homogenization intensity
during the second step was sufficiently small. The pfg-NMR technique enabled water droplet
size estimation in w/o-emulsions, whereas laser diffraction enabled oil droplet size analysis in
w/o/w-emulsions. T2-relaxation analysis upon MnCl2 addition permitted resolution of internal
and external water in a double emulsion. Whereas a low fat cream substitute could be
obtained, still additional research is needed to obtain good whipping properties.

140
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149
Appendices

Appendix A: Part of a data sheet in Excel that represents the experimental echo attenuation ratios Ep(δ)δ) of the emulsion H0.75/1.50 (sample1) and the matrix of the
calculated echo attenuation ratios as a function of the radius and δ (E(R,δ)). which is used in Equation 2.8.

Measured
r (µm) = 0.25 0.75 1.25 1.75 2.25 2.75 3.25 3.75 4.25 4.75 data
δi (s) I E (R,δ) (-) E (R,δ) (-) E (R,δ) (-) E (R,δ) (-) E (R,δ) (-) E (R,δ) (-) E (R,δ) (-) E (R,δ) (-) E (R,δ) (-) E (R,δ) (-) Ep (δ) (-)
0 0 1.000000000 1.000000000 1.000000000 1.000000000 1.000000000 1.000000000 1.000000000 1.000000000 1.000000000 1.000000000 1.000000000
0.00050 1 0.999920118 0.995029754 0.976540817 0.945182665 0.903409649 0.853163221 0.796254846 0.734492386 0.669667789 0.603504387 1.000000000
0.00075 2 0.999879004 0.991751185 0.956117767 0.891857398 0.807447278 0.710896991 0.609193864 0.508326465 0.413106987 0.327015061 0.946901647
0.00100 3 0.999837891 0.988457858 0.933975575 0.831338885 0.699952594 0.559842328 0.426335290 0.309471802 0.214258862 0.141531783 0.908592357
0.00125 4 0.999796781 0.985171815 0.911305940 0.768339228 0.592004808 0.419866002 0.275331087 0.167344061 0.094395864 0.049455640 0.855941678
0.00150 5 0.999755672 0.981896174 0.888682525 0.705882748 0.490863177 0.301853464 0.165386863 0.081090964 0.035668912 0.014095704 0.800259266
0.00175 6 0.999714531 0.978628741 0.866358882 0.645749324 0.400474245 0.209150413 0.093018932 0.035478947 0.011653751 0.003304763 0.749705747
0.00200 7 0.999673459 0.975377369 0.844512188 0.589096807 0.322652743 0.140486672 0.049372523 0.014151844 0.003329736 0.000645634 0.697604728
0.00225 8 0.999632355 0.972134207 0.823142295 0.536275279 0.257223367 0.091785850 0.024838496 0.005171743 0.000836289 0.000105639 0.646689514
0.00250 9 0.999591253 0.968901829 0.802285398 0.487490123 0.203352112 0.058561830 0.011911540 0.001744079 0.000186201 0.000014615 0.610694831
0.00275 10 0.999550152 0.965680199 0.781943530 0.442698176 0.159682026 0.036604751 0.005470874 0.000546021 0.000037014 0.000001723 0.569354188
0.00300 11 0.999509053 0.962469280 0.762110915 0.401739339 0.124710015 0.022477263 0.002416765 0.000159580 0.000006613 0.000000175 0.526042981
0.00325 12 0.999467956 0.959269038 0.742778164 0.364390712 0.096971259 0.013591597 0.001030772 0.000043763 0.000001069 0.000000015 0.502250107
0.00350 13 0.999426860 0.956079437 0.723934306 0.330400471 0.075136439 0.008109927 0.000425931 0.000011315 0.000000157 0.000000001 0.466485492
0.00400 14 0.999344675 0.949732016 0.687666834 0.271459273 0.044750371 0.002793661 0.000066944 0.000000645 0.000000003 0.000000000 0.401911725
0.00450 15 0.999262496 0.943426735 0.653215517 0.222924040 0.026454108 0.000929525 0.000009586 0.000000031 0.000000000 0.000000000 0.349155834
0.00500 16 0.999180323 0.937163315 0.620489993 0.183022867 0.015562489 0.000301343 0.000001273 0.000000001 0.000000000 0.000000000 0.318035649
0.00600 17 0.999015999 0.924760949 0.559875275 0.123331963 0.005340957 0.000030020 0.000000019 0.000000000 0.000000000 0.000000000 0.256617697
0.00800 18 0.998687431 0.900446440 0.455831443 0.055987662 0.000620630 0.000000266 0.000000000 0.000000000 0.000000000 0.000000000 0.136287291

150
Appendices

Appendix B: scripts in Matlab

SIMULATION_DATA.M

% simulation of pfg-NMR decay graphs for W/O als a function of droplet size
(lognormal
distribution)

clc;
clear all;

Ds=1.31e-9; %free diffusion coefficient of water in aqueous


phase
ldelta = xlsread('emulsie1-Maran-data17.xls',1,'c2') %diffusion time
(big delta) [s]
gamma=267522128; %gyromagnetic constant in
1/(T.s)
g = xlsread('emulsie1-Maran-data17.xls', 1, 'e2') %magnetic field
gradient [T/m]
%sdelta=(0.0:0.0001:0.01); %pulse gradient widths
[s]
%ndelta=1+(0.01-0.0)/0.0001 %number of small delta
values
sdelta = xlsread('emulsie1-Maran-data17.xls', 1, 'b2:b18');
ndelta=length(sdelta)
I=xlsread('emulsie1-Maran-data17.xls', 1, 'f2:f18'); %primary data of
Maran
Io=I(1,1); %first experimental point (good estimate for
Io)
pFW=0; %percentage of free water
(%)

nra=25; %25 contributions in Bessel-


series
besselroots=[1.84;5.33;8.54;11.71;14.86;18.02;21.16;24.31;27.46;30.60;33.75
;36.89;40.03;43.18;46.32;49.46;52.61;55.75;58.89;62.03;65.17;68.32;71.46;74
.60;77.74] %roots of Bessel
relations
mean=0.7e-6; %mean particle radius
[m]
stdev=0.2e-6; %st.dev. of
particle radius distribution [m]
mu=log(mean^2/sqrt(stdev^2+mean^2)) %geometric mean radius (in fact:
ln) of lognormal distribution
sigma=sqrt(log(stdev^2/mean^2+1)) %geometric standard deviation (in fact:
ln)
p=(0.01:0.01:0.99); %cumulative
probability from ... in steps of .... up to ....
r=logninv(p,mu,sigma); %radii corresponding
to cumulative probability (according to lognormal)
d=r.*2e6 %diameters in
micron
nr=length(r); %number of particle size
classes

% calculation of nra zero points alfa per size class;


alfa = 0;
for i = 1:nr;
for j = 1:nra;
alfa(i,j)=besselroots(j)/r(i);

151
Appendices

end;
end;

% calculation of NMR signal R per size class for every small delta +
overall NMR signal Raccum for the particle size distribution for every
small delta;
R=0;
FW=0; %contribution of
free water (FW) in signal at each small delta
for j = 1:ndelta; %for every individual value of small
delta
Raccum(j)=0
for i = 1:nr;
som = 0;
for k = 1:nra;
som=som+[(2*sdelta(j,1)/(Ds*alfa(i,k)^2))-((2+exp(-
alfa(i,k)^2*Ds*(ldelta-sdelta(j,1)))-2*exp(-alfa(i,k)^2*Ds*ldelta)-2*exp(-
alfa(i,k)^2*Ds*sdelta(j,1))+exp(-
alfa(i,k)^2*Ds*(ldelta+sdelta(j,1))))/(alfa(i,k)^2*Ds)^2)]/(alfa(i,k)^2*(al
fa(i,k)^2*r(i)^2-2));
end;
R(i,j) = Io*exp(-2*gamma^2*g^2*som);
Raccum(j)=Raccum(j)+R(i,j)/nr % NMR decay point for
the lognormal distribution
end;
FW(j)=Io*exp(-gamma^2*g^2*Ds*sdelta(j,1)^2*(ldelta-sdelta(j,1)/3));
RaccumFW(j) = Raccum(j)*(100-pFW)/100 + FW(j)*pFW/100; %calculated
NMR signal with free water contribution
end;

figure (1)
plot (d,p) % plot of particle diameter distribution (in
micron)

Rmin=R(1,:); % NMR decay curve


for smallest particle radius of the lognormal distribution
Rmax=R(nr,:); % NMR decay curve
for largest particle radius of the lognormal distribution

figure (2)
plot
(sdelta,Rmin,sdelta,Rmax,sdelta,Raccum,sdelta,RaccumFW,'db',sdelta,I,'dm')

152
Appendices

FITLIEN.M

% Calculation of the SQD for measured <-> theoretical NMR signal (XX)

function [MLH] = fitlien(XX);

%XX(1)=1
%XX(2)=0.2
%XX(3)=0
%XX(1) = mean radius (in micron)
%XX(2) = standard deviation of radius(in micron)
%XX(3) = percentage of free water
%XX(4) = Io

% import necessary parameters from the workspace


%rawdata = evalin('base','rawdata');
%nr = evalin('base','nr');
%R = evalin('base','R');
%r = evalin('base','r');
gamma = evalin('base','gamma'); %gyromagnetic constant in
1/(T.s)
g = evalin('base','g'); %magnetic field gradient
[T/m]
Ds = evalin('base','Ds');%free diffusion coefficient of water in aqueous
phase
ldelta = evalin('base','ldelta'); %diffusion time (big delta)
[s]
sdelta = evalin('base','sdelta');
ndelta=length(sdelta);
I=evalin('base','I'); %primary data of
Maran
I0=XX(3); %first experimental point (good estimate for
Io)
pFW=0;
%pFW=XX(4); %if percentage of
free water (%) has to be an adjustable parameter

nra=25; %25 contributions in Bessel-


series
besselroots=[1.84;5.33;8.54;11.71;14.86;18.02;21.16;24.31;27.46;30.60;33.75
;36.89;40.03;43.18;46.32;49.46;52.61;55.75;58.89;62.03;65.17;68.32;71.46;74
.60;77.74]; %roots of Bessel
relations
mean=XX(1)*1e-6; %mean particle radius
[m]
stdev=XX(2)*1e-6; %st.dev. of particle radius distribution
[m]
mu=log(mean^2/sqrt(stdev^2+mean^2)); %geometric mean radius
(in fact: ln) of lognormal distribution
sigma=sqrt(log(stdev^2/mean^2+1)); %geometric standard deviation (in fact:
ln)
p=(0.01:0.01:0.99); %cumulative
probability from ... in steps of .... up to ....
r=logninv(p,mu,sigma); %radii corresponding
to cumulative probability (according to lognormal)
%d=r.*2e6 %diameters in
micron
nr=length(r); %number of particle size
classes

153
Appendices

% calculation of nra zero points alfa per size class;


alfa = 0;
for i = 1:nr;
for j = 1:nra;
alfa(i,j)=besselroots(j)/r(i);
end;
end;

% calculation of theoretical NMR signal Raccum for the particle size


distribution XX for every small delta;
FW=0; %contribution of free water (FW)
in signal at each small delta
for j = 1:ndelta; %for every individual value of small
delta
Raccum(j)=0;
for i = 1:nr;
som = 0;
for k = 1:nra;
som=som+[(2*sdelta(j,1)/(Ds*alfa(i,k)^2))-((2+exp(-
alfa(i,k)^2*Ds*(ldelta-sdelta(j,1)))-2*exp(-alfa(i,k)^2*Ds*ldelta)-2*exp(-
alfa(i,k)^2*Ds*sdelta(j,1))+exp(-
alfa(i,k)^2*Ds*(ldelta+sdelta(j,1))))/(alfa(i,k)^2*Ds)^2)]/(alfa(i,k)^2*(al
fa(i,k)^2*r(i)^2-2));
end;
R(i,j) = I0*exp(-2*gamma^2*g^2*som);
Raccum(j)=Raccum(j)+R(i,j)/nr; % NMR decay point for
the lognormal distribution
end;
FW(j)=I0*exp(-gamma^2*g^2*Ds*sdelta(j,1)^2*(ldelta-sdelta(j,1)/3));
RaccumFW(j) = Raccum(j)*(100-pFW)/100 + FW(j)*pFW/100;%calculated NMR
signal with free water contribution
end;

assignin('base','RaccumFW',RaccumFW);

% Calculation of the SQD of the difference between the theoretical and


measured NMR signal
SQD=0;
for i=1:ndelta;
SQD = SQD + (I(i)-RaccumFW(i))^2;
end;
MLH = 0.5*8*log10(SQD/8)
assignin('base','MLH',MLH);
assignin('base','SQD',SQD);
assignin('base','sdelta',sdelta);
assignin('base','I',I);

% plot van theoretisch (blauw) en waargenomen NMR signaal (roze)


plot(sdelta,I,'dm',sdelta,RaccumFW,'b')

154
Appendices

MINIM_LIEN.M

% start value XX2, varies until max likelihood (fit), saved as ZZZ
XX2(1)=0.8;
XX2(2)=0.1;
XX2(3)=I(1,1)*1.05;
%XX2(4)=2;%this parameter may be used to enable adjustment of percentage of
free water
pFW=0;
[ZZZ,fval,exitflag,output,grad,hessian] = fminunc(@fitlien,XX2);
%lognormal(ZZZ);
display(ZZZ);
display(hessian);

SIMULATION.M

% simulation of pfg-NMR decay graphs for W/O emulsions as a function of


droplet size

clc;
clear all;

nr=10; %amount of particle size


classes
%factor=evalin('base','factor');
%kb=evalin('base','kb');
%rawdata=evalin('base','rawdata');
Ds=1.31e-9; %free diffusion coefficient
of water in aqueous phase
ldelta=0.21; %diffusion time (big delta)
[s]
gamma=267522128; %gyromagnetische constante in
1/(T.s)
g=2; %magnetic field gradient
[T/m]
nra=25; %25 contributions in the Bessel-
series
sdelta=[0.0000 0.001 0.002 0.003 0.004 0.005 0.006 0.008];%pulse gradient
widths [s]
ndelta=8; %number of small delta
values
%sdelta=(0.0:0.0001:0.01)
%ndelta=1+(0.01-0.0)/0.0001
besselroots=[1.84;5.33;8.54;11.71;14.86;18.02;21.16;24.31;27.46;30.60;33.75
;36.89;40.03;43.18;46.32;49.46;52.61;55.75;58.89;62.03;65.17;68.32;71.46;74
.60;77.74]
%roots of the Bessel relations
r=[0.5;1.5;2.5;3.5;4.5;5.5;6.5;7.5;8.5;9.5]; %particle radii in
micron
% setting of the class averages;
%for i = 2:nr;
% r(i) = r(i-1)+kb;
%end;

% calculation of nra zero points alfa for the class averages;

155
Appendices

alfa = 0;
for i = 1:nr;
r(i)=r(i)/1e6; %particle radii
[m]
for j = 1:nra;
alfa(i,j)=besselroots(j)/r(i);
end;
end;

% calculation of the NMR signal R for each class for each small delta from
the rawdata(x,1);
R=0;
for j = 1:ndelta;
for i = 1:nr;
som = 0;
for k = 1:nra;
som=som+[(2*sdelta(1,j)/(Ds*alfa(i,k)^2))-((2+exp(-
alfa(i,k)^2*Ds*(ldelta-sdelta(1,j)))-2*exp(-alfa(i,k)^2*Ds*ldelta)-2*exp(-
alfa(i,k)^2*Ds*sdelta(1,j))+exp(-
alfa(i,k)^2*Ds*(ldelta+sdelta(1,j))))/(alfa(i,k)^2*Ds)^2)]/(alfa(i,k)^2*(al
fa(i,k)^2*r(i)^2-2));
end;
R(i,j) = exp(-2*gamma^2*g^2*som);
end;
end;

R05=R(1,:);
R15=R(2,:);
R25=R(3,:);
R35=R(4,:);

figure (1)
plot (sdelta,R05,'r',sdelta,R15,'b',sdelta,R25,'g',sdelta,R35,'y')

SIMULATION_LOG.M

% simulation of pfg-NMR decay graphs for W/O emulsions as a function of


% the droplet size (lognormal distribution)

clc;
clear all;

%factor=evalin('base','factor');
%rawdata=evalin('base','rawdata');
Ds=1.31e-9; %free diffusion coefficient
of water in aqueous phase
ldelta=0.21; %diffusion time (big delta)
[s]
gamma=267522128; %gyromagnetische constante in
1/(T.s)
g=2; %magnetic field gradient
[T/m]
nra=25; %25 contributions of the Bessel-
series

156
Appendices

%sdelta=[0.0000 0.001 0.002 0.003 0.004 0.005 0.006 0.008]; %pulse


gradient widths [s]
%ndelta=8; %number of small delta
values
sdelta=(0.0:0.0001:0.01);
ndelta=1+(0.01-0.0)/0.0001

besselroots=[1.84;5.33;8.54;11.71;14.86;18.02;21.16;24.31;27.46;30.60;33.75
;36.89;40.03;43.18;46.32;49.46;52.61;55.75;58.89;62.03;65.17;68.32;71.46;74
.60;77.74] %roots of the Bessel
relations
mean=3e-6; %mean particle radius
in m
stdev=1e-6; %stdev of particle radius distribution
in m
mu=log(mean^2/sqrt(stdev^2+mean^2))
sigma=sqrt(log(stdev^2/mean^2+1))
p=(0.1:0.1:0.9); %cumulative probability from ...
in steps of .... up to ....
r=logninv(p,mu,sigma);
nr=length(r); %amount of particle size-
classes

%r=[0.5;1.5;2.5;3.5;4.5;5.5;6.5;7.5;8.5;9.5]; %particle radii in


micron

% calculation of nra zero points alfa for each class averga;


alfa = 0;
for i = 1:nr;
for j = 1:nra;
alfa(i,j)=besselroots(j)/r(i);
end;
end;

% calculation of the NMR signal R for each class for each small delta;
R=0;
for j = 1:ndelta; %8 different values for small
delta
for i = 1:nr;
som = 0;
for k = 1:nra;
som=som+[(2*sdelta(1,j)/(Ds*alfa(i,k)^2))-((2+exp(-
alfa(i,k)^2*Ds*(ldelta-sdelta(1,j)))-2*exp(-alfa(i,k)^2*Ds*ldelta)-2*exp(-
alfa(i,k)^2*Ds*sdelta(1,j))+exp(-
alfa(i,k)^2*Ds*(ldelta+sdelta(1,j))))/(alfa(i,k)^2*Ds)^2)]/(alfa(i,k)^2*(al
fa(i,k)^2*r(i)^2-2));
end;
R(i,j) = exp(-2*gamma^2*g^2*som);
end;
end;

figure (1)
plot (r,p)

Rmin=R(1,:);
Rmax=R(nr,:);
figure (2)
plot (sdelta,Rmin,sdelta,Rmax)

157
Appendices

Appendix C

Commands T2 relaxation measurements (RINMR, Maran Ultra 23 spectrometer )

Load CPMG
Load Parameters (or REP)
CPMG_paolo.RiPar
Tau 200
Nech 8K
DS 0
NS 2
.AUTOO1
.AUTORG
DS 2
NS 8
GO

Parameters
ID Value TRIM3 0
P90 7.90 TRIM4 0
P180 15.80 TRIM5 0
P1 1.00 TRIM6 157
P2 1.00 TRIM7 159
P3 1.00 TRIM8 0
P4 1.00 TRIM9 158
P5 1.00 TRIMA 0
DEAD1 5.00 TRIMB 173
DEAD2 3.00 TRIMC 0
DW 1.00 TRIMD 167
RD 2000000.00 TRIME 0
TAU 57500.00 TRIMF 170
D1 100.00 TRIMG 0
D2 7000.00 TRIMH 0
D3 100.00 TRIMI 0
D4 20000.00 TRIMJ 0
D5 1000.00 TRIMK 0
D6 1000000.00 TRIML 0
D7 1000000.00 TRIMM 0
D8 1000000.00 TRIMN 0
D9 1000000.00 TRIMO 0
D10 1000000.00 TRIMP 0
D11 1000000.00 TRIMQ 0
D12 1000000.00 TRIMR 0
SI 1024 TRIMS 0
NECH 256 TRIMT 0
NS 4 TRIMU 0
SF 23.400000 TRIMV 0
O1 26911.60 TRIMW 75
SF2 0.000000 TRIMX 0
O2 0.00 SW 1000000.0
FW 1.0 DB 35
RG 4.45 BES 1000000
PH1 0213 BUT 100000
PH2 0213 RFA0 100.0
PH3 2031 RFA1 0.0
PH4 0213 RFA2 0.0
PH5 1122 RFA3 0.0
LB 0.00 RFA4 0.0
PA 0.00 RFA5 0.0
PB 0.00 RF2A0 0.0
DP 0.00 RF2A1 0.0
TRIM0 49 RF2A2 0.0
TRIM1 45 RF2A3 0.0
TRIM2 0 RF2A4 0.0

158
Appendices

RF2A5 0.0 %GOSTATE 6


WW 5.0 %SCANSDONE 1
C1 0 %CAL1 0.000000
C2 0 %CAL2 0.000000
C3 0 %CAL3 0.000000
C4 0 %CAL4 0.000000
C5 0 %CALIBDIR C:\Program
TI ???? Files\Resonance\RINMR\DATA\
SMP 0 %1
GX 0 %2
GY 32767 %3
GZ 0 %4
G1 10000 %5
G2 50 PREXA1 0.000
G3 0 PREXK1 100000000.00
G4 0 PREXA2 0.000
G5 0 PREXK2 100000000.00
G6 0 PREXA3 0.000
G7 0 PREXK3 100000000.00
G8 0 PREXA4 0.000
G9 0 PREXK4 100000000.00
IG1 1 PREYA1 0.000
IG2 1 PREYK1 100000000.00
IG3 1 PREYA2 0.000
IG4 1 PREYK2 100000000.00
IG5 1 PREYA3 0.000
IG6 1 PREYK3 100000000.00
IG7 1 PREYA4 0.000
IG8 1 PREYK4 100000000.00
IG9 1 PREZA1 0.000
MAC1 0.0 PREZK1 100000000.00
MAC2 0.0 PREZA2 0.000
SH1 PREZK2 100000000.00
SH2 PREZA3 0.000
SH3 PREZK3 100000000.00
SH4 PREZA4 0.000
SH5 PREZK4 100000000.00
DS 4 XB0A 0.000
NA 1 XB0K 100000000.00
SEQ DIFFTRIG YB0A 0.000
GRS X YB0K 100000000.00
GRP Y ZB0A 0.000
GRR Z ZB0K 100000000.00
%TEMPDIR Z PREBA4 0.000
%ROOTDIR Z PREBK4 0.00
%DATADIR C:\Daan\slib.00001.RiDat PPTH 4.0
%EXPORTDIR C:\Bart PPRF 50
Heyman\guarsaus3c_diepvries_zonderstaafje.00001 PPAF 1
%LISTFILE C:\Program PPPF 0
Files\Resonance\RINMR\list\DIFFLIST_ps DEC90 5000000.00
%LOGFILE C:\Program CPD WALTZ16
Files\Resonance\RINMR\DATA\SLIB.00001.RiLog GSLICEX 1.000
%HARDWARE 3 GSLICEY 0.000
GSH1 GSLICEZ 0.000
GSH2 GPHASEX 0.000
GSH3 GPHASEY 1.000
GSH4 GPHASEZ 0.000
GSH5 GREADX 0.000
SNR 100 GREADY 0.000
%SEQFILE C:\PROGRAM GREADZ 1.000
FILES\RESONANCE\RINMR\SEQ\BIN\DIFFTRIG.Exe GREAD 0
PP 0 GPHASE 32767
NOBC 0 GSLICE 0
C6 1 %SNR 0
C7 1 GD 0.00
C8 1 %ERROR 1002
C9 1 %CALCERROR 0.000
C10 1 INC2D 0.00
C11 1 SF2D 0.000000
C12 1 %TP 0
FP1 1.00000 %TS 0
FP2 1.00000 %R0 900.000
FP3 1.00000 %R1 0.000
FP4 1.00000 %R2 0.000
FP5 1.00000 %R3 0.000
%LASTWEIGHT 0.000 %R4 0.000

159
Appendices

%R5 0.000 G30 0


%R6 0.000 G31 0
%R7 0.000 G32 0
%R8 0.000 ACQUISITION 3
%R9 0.000 RG1 0.00
%ADCSCALE 2048 RG2 0.00
PREAMP 0 RG3 0.00
TRIGGER 0 RG4 0.00
MATRIX 0 RG5 0.00
VER Cï RG6 0.00
XB0 0.000 RG7 0.00
YB0 0.000 RG8 0.00
ZB0 0.000 RG9 0.00
XOFFSET 0.00000000 %TOTALSTEPS 0
YOFFSET 0.00000000 %HIDEERROR 0
ZOFFSET 0.00000000 %LASTSEQERROR 0
D13 0.00 SH6
D14 0.00 SH7
D15 0.00 SH8
D16 0.00 SH9
D17 0.00 SH10
D18 0.00 FP6 0.00000
D19 0.00 FP7 0.00000
D20 0.00 FP8 0.00000
D21 0.00 FP9 0.00000
D22 0.00 FP10 0.00000
D23 0.00 %DISPLAYBUFFER0
D24 0.00 QCSHIM1 0
D25 0.00 QCSHIM2 0
D26 0.00 QCSHIM3 0
D27 0.00 QCSHIM4 0
D28 0.00 QCSHIM5 0
D29 0.00 QCSHIM6 0
D30 0.00 QCSHIM7 0
D31 0.00 QCSHIM8 0
D32 0.00 QCLEDS 0
C13 0 QCVAR1 0.0
C14 0 QCVAR2 0.0
C15 0 QCVAR3 0.0
C16 0 QCVAR4 0.0
C17 0 QCVAR5 0.0
C18 0 QCVAR6 0.0
C19 0 QCVAR7 0.0
C20 0 QCVAR8 0.0
C21 0 QCVAR9 0.0
C22 0 QCVAR10 0.0
C23 0 QCVAR11 0.0
C24 0 QCVAR12 0.0
C25 0 QCVAR13 0.0
C26 0 QCVAR14 0.0
C27 0 QCVAR15 0.0
C28 0 QCVAR16 0.0
C29 0 QCVAR17 0.000
C30 0 QCVAR18 0.000
C31 0 QCVAR19 0
C32 0 QCVAR20 0
G10 0
G11 0
G12 0
G13 0
G14 0
G15 0
G16 0
G17 0
G18 0
G19 0
G20 0
G21 0
G22 0
G23 0
G24 0
G25 0
G26 0
G27 0
G28 0
G29 0

160
Appendices

Appendix D:

Exported data from WinDXP to Excel (W/o/w emulsion with fat phase of soft PMF and Hozol in a ratio of
3:1, 1st repetition, production 09/03/11; analysis 10/3/11.

NMR Data Best Fit Data Distribution


Time Magnitude Time DDAT DCALC DERR TC CCALC
403.95 13765.22 403.95 13765.22 13813.14 -47.92 100.00 0.00
803.95 13733.46 803.95 13733.46 13694.11 39.34 105.57 0.00
1203.95 13583.12 1203.95 13583.12 13577.43 5.70 111.44 0.00
1603.95 13454.82 1603.95 13454.82 13462.97 -8.15 117.65 0.00
2003.95 13376.35 2003.95 13376.35 13350.65 25.70 124.20 0.00
2403.95 13207.53 2403.95 13207.53 13240.36 -32.83 131.11 0.00
2803.95 13162.25 2803.95 13162.25 13132.01 30.23 138.41 0.00
3203.95 13008.36 3203.95 13008.36 13025.53 -17.17 146.12 0.00
3603.95 12940.85 3603.95 12940.85 12920.83 20.02 154.25 0.00
4003.95 12800.83 4003.95 12800.83 12817.85 -17.02 162.84 0.00
4403.95 12731.37 4403.95 12731.37 12716.50 14.87 171.91 0.00
4803.95 12625.24 4803.95 12625.24 12616.74 8.50 181.48 0.00
5203.95 12516.68 5203.95 12516.68 12518.49 -1.82 191.58 0.00
5603.95 12426.43 5603.95 12426.44 12421.71 4.72 202.25 0.00
6003.95 12313.33 6003.95 12313.33 12326.34 -13.00 213.51 0.00
6403.95 12238.05 6403.95 12238.05 12232.32 5.73 225.39 0.00
6803.95 12123.42 6803.95 12123.42 12139.62 -16.20 237.94 0.00
7203.95 12057.64 7203.95 12057.65 12048.18 9.46 251.19 0.00
7603.95 11957.20 7603.95 11957.20 11957.97 -0.77 265.17 0.00
8003.95 11879.82 8003.95 11879.82 11868.95 10.87 279.94 0.00
8403.95 11768.27 8403.95 11768.27 11781.08 -12.81 295.52 0.00
8803.95 11696.46 8803.95 11696.46 11694.32 2.14 311.97 0.00
9203.95 11604.71 9203.95 11604.71 11608.64 -3.93 329.34 0.00
9603.95 11513.35 9603.95 11513.35 11524.02 -10.67 347.68 0.00
10004 11459.54 10003.95 11459.54 11440.42 19.12 367.03 0.00
10404 11351.85 10403.95 11351.85 11357.81 -5.96 387.47 0.00
10804 11288.17 10803.95 11288.17 11276.17 12.00 409.04 0.00
11204 11194.33 11203.95 11194.33 11195.48 -1.15 431.81 0.00
11604 11126.48 11603.95 11126.48 11115.70 10.78 455.85 0.00
12004 11039.83 12003.95 11039.83 11036.82 3.01 481.23 0.00
12404 10952.29 12403.95 10952.29 10958.82 -6.52 508.02 0.00
12804 10889.16 12803.95 10889.16 10881.67 7.49 536.30 0.00
13204 10800.59 13203.95 10800.59 10805.36 -4.77 566.16 0.00
13604 10731.18 13603.95 10731.18 10729.86 1.32 597.68 0.00
14004 10645.42 14003.95 10645.43 10655.17 -9.75 630.96 0.00
14404 10582.34 14403.95 10582.34 10581.27 1.07 666.08 0.00
14804 10496.65 14803.95 10496.65 10508.13 -11.48 703.17 0.00
15204 10432.04 15203.95 10432.04 10435.74 -3.70 742.32 0.00
15604 10355.46 15603.95 10355.46 10364.10 -8.64 783.64 0.00
16004 10287.65 16003.95 10287.65 10293.18 -5.53 827.27 0.00
16404 10210.28 16403.95 10210.28 10222.97 -12.69 873.33 0.00
16804 10156.05 16803.95 10156.05 10153.46 2.59 921.95 0.00
17204 10080.29 17203.95 10080.29 10084.64 -4.35 973.27 0.00
17604 10016.66 17603.95 10016.66 10016.50 0.17 1027.46 0.00
18004 9952.79 18003.95 9952.79 9949.01 3.77 1084.66 0.00
18404 9882.52 18403.95 9882.52 9882.19 0.33 1145.05 0.00
18804 9818.91 18803.95 9818.91 9816.00 2.91 1208.80 0.00

161
Appendices

19204 9749.55 19203.95 9749.55 9750.45 -0.90 1276.09 0.00


19604 9677.36 19603.95 9677.36 9685.52 -8.16 1347.14 0.00
20004 9623.89 20003.95 9623.89 9621.21 2.68 1422.14 0.00
20404 9554.23 20403.95 9554.23 9557.50 -3.27 1501.31 0.00
20804 9502.05 20803.95 9502.05 9494.39 7.66 1584.89 0.00
21204 9432.40 21203.95 9432.40 9431.87 0.53 1673.13 0.00
21604 9373.50 21603.95 9373.50 9369.92 3.58 1766.28 0.00
22004 9302.43 22003.95 9302.43 9308.55 -6.12 1864.61 0.00
22404 9241.87 22403.95 9241.87 9247.74 -5.87 1968.42 0.00
22804 9180.96 22803.95 9180.96 9187.49 -6.53 2078.01 0.00
23204 9126.05 23203.95 9126.05 9127.79 -1.74 2193.70 0.00
23604 9066.78 23603.95 9066.78 9068.63 -1.85 2315.83 0.00
24004 9014.12 24003.95 9014.12 9010.01 4.11 2444.75 0.00
24404 8948.39 24403.95 8948.39 8951.91 -3.52 2580.86 0.00
24804 8888.87 24803.95 8888.87 8894.34 -5.47 2724.55 0.00
25204 8829.93 25203.95 8829.93 8837.29 -7.35 2876.23 0.00
25604 8778.37 25603.95 8778.37 8780.74 -2.37 3036.36 0.00
26004 8727.47 26003.95 8727.47 8724.70 2.78 3205.40 0.00
26404 8664.18 26403.95 8664.18 8669.16 -4.97 3383.86 0.00
26804 8607.43 26803.95 8607.43 8614.10 -6.67 3572.24 0.00
27204 8554.44 27203.95 8554.44 8559.54 -5.10 3771.12 0.00
27604 8494.23 27603.95 8494.23 8505.46 -11.22 3981.07 0.00
28004 8450.95 28003.95 8450.95 8451.85 -0.90 4202.71 0.00
28404 8398.79 28403.95 8398.79 8398.71 0.08 4436.69 1.19
28804 8344.88 28803.95 8344.88 8346.04 -1.16 4683.69 18.87
29204 8301.26 29203.95 8301.26 8293.82 7.44 4944.45 33.09
29604 8237.97 29603.95 8237.97 8242.07 -4.09 5219.72 43.54
30004 8196.44 30003.95 8196.44 8190.76 5.68 5510.32 49.97
30404 8132.53 30403.95 8132.54 8139.90 -7.36 5817.09 52.19
30804 8070.77 30803.95 8070.77 8089.47 -18.70 6140.95 50.06
31204 8038.22 31203.95 8038.22 8039.49 -1.27 6482.83 43.54
31604 7996.35 31603.95 7996.35 7989.93 6.42 6843.75 32.68
32004 7934.90 32003.95 7934.90 7940.81 -5.90 7224.76 17.64
32404 7891.21 32403.95 7891.21 7892.10 -0.89 7626.99 0.00
32804 7850.01 32803.95 7850.01 7843.81 6.20 8051.60 0.00
33204 7799.76 33203.95 7799.76 7795.94 3.82 8499.86 0.00
33604 7747.47 33603.95 7747.47 7748.48 -1.00 8973.07 0.00
34004 7702.40 34003.95 7702.40 7701.42 0.99 9472.63 0.00
34404 7663.82 34403.95 7663.81 7654.76 9.06 10000.00 0.00
34804 7620.12 34803.95 7620.12 7608.50 11.62 10556.73 0.00
35204 7554.68 35203.95 7554.68 7562.63 -7.95 11144.46 0.00
35604 7518.90 35603.95 7518.90 7517.15 1.75 11764.90 0.00
36004 7457.35 36003.95 7457.35 7472.06 -14.71 12419.89 0.00
36404 7418.27 36403.95 7418.27 7427.35 -9.08 13111.34 0.00
36804 7389.85 36803.95 7389.85 7383.02 6.83 13841.29 0.00
37204 7335.53 37203.95 7335.54 7339.06 -3.53 14611.87 0.00
37604 7290.79 37603.95 7290.79 7295.48 -4.69 15425.36 0.00
38004 7246.31 38003.95 7246.31 7252.25 -5.94 16284.14 0.00
38404 7214.14 38403.95 7214.14 7209.40 4.74 17190.72 0.00
38804 7167.19 38803.95 7167.19 7166.90 0.29 18147.78 0.00
39204 7124.46 39203.95 7124.46 7124.76 -0.30 19158.12 0.00
39604 7097.55 39603.95 7097.55 7082.97 14.58 20224.71 0.00
40004 7043.81 40003.95 7043.81 7041.53 2.27 21350.68 0.00
40404 7005.69 40403.95 7005.69 7000.44 5.25 22539.34 0.00

162
Appendices

40804 6966.77 40803.95 6966.77 6959.69 7.08 23794.17 0.00


41204 6916.45 41203.95 6916.45 6919.28 -2.83 25118.87 61.76
41604 6889.40 41603.95 6889.40 6879.21 10.19 26517.31 130.23
42004 6839.68 42003.95 6839.68 6839.47 0.21 27993.61 201.07
42404 6804.17 42403.95 6804.17 6800.06 4.11 29552.09 272.82
42804 6762.64 42803.95 6762.65 6760.98 1.67 31197.35 343.93
43204 6723.67 43203.95 6723.67 6722.22 1.45 32934.20 412.83
43604 6690.10 43603.95 6690.10 6683.78 6.32 34767.74 477.92
44004 6651.45 44003.95 6651.45 6645.66 5.79 36703.36 537.69
44404 6606.52 44403.95 6606.52 6607.85 -1.33 38746.75 590.68
44804 6580.67 44803.95 6580.67 6570.36 10.31 40903.90 635.57
45204 6544.54 45203.95 6544.54 6533.17 11.37 43181.14 671.20
45604 6506.04 45603.95 6506.04 6496.30 9.74 45585.16 696.61
46004 6463.99 46003.95 6463.99 6459.72 4.27 48123.03 711.05
46404 6433.49 46403.95 6433.49 6423.44 10.05 50802.18 714.02
46804 6395.06 46803.95 6395.06 6387.47 7.60 53630.49 705.28
47204 6356.06 47203.95 6356.06 6351.78 4.28 56616.26 684.85
47604 6324.24 47603.95 6324.24 6316.39 7.85 59768.26 653.01
48004 6291.07 48003.95 6291.07 6281.29 9.78 63095.73 610.31
48404 6248.44 48403.95 6248.44 6246.48 1.96 66608.46 557.54
48804 6223.60 48803.95 6223.60 6211.95 11.66 70316.76 495.72
49204 6177.73 49203.95 6177.73 6177.70 0.04 74231.50 426.04
49604 6142.95 49603.95 6142.95 6143.73 -0.78 78364.19 349.88
50004 6125.86 50003.95 6125.86 6110.03 15.82 82726.96 268.73
50404 6084.52 50403.95 6084.52 6076.61 7.91 87332.62 184.19
50804 6048.58 50803.95 6048.58 6043.46 5.12 92194.69 97.87
51204 6022.61 51203.95 6022.61 6010.58 12.03 97327.44 11.42
51604 5980.81 51603.95 5980.81 5977.97 2.84 102745.95 0.00
52004 5951.21 52003.95 5951.21 5945.62 5.60 108466.13 0.00
52404 5915.88 52403.95 5915.88 5913.53 2.35 114504.76 0.00
52804 5874.35 52803.95 5874.35 5881.70 -7.35 120879.58 0.00
53204 5857.35 53203.95 5857.35 5850.13 7.23 127609.30 0.00
53604 5835.63 53603.95 5835.63 5818.81 16.82 134713.70 0.00
54004 5790.76 54003.95 5790.76 5787.74 3.02 142213.61 0.00
54404 5761.21 54403.95 5761.21 5756.93 4.28 150131.08 0.00
54804 5722.16 54803.95 5722.16 5726.36 -4.19 158489.31 0.00
55204 5699.70 55203.95 5699.70 5696.03 3.66 167312.89 0.00
55604 5668.54 55603.95 5668.54 5665.95 2.59 176627.70 0.00
56004 5644.55 56003.95 5644.55 5636.11 8.43 186461.09 0.00
56404 5609.90 56403.95 5609.90 5606.51 3.39 196841.94 0.00
56804 5580.37 58403.95 5480.99 5462.01 18.98 207800.72 0.00
57204 5545.81 60803.95 5280.49 5296.05 -15.56 219369.61 0.00
57604 5519.98 62803.95 5159.25 5163.66 -4.41 231582.58 0.00
58004 5497.46 65203.95 5015.17 5011.53 3.64 244475.47 0.00
58404 5480.99 67603.95 4865.27 4866.39 -1.12 258086.16 0.00
58804 5449.19 70003.95 4724.24 4727.88 -3.64 272454.59 0.00
59204 5420.94 72403.95 4592.80 4595.66 -2.86 287622.94 0.00
59604 5382.42 75203.95 4443.30 4448.91 -5.62 303635.78 0.00
60004 5344.91 77603.95 4330.12 4329.23 0.89 320540.09 0.00
60404 5332.40 80403.95 4187.15 4196.31 -9.15 338385.50 0.00
60804 5280.49 83203.95 4072.83 4070.21 2.62 357224.44 0.00
61204 5277.95 86403.95 3930.26 3933.94 -3.68 377112.22 0.00
61604 5243.27 89603.95 3805.16 3805.50 -0.34 398107.16 0.00
62004 5226.78 92803.95 3681.22 3684.38 -3.17 420271.00 17.77

163
Appendices

62404 5200.83 96003.95 3561.41 3570.11 -8.69 443668.72 56.86


62804 5159.25 99603.95 3439.43 3449.17 -9.75 468369.09 91.49
63204 5133.06 103203.95 3328.51 3335.75 -7.23 494444.63 121.23
63604 5123.35 106803.95 3215.61 3229.29 -13.69 521971.81 145.74
64004 5100.13 110803.95 3111.90 3118.58 -6.68 551031.56 164.75
64404 5067.89 114403.95 3011.66 3025.25 -13.58 581709.13 178.12
64804 5035.69 118803.95 2903.72 2918.66 -14.94 614094.63 185.77
65204 5015.17 122803.95 2801.45 2828.36 -26.91 648283.13 187.72
65604 4989.43 127203.95 2722.03 2735.69 -13.66 684375.00 184.04
66004 4961.58 132003.95 2632.57 2641.89 -9.32 722476.19 174.88
66404 4939.68 136803.95 2541.61 2555.03 -13.42 762698.56 160.46
66804 4918.99 141603.95 2451.01 2474.49 -23.48 805160.31 141.02
67204 4900.47 146803.95 2372.46 2393.73 -21.27 849986.00 116.89
67604 4865.27 152003.95 2304.48 2319.09 -14.61 897307.25 88.39
68004 4857.08 157603.95 2226.26 2244.90 -18.63 947263.00 55.89
68404 4826.04 163203.95 2164.15 2176.50 -12.35 1000000.00 19.77
68804 4798.83 168803.95 2101.72 2113.32 -11.60 1055673.00 0.00
69204 4769.75 175203.95 2026.44 2046.84 -20.41 1114445.50 0.00
69604 4754.56 181203.95 1988.95 1989.51 -0.56 1176490.00 0.00
70004 4724.24 188003.95 1921.30 1929.73 -8.43 1241988.80 0.00
70404 4715.29 194403.95 1867.97 1877.97 -10.00 1311133.90 0.00
70804 4682.62 201603.95 1825.78 1824.37 1.41 1384128.80 0.00
71204 4662.75 208803.95 1775.04 1775.12 -0.08 1461187.30 0.00
71604 4635.32 216403.95 1727.00 1727.30 -0.30 1542536.00 0.00
72004 4614.75 224003.95 1688.79 1683.25 5.54 1628413.50 0.00
72404 4592.80 232003.95 1655.04 1640.47 14.57 1719072.30 0.00
72804 4582.22 240403.95 1602.48 1599.02 3.46 1814778.10 0.00
73204 4555.72 249203.95 1566.34 1558.94 7.39 1915812.30 0.00
73604 4528.33 258003.95 1522.41 1521.85 0.56 2022471.30 0.00
74004 4511.78 267203.94 1494.97 1485.84 9.13 2135068.30 0.00
74404 4476.46 276803.94 1459.31 1450.91 8.40 2253934.00 0.00
74804 4465.04 286803.94 1431.38 1417.01 14.37 2379417.30 0.00
75204 4443.30 297203.94 1407.32 1384.09 23.23 2511886.50 0.00
75604 4438.54 308003.94 1368.06 1352.08 15.97 2651730.80 0.00
76004 4411.46 318803.94 1340.95 1322.00 18.95 2799360.50 0.00
76404 4390.43 330403.94 1299.98 1291.56 8.42 2955209.30 0.00
76804 4368.62 342403.94 1270.12 1261.82 8.30 3119734.50 0.00
77204 4333.96 354403.94 1255.04 1233.63 21.40 3293419.50 0.00
77604 4330.12 367203.94 1222.80 1205.06 17.75 3476774.00 0.00
78004 4314.82 380403.94 1197.63 1177.00 20.63 3670336.50 0.00
78404 4295.77 394003.94 1159.00 1149.39 9.61 3874675.00 0.00
78804 4257.78 408403.94 1137.38 1121.43 15.95 4090390.00 0.00
79204 4252.39 422803.94 1108.57 1094.63 13.95 4318114.00 0.00
79604 4232.42 438003.94 1074.79 1067.45 7.35 4558516.50 0.00
80004 4204.33 454003.94 1051.15 1039.94 11.21 4812302.50 0.00
80404 4187.15 470403.94 1015.79 1012.81 2.98 5080218.00 0.00
80804 4181.66 487203.94 983.08 986.02 -2.94 5363049.00 0.00
81204 4166.34 504803.94 968.23 958.95 9.28 5661626.00 0.00
81604 4142.49 522803.94 927.81 932.24 -4.43 5976825.50 0.00
82004 4114.34 541603.94 911.52 905.32 6.20 6309573.50 0.00
82404 4096.67 560803.94 872.59 878.77 -6.18 6660846.50 0.00
82804 4083.22 581203.94 852.87 851.54 1.32 7031675.50 0.00
83204 4072.83 602003.94 817.51 824.77 -7.25 7423150.00 0.00
83604 4055.30 623603.94 797.14 797.95 -0.81 7836419.00 0.00

164
Appendices

84004 4043.56 646003.94 767.48 771.15 -3.67 8272696.00 0.00


84404 4007.79 669203.94 739.90 744.43 -4.53 8733262.00 0.00
84804 4002.46 693203.94 712.07 717.82 -5.75 9219468.00 0.00
85204 3984.78 718403.94 690.64 690.97 -0.33 9732744.00 0.00
85604 3974.14 744003.94 652.90 664.79 -11.89 10274595.00 0.00
86004 3940.98 770803.94 629.25 638.49 -9.25 10846612.00 0.00
86404 3930.26 798403.94 606.78 612.56 -5.78 11450476.00 0.00
86804 3923.15 827203.94 585.51 586.67 -1.16 12087958.00 0.00
87204 3899.31 856803.94 558.08 561.25 -3.17 12760931.00 0.00
87604 3871.86 887603.94 541.60 536.02 5.58 13471370.00 0.00
88004 3880.11 919603.94 490.83 511.06 -20.22 14221362.00 0.00
88404 3844.71 952403.94 480.73 486.72 -5.99 15013107.00 0.00
88804 3832.53 986803.94 468.22 462.50 5.72 15848932.00 0.00
89204 3818.26 1022403.90 438.88 438.75 0.13 16731289.00 0.00
89604 3805.16 1058804.00 411.49 415.77 -4.28 17662770.00 0.00
90004 3797.18 1097204.00 383.19 392.89 -9.70 18646110.00 0.00
90404 3766.32 1136404.00 355.42 370.88 -15.46 19684194.00 0.00
90804 3758.62 1177204.00 334.64 349.33 -14.69 20780072.00 0.00
91204 3731.92 1219604.00 323.30 328.31 -5.00 21936962.00 0.00
91604 3727.14 1263604.00 304.89 307.88 -2.99 23158258.00 0.00
92004 3702.58 1308804.00 284.18 288.27 -4.08 24447548.00 0.00
92404 3694.90 1356004.00 276.73 269.17 7.56 25808616.00 0.00
92804 3681.22 1404404.00 251.08 250.95 0.13 27245458.00 0.00
93204 3667.97 1454804.00 229.49 233.33 -3.84 28762294.00 0.00
93604 3657.06 1507204.00 231.85 216.37 15.48 30363578.00 0.00
94004 3641.76 1561604.00 197.40 200.11 -2.72 32054008.00 0.00
94404 3619.31 1617604.00 183.85 184.70 -0.85 33838552.00 0.00
94804 3611.04 1675604.00 165.65 170.03 -4.38 35722444.00 0.00
95204 3580.92 1736004.00 152.20 156.03 -3.83 37711220.00 0.00
95604 3579.61 1798404.00 133.26 142.83 -9.57 39810716.00 0.00
96004 3561.41 1862804.00 127.32 130.41 -3.09 42027100.00 0.00
96404 3547.45 1929604.00 127.42 118.70 8.72 44366872.00 0.00
96804 3524.88 1999204.00 93.06 107.66 -14.60 46836908.00 0.00
97204 3522.61 2070804.00 92.28 97.41 -5.13 49444460.00 0.00
97604 3529.93 2145204.00 92.13 87.82 4.31 52197184.00 0.00
98004 3499.69 2222404.00 78.60 78.91 -0.31 55103156.00 0.00
98404 3474.71 2302404.00 70.21 70.65 -0.44 58170912.00 0.00
98804 3464.56 2384804.00 65.46 63.08 2.38 61409464.00 0.00
99204 3467.90 2470804.00 59.91 56.06 3.84 64828312.00 0.00
99604 3439.43 2559604.00 31.14 49.67 -18.52 68437496.00 0.00
100004 3420.07 2651204.00 46.53 43.85 2.68 72247616.00 0.00
100404 3418.11 2746804.00 35.85 38.53 -2.69 76269856.00 0.00
100804 3410.61 2845204.00 40.50 33.75 6.75 80516032.00 0.00
101204 3385.87 2947604.00 41.76 29.42 12.34 84998600.00 0.00
101604 3375.54 3053604.00 21.38 25.54 -4.16 89730728.00 0.00
102004 3365.58 3163204.00 25.08 22.07 3.01 94726304.00 0.00
102404 3347.50 3276804.00 27.59 18.99 8.59 100000000.00 0.00

3276804 27.59

165
Appendices

Appendix E

Appendix Commands Profilometry (RINMR, Maran Ultra 23 spectrometer)

Acquisition
Sequence
Load All Profile
Parameters Load all (or REP)
Profilometry.RiPar
GO
FT
MAG
Export to Excel

New sample:
Go
FT
MAG
Export to Excel

Parameters in Profilometry.RiPar
ID Value TRIM1 45
P90 7.50 TRIM2 0
P180 15.00 TRIM3 0
P1 1.00 TRIM4 0
P2 1.00 TRIM5 0
P3 1.00 TRIM6 157
P4 1.00 TRIM7 159
P5 1.00 TRIM8 0
DEAD1 7.00 TRIM9 158
DEAD2 2.80 TRIMA 0
DW 10.00 TRIMB 173
RD 10000000.00 TRIMC 0
TAU 6000.00 TRIMD 167
D1 100.00 TRIME 0
D2 3562.00 TRIMF 170
D3 1000.00 TRIMG 0
D4 1.00 TRIMH 0
D5 1.00 TRIMI 0
D6 1000000.00 TRIMJ 0
D7 1000000.00 TRIMK 0
D8 1000000.00 TRIML 0
D9 1000000.00 TRIMM 0
D10 1000000.00 TRIMN 0
D11 1000000.00 TRIMO 0
D12 1000000.00 TRIMP 0
SI 512 TRIMQ 0
NECH 256 TRIMR 0
NS 16 TRIMS 0
SF 23.400000 TRIMT 0
O1 27932.39 TRIMU 0
SF2 0.000000 TRIMV 0
O2 0.00 TRIMW 75
FW 1.0 TRIMX 0
RG 1.20 SW 100000.0
PH1 0213 DB 24
PH2 0213 BES 1000000
PH3 0011 BUT 1000000
PH4 0213 RFA0 100.0
PH5 0213 RFA1 0.0
LB 0.00 RFA2 0.0
PA 0.00 RFA3 0.0
PB 0.00 RFA4 0.0
DP 0.00 RFA5 0.0
TRIM0 49 RF2A0 0.0

166
Appendices

RF2A1 0.0 %CAL4 0.000000


RF2A2 0.0 %CALIBDIR C:\Program Files\Resonance\RINMR\DATA\
RF2A3 0.0 %1
RF2A4 0.0 %2
RF2A5 0.0 %3
WW 5.0 %4
C1 0 %5
C2 0 PREXA1 0.000
C3 0 PREXK1 100000000.00
C4 0 PREXA2 0.000
C5 0 PREXK2 100000000.00
TI ???? PREXA3 0.000
SMP 0 PREXK3 100000000.00
GX 0 PREXA4 0.000
GY 3000 PREXK4 100000000.00
GZ 0 PREYA1 0.000
G1 3500 PREYK1 100000000.00
G2 3500 PREYA2 0.000
G3 0 PREYK2 100000000.00
G4 0 PREYA3 0.000
G5 0 PREYK3 100000000.00
G6 0 PREYA4 0.000
G7 0 PREYK4 100000000.00
G8 0 PREZA1 0.000
G9 0 PREZK1 100000000.00
IG1 1 PREZA2 0.000
IG2 1 PREZK2 100000000.00
IG3 1 PREZA3 0.000
IG4 1 PREZK3 100000000.00
IG5 1 PREZA4 0.000
IG6 1 PREZK4 100000000.00
IG7 1 XB0A 0.000
IG8 1 XB0K 100000000.00
IG9 1 YB0A 0.000
MAC1 0.0 YB0K 100000000.00
MAC2 0.0 ZB0A 0.000
SH1 ZB0K 100000000.00
SH2 PREBA4 0.000
SH3 PREBK4 0.00
SH4 PPTH 4.0
SH5 PPRF 50
DS 0 PPAF 1
NA 1 PPPF 0
SEQ PROFILE DEC90 5000000.00
GRS X CPD WALTZ16
GRP Y GSLICEX 1.000
GRR Z GSLICEY 0.000
%TEMPDIR Z GSLICEZ 0.000
%ROOTDIR Z GPHASEX 0.000
%DATADIR E:\parameters\CPMG_daan20090501.RiPar GPHASEY 1.000
%EXPORTDIR C:\Documents and GPHASEZ 0.000
Settings\Jan\Desktop\Dodac0.5percent_0.1Mn_tau50.00001 GREADX 0.000
%LISTFILEC:\Documents and Settings\Jan\My GREADY 0.000
Documents\PhD\Enclosed volume in DODAC\Maran GREADZ 1.000
measurements\lists#\deltaGlist.txt GREAD 0
%LOGFILE C:\Program GPHASE 3000
Files\Resonance\RINMR\DATA\DODAC_0.1PERC+MNCL2.00001.RiL GSLICE 0
og %SNR 0
%HARDWARE 0 GD 0.00
GSH1 %ERROR 0
GSH2 %CALCERROR 0.000
GSH3 INC2D 0.00
GSH4 SF2D 0.000000
GSH5 %TP 0
SNR 100 %TS 0
%SEQFILE C:\Program Files\Resonance\RINMR\bak\bin\CPMG.exe %R0 0.000
PP 0 %R1 0.000
NOBC 0 %R2 0.000
C6 1 %R3 0.000
C7 1 %R4 0.000
C8 1 %R5 0.000
C9 1 %R6 0.000
C10 1 %R7 0.000
C11 1 %R8 0.000
C12 1 %R9 0.000
FP1 1.00000 %ADCSCALE 2048
FP2 1.00000 PREAMP 0
FP3 1.00000 TRIGGER 0
FP4 1.00000 MATRIX 0
FP5 1.00000 VER äeú
%LASTWEIGHT 0.000 XB0 0.000
%GOSTATE 0 YB0 0.000
%SCANSDONE 0 ZB0 0.000
%CAL1 0.000000 XOFFSET 0.00000000
%CAL2 0.000000 YOFFSET 0.00000000
%CAL3 0.000000 ZOFFSET 0.00000000

167
Appendices

D13 1000000.00 G28 0


D14 1000000.00 G29 0
D15 1000000.00 G30 0
D16 1000000.00 G31 0
D17 1000000.00 G32 0
D18 1000000.00 ACQUISITION 3
D19 1000000.00 RG1 0.00
D20 1000000.00 RG2 0.00
D21 1000000.00 RG3 0.00
D22 1000000.00 RG4 0.00
D23 1000000.00 RG5 0.00
D24 1000000.00 RG6 0.00
D25 1000000.00 RG7 0.00
D26 1000000.00 RG8 0.00
D27 1000000.00 RG9 0.00
D28 1000000.00 %TOTALSTEPS 0
D29 1000000.00 %HIDEERROR 0
D30 1000000.00 %LASTSEQERROR 0
D31 1000000.00 SH6
D32 1000000.00 SH7
C13 0 SH8
C14 0 SH9
C15 0 SH10
C16 0 FP6 1.00000
C17 0 FP7 1.00000
C18 0 FP8 1.00000
C19 0 FP9 1.00000
C20 0 FP10 1.00000
C21 0 %DISPLAYBUFFER 0
C22 0 QCSHIM1 0
C23 0 QCSHIM2 0
C24 0 QCSHIM3 0
C25 0 QCSHIM4 0
C26 0 QCSHIM5 0
C27 0 QCSHIM6 0
C28 0 QCSHIM7 0
C29 0 QCSHIM8 0
C30 0 QCLEDS 0
C31 0 QCVAR1 0.0
C32 0 QCVAR2 0.0
G10 0 QCVAR3 0.0
G11 0 QCVAR4 0.0
G12 0 QCVAR5 0.0
G13 0 QCVAR6 0.0
G14 0 QCVAR7 0.0
G15 0 QCVAR8 0.0
G16 0 QCVAR9 0.0
G17 0 QCVAR10 0.0
G18 0 QCVAR11 0.0
G19 0 QCVAR12 0.0
G20 0 QCVAR13 0.0
G21 0 QCVAR14 0.0
G22 0 QCVAR15 0.0
G23 0 QCVAR16 0.0
G24 0 QCVAR17 0.000
G25 0 QCVAR18 0.000
G26 0 QCVAR19 0
G27 0 QCVAR20 0

168
Appendices

Appendix F

Table: The experimental numerical data of the echo intensities as a function of small
delta of different emulsions
Measured Echo Intensity (I)
H0/1 Prod. date 8/9/10 M0/1 Prod. date 9/9/10 P0/1 Prod. date 9/9/10
∆ (s) sample 1 sample 2 Sample 3 sample 1 Sample 2 sample 3 sample 1 sample 2 sample 3
4.00E-04 1.65E+03 1.71E+03 1.87E+03 1.45E+03 1.31E+03 1.38E+03 4.30E+03 3.52E+03 3.97E+03
6.00E-04 1.71E+03 1.87E+03 2.11E+03 1.39E+03 1.25E+03 1.33E+03 3.99E+03 3.34E+03 3.72E+03
8.00E-04 1.70E+03 2.01E+03 2.28E+03 1.33E+03 1.23E+03 1.29E+03 3.76E+03 3.22E+03 3.54E+03
1.00E-03 1.62E+03 2.03E+03 2.21E+03 1.30E+03 1.20E+03 1.26E+03 3.58E+03 3.11E+03 3.39E+03
1.25E-03 1.45E+03 1.86E+03 1.97E+03 1.24E+03 1.13E+03 1.19E+03 3.38E+03 2.98E+03 3.21E+03
1.50E-03 1.28E+03 1.66E+03 1.70E+03 1.17E+03 1.08E+03 1.14E+03 3.20E+03 2.83E+03 3.07E+03
1.75E-03 1.10E+03 1.40E+03 1.46E+03 1.10E+03 1.02E+03 1.08E+03 3.07E+03 2.74E+03 2.94E+03
2.00E-03 9.68E+02 1.24E+03 1.27E+03 1.02E+03 9.59E+02 1.02E+03 2.94E+03 2.63E+03 2.85E+03
2.25E-03 8.03E+02 1.04E+03 1.11E+03 9.49E+02 9.05E+02 9.69E+02 2.85E+03 2.56E+03 2.77E+03
2.50E-03 7.09E+02 9.46E+02 9.63E+02 8.82E+02 8.39E+02 8.85E+02 2.74E+03 2.48E+03 2.70E+03
2.75E-03 6.25E+02 8.56E+02 8.38E+02 8.04E+02 7.90E+02 8.18E+02 2.66E+03 2.42E+03 2.64E+03
3.00E-03 5.42E+02 7.44E+02 7.51E+02 7.45E+02 7.27E+02 7.75E+02 2.57E+03 2.35E+03 2.57E+03
3.25E-03 4.60E+02 6.57E+02 6.49E+02 6.86E+02 6.73E+02 7.26E+02 2.50E+03 2.29E+03 2.51E+03
3.50E-03 4.05E+02 5.89E+02 5.84E+02 6.27E+02 6.30E+02 6.58E+02 2.41E+03 2.22E+03 2.45E+03
3.75E-03 3.12E+02 5.07E+02 4.52E+02 5.64E+02 5.89E+02 6.21E+02 2.35E+03 2.18E+03 2.39E+03
4.00E-03 2.90E+02 4.75E+02 4.62E+02 5.21E+02 5.45E+02 5.82E+02 2.28E+03 2.14E+03 2.32E+03
4.50E-03 2.00E+02 3.23E+02 3.45E+02 4.47E+02 4.82E+02 5.14E+02 2.15E+03 2.03E+03 2.23E+03

Measured Echo Intensity (I)


H0.5/1 Prod. date 22/9/10
∆ (s) sample 1 sample 2 Sample 3
5.00E-04 3.68E+03 3.68E+03 3.55E+03
7.50E-04 3.36E+03 3.36E+03 3.23E+03
1.00E-03 3.20E+03 3.20E+03 3.10E+03
1.25E-03 3.04E+03 3.04E+03 2.95E+03
1.50E-03 2.91E+03 2.91E+03 2.77E+03
1.75E-03 2.74E+03 2.74E+03 2.60E+03
2.00E-03 2.54E+03 2.54E+03 2.43E+03
2.25E-03 2.37E+03 2.37E+03 2.28E+03
2.50E-03 2.20E+03 2.20E+03 2.11E+03
2.75E-03 2.02E+03 2.02E+03 1.95E+03
3.00E-03 1.90E+03 1.90E+03 1.83E+03
3.25E-03 1.72E+03 1.72E+03 1.67E+03
3.50E-03 1.64E+03 1.64E+03 1.56E+03
4.00E-03 1.34E+03 1.34E+03 1.25E+03
4.50E-03 1.16E+03 1.16E+03 1.09E+03
5.00E-03 1.02E+03 1.02E+03 9.65E+02
6.00E-03 6.86E+02 6.86E+02 7.68E+02
8.00E-03 4.20E+02 4.20E+02 4.03E+02

169
Appendices

Details H0/1 sample 1


g
δ (s) ∆ (s) G1 (Tesla/m) I g^2.δ^2.(∆-δ/3)
4.00E-04 2.00E-01 18000 1.74E+00 1.65E+03 9.68E-08
6.00E-04 2.00E-01 18000 1.74E+00 1.71E+03 2.18E-07
8.00E-04 2.00E-01 18000 1.74E+00 1.70E+03 3.87E-07
1.00E-03 2.00E-01 18000 1.74E+00 1.62E+03 6.04E-07
1.25E-03 2.00E-01 18000 1.74E+00 1.45E+03 9.44E-07
1.50E-03 2.00E-01 18000 1.74E+00 1.28E+03 1.36E-06
1.75E-03 2.00E-01 18000 1.74E+00 1.10E+03 1.85E-06
2.00E-03 2.00E-01 18000 1.74E+00 9.68E+02 2.41E-06
2.25E-03 2.00E-01 18000 1.74E+00 8.03E+02 3.05E-06
2.50E-03 2.00E-01 18000 1.74E+00 7.09E+02 3.77E-06
2.75E-03 2.00E-01 18000 1.74E+00 6.25E+02 4.56E-06
3.00E-03 2.00E-01 18000 1.74E+00 5.42E+02 5.42E-06
3.25E-03 2.00E-01 18000 1.74E+00 4.60E+02 6.36E-06
3.50E-03 2.00E-01 18000 1.74E+00 4.05E+02 7.37E-06
3.75E-03 2.00E-01 18000 1.74E+00 3.12E+02 8.46E-06
4.00E-03 2.00E-01 18000 1.74E+00 2.90E+02 9.62E-06
4.50E-03 2.00E-01 18000 1.74E+00 2.00E+02 1.22E-05

170
Appendices

Appendix G: output of a profilometric analysis

Frequency (MHz) Signal intensity


0.049804688 273.2489624 Frequency 23427932
0.049609375 235.45047 P90 7.5E-06
0.049414063 256.2937927 P180 1.5E-05
0.04921875 286.750885 P1 1E-06
0.049023438 901.8967896 P2 1E-06
0.048828125 637.4200439 P3 1E-06
0.048632813 407.6343079 P4 1E-06
0.0484375 178.0191498 P5 1E-06
0.048242188 155.5707703 DEAD1 7E-06
0.048046875 296.5934143 DEAD2 2.8E-06
0.047851563 778.2650757 DW 1E-05
0.04765625 296.4411621 RD 10
0.047460938 558.7873535 TAU 0.006
0.047265625 600.5683594 D1 1E-04
0.047070313 139.3050842 D2 0.003562
0.046875 455.2594299 D3 0.001
0.046679688 466.1853943 D4 1E-06
0.046484375 145.8015747 D5 1E-06
0.046289063 664.8319092 D6 1
0.04609375 510.8852539 D7 1
0.045898438 460.7337341 D8 1
0.045703125 162.0378113 D9 1
0.045507813 297.1578674 D10 1
0.0453125 599.9174805 D11 1
0.045117188 631.8066406 D12 1
0.044921875 271.763092 SI 512
0.044726563 414.2615662 NECH 256
0.04453125 402.9092712 NS 16
0.044335938 544.3898926 SF 23400000
0.044140625 760.6894531 O1 27932.39
0.043945313 287.5783691 SF2 0
0.04375 156.998291 O2 0
0.043554688 321.2329712 FW 1
0.043359375 392.8141479 RG 1.2
0.043164063 214.7452393 PH1 0213
0.04296875 238.5203247 PH2 0213
0.042773438 618.4665527 PH3 0011
0.042578125 691.7803345 PH4 0213
0.042382813 326.994751 PH5 0213
0.0421875 478.6191406 LB 0
0.041992188 133.2767792 PA 0
0.041796875 435.1164551 PB 0
0.041601563 655.3277588 DP 1.034583
0.04140625 270.3832397 TRIM0 49
0.041210938 59.87940216 TRIM1 45
0.041015625 506.2037048 TRIM2 0
0.040820313 1022.53894 TRIM3 0
0.040625 293.696228 TRIM4 0
0.040429688 113.4191132 TRIM5 0
0.040234375 465.0850525 TRIM6 157
0.040039063 776.0014648 TRIM7 159
0.03984375 603.4848633 TRIM8 0

171
Appendices

0.039648438 286.8362732 TRIM9 158


0.039453125 152.5377045 TRIMA 0
0.039257813 524.3954468 TRIMB 173
0.0390625 420.5890808 TRIMC 0
0.038867188 222.1972809 TRIMD 167
0.038671875 347.4411621 TRIME 0
0.038476563 222.091156 TRIMF 170
0.03828125 399.2488403 TRIMG 0
0.038085938 371.6633606 TRIMH 0
0.037890625 299.5723877 TRIMI 0
0.037695313 526.5463867 TRIMJ 0
0.0375 230.3275604 TRIMK 0
0.037304688 233.6271362 TRIML 0
0.037109375 190.8079681 TRIMM 0
0.036914063 512.5438843 TRIMN 0
0.03671875 904.374939 TRIMO 0
0.036523438 160.8424988 TRIMP 0
0.036328125 112.9999237 TRIMQ 0
0.036132813 221.906723 TRIMR 0
0.0359375 596.473938 TRIMS 0
0.035742188 117.9434891 TRIMT 0
0.035546875 463.7452393 TRIMU 0
0.035351563 384.6939087 TRIMV 0
0.03515625 295.9091187 TRIMW 75
0.034960938 407.1180115 TRIMX 0
0.034765625 117.0415649 SW 100000
0.034570313 503.1583862 DB 24
0.034375 202.5902405 BES 1000000
0.034179688 227.3736115 BUT 1000000
0.033984375 330.9534912 RFA0 100
0.033789063 186.7667847 RFA1 0
0.03359375 607.4312744 RFA2 0
0.033398438 234.3165131 RFA3 0
0.033203125 492.7983704 RFA4 0
0.033007813 508.1192017 RFA5 0
0.0328125 475.6696167 RF2A0 0
0.032617188 816.7148438 RF2A1 0
0.032421875 361.7311096 RF2A2 0
0.032226563 846.8509521 RF2A3 0
0.03203125 337.5320129 RF2A4 0
0.031835938 328.0878601 RF2A5 0
0.031640625 238.6884155 WW 5
0.031445313 224.521698 C1 0
0.03125 611.2073975 C2 0
0.031054688 559.6374512 C3 0
0.030859375 270.987915 C4 0
0.030664063 92.80229187 C5 0
0.03046875 389.1214905 TI ????
0.030273438 284.5443115 SMP 0
0.030078125 531.8380737 GX 0
0.029882813 206.9532471 GY 3000
0.0296875 386.1412354 GZ 0
0.029492188 454.256958 G1 3500
0.029296875 126.819603 G2 3500

172
Appendices

0.029101563 374.5834351 G3 0
0.02890625 408.7837219 G4 0
0.028710938 374.0313721 G5 0
0.028515625 497.9112244 G6 0
0.028320313 534.1647339 G7 0
0.028125 334.4261169 G8 0
0.027929688 345.5342712 G9 0
0.027734375 147.9359436 IG1 1
0.027539063 50.90776443 IG2 1
0.02734375 215.4097748 IG3 1
0.027148438 637.8907471 IG4 1
0.026953125 308.1389771 IG5 1
0.026757813 312.6181335 IG6 1
0.0265625 366.6786499 IG7 1
0.026367188 468.5193787 IG8 1
0.026171875 467.3326111 IG9 1
0.025976563 647.7133789 MAC1 0
0.02578125 190.9958038 MAC2 0
0.025585938 341.8713989 SH1
0.025390625 161.2305756 SH2
0.025195313 541.5947876 SH3
0.025 419.7670593 SH4
0.024804688 298.8596497 SH5
0.024609375 635.3144531 DS 0
0.024414063 408.7278137 NA 1
0.02421875 401.7145996 SEQ PROFILE
0.024023438 420.7365723 GRS X
0.023828125 767.1320801 GRP Y
0.023632813 216.095993 GRR Z
0.0234375 179.8995361
0.023242188 523.973999
0.023046875 390.7264099
0.022851563 212.7952881
0.02265625 418.6812744
0.022460938 300.6339111
0.022265625 211.0618744
0.022070313 285.1594238 GSH1
0.021875 214.8972626 GSH2
0.021679688 410.0625 GSH3
0.021484375 81.38557434 GSH4
0.021289063 98.58622742 GSH5
0.02109375 305.0961304 SNR 100
0.020898438 217.923996
0.020703125 386.3705444 PP 0
0.020507813 455.2966309 NOBC 0
0.0203125 704.4728394 C6 1
0.020117188 330.5545044 C7 1
0.019921875 89.52977753 C8 1
0.019726563 456.6725769 C9 1
0.01953125 364.1968689 C10 1
0.019335938 220.3947906 C11 1
0.019140625 267.0543518 C12 1
0.018945313 337.0260925 FP1 1
0.01875 246.9345703 FP2 1

173
Appendices

0.018554688 460.9109802 FP3 1


0.018359375 290.5318298 FP4 1
0.018164063 368.2280579 FP5 1
0.01796875 233.395462
0.017773438 218.7448425
0.017578125 81.34256744
0.017382813 426.1618958
0.0171875 207.6192932
0.016992188 319.343811
0.016796875 457.0608215
0.016601563 281.1471863
0.01640625 258.8962708
0.016210938 390.9786682
0.016015625 420.4814453
0.015820313 499.5676575
0.015625 240.9592285
0.015429688 522.4503784 PREXA1 0
0.015234375 312.4052429 PREXK1 100
0.015039063 110.3498611 PREXA2 0
0.01484375 677.1757202 PREXK2 100
0.014648438 618.7169189 PREXA3 0
0.014453125 511.7521362 PREXK3 100
0.014257813 98.89109802 PREXA4 0
0.0140625 435.5617676 PREXK4 100
0.013867188 238.7025604 PREYA1 0
0.013671875 195.4999695 PREYK1 100
0.013476563 135.7706757 PREYA2 0
0.01328125 566.0339966 PREYK2 100
0.013085938 545.1677246 PREYA3 0
0.012890625 303.1686096 PREYK3 100
0.012695313 798.43573 PREYA4 0
0.0125 88.90397644 PREYK4 100
0.012304688 49.86899567 PREZA1 0
0.012109375 328.4577942 PREZK1 100
0.011914063 438.9898987 PREZA2 0
0.01171875 385.4032288 PREZK2 100
0.011523438 132.2007446 PREZA3 0
0.011328125 408.8601685 PREZK3 100
0.011132813 369.9115601 PREZA4 0
0.0109375 565.9788208 PREZK4 100
0.010742188 462.2780151 XB0A 0
0.010546875 270.3238525 XB0K 100
0.010351563 87.88369751 YB0A 0
0.01015625 202.5294037 YB0K 100
0.009960938 316.1564636 ZB0A 0
0.009765625 189.2915039 ZB0K 100
0.009570313 906.8520508 PREBA4 0
0.009375 486.893219 PREBK4 0
0.009179688 610.1662598 PPTH 4
0.008984375 215.4837189 PPRF 50
0.008789063 447.9555969 PPAF 1
0.00859375 310.1137695 PPPF 0
0.008398438 74.51745605 DEC90 5
0.008203125 210.2084503 CPD WALTZ16

174
Appendices

0.008007813 437.8165283 GSLICEX 1


0.0078125 594.2328491 GSLICEY 0
0.007617188 574.9415894 GSLICEZ 0
0.007421875 465.2231445 GPHASEX 0
0.007226563 393.682373 GPHASEY 1
0.00703125 187.5591888 GPHASEZ 0
0.006835938 257.3603516 GREADX 0
0.006640625 175.2765808 GREADY 0
0.006445313 710.3255005 GREADZ 1
0.00625 565.4083252 GREAD 0
0.006054688 729.3746338 GPHASE 3000
0.005859375 577.6845093 GSLICE 0
0.005664063 794.4776611
0.00546875 1312.459351 GD 0
0.005273438 343.3006897
0.005078125 479.5255127
0.004882813 541.0355835 INC2D 0
0.0046875 252.5053253 SF2D 0
0.004492188 457.0461731
0.004296875 63.99576187
0.004101563 804.342041
0.00390625 1104.436768
0.003710938 238.9017792
0.003515625 509.0552673
0.003320313 606.7443848
0.003125 296.3849182
0.002929688 938.7177124
0.002734375 668.4386597
0.002539063 805.7462769
0.00234375 487.1318054
0.002148438 159.5255127
0.001953125 350.0638123 PREAMP 0
0.001757813 932.3417358 TRIGGER 0
0.0015625 1205.988403 MATRIX 0
0.001367188 1292.137329 VER
0.001171875 1034.407227 XB0 0
0.000976563 1579.183472 YB0 0
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