wax encapsulation of water-soluble compounds for application in foods
TRANSCRIPT
Journal of Microencapsulation, November 2006; 23(7): 729–740
Wax encapsulation of water-soluble compounds forapplication in foods
M. MELLEMA, W. A. J. VAN BENTHUM, B. BOER,
J. VON HARRAS, & A. VISSER
Unilever R&D Vlaardingen, Vlaardingen, The Netherlands
(Received 24 October 2005; revised 16 February 2006; accepted 10 March 2006)
AbstractWater-soluble ingredients have been successfully encapsulated in wax using two preparationtechniques. The first technique (‘solid preparation’) leads to relatively large wax particles. Thesecond technique (‘liquid preparation’) leads to relatively small wax particles immersed in vegetableoil. On the first technique: stable encapsulation of water-soluble colourants (dissolved at lowconcentration in water) has been achieved making use of beeswax and PGPR. The leakage from thecapsules, for instance of size 2mm, is about 30% after 16 weeks storage in water at room temperature.To form such capsules a minimum wax mass of 40% relative to the total mass is needed. High amountsof salt or acids at the inside water phase causes more leaking, probably because of the osmotic pressuredifference. Osmotic matching of inner and outer phase can lead to a dramatic reduction in leakage. Fatcapsules are less suitable to incorporate water soluble colourants. The reason for this could be adifference in crystal structure (fat is less ductile and more brittle). On the second technique: stableencapsulation of water-soluble colourants (encapsulated in solid wax particles) has been achievedmaking use of carnauba wax. The leakage from the capsules, for instance of size 250mm, is about 40%after 1 weeks storage in water at room temperature.
Keywords: Food, water-soluble compounds, wax, double emulsion, crystal structure
Introduction
Functional foods exhibit a functional benefit beyond basic nutrition, usually obtained by
fortification with a functional ingredient. The functional ingredient may impart negative
properties like taste (bitterness, oxidation) or physical texture (sedimentation, phase
separation). One of the challenges in developing functional foods is to achieve incorporation
of the functional ingredient with acceptable bioavailability, without interference with
product quality. Microencapsulation can be a suitable technique for incorporation of some
types of functional ingredients. For foods the crucial criterion is that the encapsulated
compound should not leak out of the capsules during shelf life and that the preparation
Correspondence: M. Mellema, Unilever R&D Vlaardingen, PO Box 114, 3130 AC Vlaardingen, The Netherlands.
Fax: þ31(0)10 460 6776. E-mail: [email protected]
ISSN 0265–2048 print/ISSN 1464–5246 online � 2006 Informa UK Ltd.
DOI: 10.1080/02652040600787900
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procedure is cheap. Moreover, the size and shape of the capsules should be such that it is not
sensed in the mouth.
Incorporation of lipids or lipophilic compounds can be achieved by encapsulation using
a wall material of gelatin-gum arabic (Jozomoto et al. 1993; Malone and Appelqvist 2003)
or whey protein-gum arabic (Weinbreck et al. 2004) complex coacervates. Using cross-
linking with e.g. glutaric dialdehyde (Friend 1992; Santinho et al. 2002; Weinbreck et al.
2004), dehydroascorbic acid (Lamprecht et al. 2001) or transglutaminase (Cho et al. 2003)
capsules with proteins as wall material can be made more robust. However, long-term
(>days/weeks) storage in such oily capsules can only be obtained if the partition coefficient
(ko/w) of the encapsulated compound is very high (in the case of lipophilic compounds).
Examples of such compounds in foods are limited (e.g. carotenoids). Only compounds
with similarly extremely low partition coefficients (hydrophilic compounds) can be retained
in a hydrophobic wall material in an aqueous environment. However, compounds with such
low partition coefficients do not exist.
Short-term (minutes/hours) encapsulation of water-soluble compounds can be obtained
by duplex or water/oil/water (W/O/W) emulsions (Moroshita et al. 1998; Pistel and Kissel
2000). However, for long-term encapsulation in an aqueous environment this technique
is less suitable. The wall material is liquid, which is not favourable for robustness and
retention. One could use solid fats instead, but this does not solve the partitioning issue and
increases the chance of crack formation. The primary emulsifier (for the water-in-oil
emulsion) may accelerate leakage by forming reverse micelles and is generally not perceived
as natural by the consumer. In short, existing encapsulation techniques can currently not be
used for the purpose of long-term retention of water-soluble compounds in aqueous
applications, like foods.
Edible waxes could be a suitable wall material to fulfil the requirements for the above
application. Waxes are esters of long-chains alcohols and fatty acids and occur naturally
in fruits (Ritter et al. 2001), seeds (Reiter and Lorbeer 2001), petroleum (Musser and
Kilpatrick 1998) or are produced by insects (beeswax) (Patel et al. 2001). Waxes are known
for their hydrophobicity. Also their rheology and microstructure are interesting. At room
temperature wax is ductile without giving cracks (Donhowe and Fennema 1993; Scheiber
and Riederer 1996; McMillan and Darvell 2000). Also there are indications that in waxes
plate-like crystals are formed which are more efficient in hampering the diffusion of small
compounds (Donhowe and Fennema 1993; Redl et al. 1996; Scheiber and Riederer 1996).
This hypothesis is schematically explained in Figure 1. All these properties suggest a high
potential for use of wax in encapsulation of water-solubles in foods.
In pharmaceutical applications, waxes have been used as carrier for various types of drugs.
Water-soluble drugs like salbutamol sulphate have been encapsulated in wax for application
in tablets to retard release in the gastro-intestinal tract (Raghuvanshi et al. 1992). Bodmeier
et al. (1992) have also explored the use of wax for encapsulation of water solubles using
a W/O/W technique. They have found that drug release (in this case pseudoephedrine) was
much faster than from traditional polymeric microspheres, possibly because wall thicknesses
were not large enough. In some more recent applications (Quaglia et al. 2001; Uddin
et al. 2001), it was also found that for small (�20mm size) wax microcapsules containing
hydrophilic compounds (developed for dry pharmaceutical applications), �25–50% of the
compounds leak out within 30min in an aqueous environment.
Capsules prepared from typical food ingredients like vegetable oils, emulsifiers and
proteins have much quicker release. For example: Rayner et al. (2004) reported the
encapsulation of dextran blue (a very hydrophilic colourant in a W/O/W emulsion, with
sunflower oil, polyglycerol polyricinoleate (PGPR, an excellent W/O emulsifier) and
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caseinate (O/W emulsifier). They succeeded to entrap dextran blue for 16 s in the droplets
in an aqueous environment. Due to the osmotic difference, the droplets exploded and
released the colourant.
In this study, the use of waxes to encapsulate water-soluble compounds for long periods
of time (comparable to shelf life of foods: days/weeks/months) is explored. Various in vitro
experiments are described using model water-soluble compounds like colourants and model
leakage tests. Various ways of preparing the capsules are tested, among which are variations
in wall thickness. Wall thickness or particle size should be large enough to obtain sufficient
retention, while at the same time it should be small enough to prevent mouthfeel issues
(sandiness) (Tyle 1993).
To summarize, three main parameters determining leakage from the capsules will be
studied/varied: Osmotic pressure (by taking different concentrations of model compounds
to encapsulate, with varying degree of relative solubility in wall material and water), diffusion
coefficient (by taking different types of fatty wall materials) and wall thickness (by varying
particle size).
Note on legal and nutritional constraints
There are two non-technological issues concerning the application of wax: legal clearance
constraints and digestibility. Although these issues are not a primary focus of this study,
it will shortly be discussed next.
Waxes (especially those derived from petroleum) are best known as surface-finishers on
leather and wood. Insect waxes like beeswax and plant waxes like candelilla wax and
carnauba wax are permitted additives in the European Union (E901-903). However, they
are only allowed as surface-finishers on confectionery (chocolate sweets) or fruits (apples).
The status of other waxes (without E-numbers) is less clear. It can be hypothesized that
waxes originating from vegetable oils (like sunflower wax) can be regarded safe because
of a long consumption history.
Most waxes are poorly digestible by most mammals (Place 1992), which could
compromise the bioavailability of encapsulated compounds. Note that some animals like
some birds (Place 1996) can digest waxes, probably because of elevated levels of bile.
Fat
Oil
Wax
Figure 1. Schematic representation of ‘labyrinth’ effect of using wax as a wall material.
Wax encapsulation of water-soluble compounds for application in foods 731
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Materials and methods
Materials
Wall materials:
. Beeswax was provided by Fagron Nieuwerkerk a/d Ijssel, Netherlands (Cera Alba,
#7323).
. Carnauba wax was provided by Koster Keunen Holland bv. Bladel, Netherlands.
. Polyglycerol polyricinoleate (PGPR) was provided by Quest International, Zwijndrecht
(trade name: Admul WOL).
. A low-HLB (hydrophobic; low in phosphatidylcholine) lecithin: Nathin 3KE was
provided by Stern Lecithin & Soja. Hamburg, Germany.
. Fully hardened rapeseed oil (Rp70), which is a vegetable fat that melts at �70�C,
Unilever, Vlaardingen, Netherlands.
Water-soluble compounds that were encapsulated:
. Citric acid was provided by Merck, Darmstadt (Germany).
. Fluoresceine isothicyanate (‘FITC’) was provided by Janssen Chimica, Beerse, Belgium.
. Rhodamine B was provided by Aldrich, Poole, United Kingdom.
. Toluidine blue was provided by ICN Biomedicals, Aurora, United States.
. Indigo Carmine was provided by Acros, Geel, Belgium.
Others:
. Refined sunflower oil was obtained from a local grocery (‘Zonnebloemolie’).
Methods
Introduction. The two preparation procedures of the capsules and the leakage test was as
follows (see also Figure 2: ‘solid’ and ‘liquid’).
Solid preparation technique. Capsules using pure beeswax as wall material were prepared
by emulsifying water with toluidine blue colourant in water, with toluidine blue in 2M NaCl
or with 33.3% citric acid into a molten wax (temperature 65�C). Polyglycerol polyricinoleate
(PGPR) or lecithin was used as a water-in-wax emulsifer. The ratio of water phase:wax
phase was 2:3. The standard concentration PGPR or lecithin in the wax were 1.8 and 1.4%,
respectively. Whilst keeping the high temperature, this mixture was stirred using an ultra
turrax for 2min. In the last step droplets of the emulsion are dropped on a solid plate
at room temperature using a pipette. The resulting solid wax capsules usually have a size in
the order of mm or cm.
In all experiments mixing or emulsifying was done using an ultra-turrax high-shear mixer
T25 with a medium (2mm) split width and diameter of 18mm.
In order to estimate the size of the water droplets in the wax, some capsules were dissolved
in warm hexane in order to dissolve the wax. Light microscopy revealed that an emulsion
of water droplets of �1–5mm was obtained. In time no coalescence was observed, hence it is
assumed that the droplets are well-stabilized by PGPR and have the same size as those
present in the wax capsules. This means that the internal phase water droplets are a factor
1000 smaller than the capsules.
In a single experiment instead of wax Rp70 vegetable fat was used as a wall material.
In this case the temperature of the emulsion was higher than 70�C.
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Note that results did not depend a lot on the concentration of emulsifier; PGPR
concentrations can be decreased to 0.3% and lecithin to 0.6% without major increases
in leakage (results not presented). The type of emulsifier is important however; A couple
of other lecithins and citric acid esters of monoglycerides were tested (results not presented),
but besides the (low HLB) Nathin lecithin and PGPR, no emulsifier was found to give
acceptable results in terms of leakage.
A quick screening of other waxes was also performed (Carnauba-, Candelilla-, Kester
K62-, en Kesterwax K80H; all from Koster Keunen—results not shown here), with similar
results as with the standard beeswax.
Liquid preparation technique. The end result of this technique is a dispersion of small wax
particles in liquid oil (hence the reference to ‘liquid’). The basic method comprises the
following steps: A hot (90�C) and well stirred suspension of carnauba wax (which was
deemed more suitable than beeswax, because the melting temperature of carnauba wax
is higher) and dry water-soluble ingredient is slowly poured into sunflower oil while stirring
rigorously (ultra-turrax with medium head, 11 000 rpm for 30 s). The reason for using
dry colourant was to simplify the preparation process and allow higher concentrations
of ingredient to be encapsulated. The pouring is done by opening a small tap underneath
a premix vessel giving a thin jet of wax that will drop on the sunflower surface close to the
ultra-turrax head. The temperature of the sunflower oil has a temperature lower than
the melting temperature of the wax. Because of the lower temperature of the sunflower oil
the wax solidifies and is cut in small particles by the turrax. Isolation of the encapsulates
from the SF oil is done by centrifugation for �30min at a speed of 5000 rpm. The upper oil
phase is decanted and a concentrated encapsulated phase remains. Various tests
Hot wax with FI
Harvest‘Solid’
‘Liquid’
Stirrer
Hot waxwith FI
Cool
& stir
Figure 2. Schematic representation of the two preparation techniques of wax capsules used in thisstudy. The ‘solid’ technique involves the deposition of hot wax with the functional ingredient ona plate, gives particles of a size in the order of 0.1–1 cm, the ‘liquid’ technique involves the injectionof hot wax with a (model) functional ingredient (FI) in cold oil while stirring with a high-shear mixergiving particles ranging of 150–500 mm.
Wax encapsulation of water-soluble compounds for application in foods 733
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were done to optimize this procedure. The following process conditions were changed: the
rate of the addition of wax to the oil, the temperature of the SF oil (20–50�C), turrax speed
(11 000, 16 000 and 24 000 rpm), turrax time (60, 90 and 120 s) and turrax head (split
widths: 1, 2 and 2.3mm).
During the process the turrax head will become hot. The oil temperature will rise, which
will influence the formation of the particles in shape and size. To keep the temperature
constant, in some experiments the head is pre-cooled with ice. In other cases the beaker with
the SF-oil is placed in a mixture of carbon dioxide ice and methanol.
Analysis of the capsules
Solid preparation technique. The leakage test of the capsules from preparation procedure A
involves storage of the capsules in (demineralized) water at room temperature in the dark.
An amount of 2.5 g of capsules was added to 100ml water. Leakage of toluidine blue was
quantified by measuring the concentration of colourant in the outer water phase by
absorbancy (UV, 635 nm), in quartz cuvettes. Leakage of citric acid was measuring the
concentration of the acid in the outer water phase by titration with 0.1M NaOH. Standard
deviation of the leakage values is �5%.
Liquid preparation technique
. Size. The size of the encapsulates is a measure for the mouthfeel of the encapsulates.
By previous experience and by literature it is estimated that the maximum allowable size
for encapsulates in spreads is �40mm. Larger particles are felt by the tongue as
powderiness or by the teeth as sandiness. This value is similar to those reported in
literature: Heath and Prinz (1999) have found that the smallest solid particles that can be
sensed are �22mm. We have measured the size of the capsules with standard laser
diffraction analysis.
. Leakage. In most of the experiments described in this report the colouring agent Indigo
carmine was used to monitor leakage. For the leakage tests the encapsulates with dry
Indigo carmine were incorporated in a 50/50 mixture of sunflower oil and water. The
emulsions were shaken in a shaker at medium speed (150 rpm) at 40�C. These
conditions are chosen to obtain an accelerated leakage to get a quick indication of
the effectiveness of the encapsulation. The leaked Indigo carmine in the water phase of
the emulsion was monitored in the time by spectroscopy. The extinction of the sample,
analysed at 610 nm, can be compared to a calibration curve to receive the percentage
leaked Indigo carmine. Standard deviation of the leakage values is �5%.
Results and discussion
Solid preparation technique
Using the solid technique (see Materials and methods) capsules were prepared with varying
composition. The capsules were stored in water and the water was analysed for presence
of toluidine blue. The results are presented in Figure 3.
In Figure 3 it can be seen that leakage is considerably faster for the vegetable-fat based
capsules compared to wax capsules. The enrichment of the internal water phase with salt
accelerated the release. This is probably due to the osmotic pressure difference that causes
cracks in the wall material (which is problematic for application in foods). Note that
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the effect of salt is more pronounced for vegetable fat, probably because it is more
brittle than wax.
Vegetable fat and wax can also be mixed as a wall material. It was found that this increases
leakage compared to the pure capsules (results not shown), probably because the two
materials don’t mix well with each other in a crystal structure and the capsules become
more brittle.
In the remainder of this section acids will be encapsulated because of the more (realistic)
effect of the osmotic value (as mentioned above). The standard is 33.3% citric acid.
To see whether aqueous phase volume could be increased to allow more material to be
incorporated, the effect of the volume fraction of internal aqueous phase in the beeswax
phase on the leakage was tested. The results of this test are shown in Figure 4.
In Figure 4 it can be seen that the internal aqueous phase volume and the leakage are
inversely related, as expected. Surprisingly this effect is only valid for relatively large values
for the internal phase volume. It can be speculated that at relatively large phase volumes
the chances of local imperfections in the capsule wall is higher, leading to a large increase in
leakage. The inner phase volume cannot exceed 40% without significantly increasing the
leakage. Hence, this value was chosen as a standard for the remainder of this study.
Figure 5 is a plot of the effect of the osmotic properties of the outer aqueous phase on the
leakage from the standard capsules. It clearly shows that osmotic matching can reduce
0
20
40
60
80
0 10 20 30
Storage time (days)
Lea
kag
e (%
)
beeswax, lecithin, no salt
beeswax, PGPR, no salt
beeswax, PGPR, salt
fat, lecithin, no salt
fat, PGPR, no salt
fat, PGPR, salt
Figure 3. Results of leakage tests of wax or vegetable fat capsules prepared by solid technique:percentage of maximum toluidine blue leaked out as a function of time, with various emulsifier (PGPRor lecithin) and with or without addition of NaCl to the internal water phase.
0
20
40
60
80
100
0 5 10 15 20Storage time (weeks)
Lea
kag
e (%
) 60% 40%
20% 5%
Figure 4. Results of leakage tests of wax capsules prepared by solid technique: percentage ofmaximum citric acid leaked out as a function of time, at various percentages of internal aqueous phaserelative to the total volume of the capsules.
Wax encapsulation of water-soluble compounds for application in foods 735
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leakage of the encapsulated acids by at least a factor 5. The reason for this is probably that
an osmotic mismatch increases the chances of crack formation.
The effect of the concentration of encapsulated acids is shown in Figure 6. It confirms the
expectation that the osmotic mismatch between inner and outer phase is a major cause
for leakage: leakage proceeds faster at higher concentrations of citric acid in the inner phase.
Note that quite high concentrations (50%) of citric acid are required to see major differences
in leakage.
One of the major parameters of interest for application of encapsulation in foods is
texture: if the capsules are too large they can give rise to a gritty or sandy mouthfeel
(see section on the ‘liquid’ preparation technique). In Figure 7 it can be seen that there is a
considerable effect of capsule size on leakage (note that internal phase droplet size was not
varied and was �2 mm for all capsules). For 1mm size capsules leakage is unacceptable over
a typical shelf life of a couple of weeks.
It can be concluded that the capsules should be at least a few mm to obtain sufficient
retention in aqueous foods. Note that under realistic food conditions the osmotic value
0
20
40
60
80
100
0 5 10 15 20
Storage time (weeks)
Lea
kag
e (%
) 40%
60%
40%, osmotic match
60%, osmotic match
Figure 5. Results of leakage tests of wax capsules prepared by solid technique: percentage ofmaximum citric acid leaked out as a function of time, at various percentages of internal aqueous phaseand presence (‘osmotic match’) or absence of salt in the outer water phase.
0
20
40
60
0 5 10 15 20
Storage time (weeks)
Lea
kag
e (%
) 5% 10%
16.70%
50%
33.30%
Figure 6. Results of leakage tests of wax capsules prepared by solid technique: percentage ofmaximum citric acid leaked out is shown as a function of time, at various conc% of encapsulatedcitric acid. Outer phase is plain water (no acids or salts).
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of the outer phase is somewhat higher than in the current situation, which may allow for
somewhat smaller capsules.
Liquid preparation technique
The solid preparation technique described in the previous section has some disadvantages.
The fracture or detachment of the solidifying capsule from the pipette could cause capsule
imperfections and cracks and, more importantly, relatively large solid particles are obtained,
giving rise to sandy mouthfeel.
Local imperfections could be prevented, for instance by using a liquid oil wherein small
particles are created under high shear. An additional advantage is that the liquid technique
allows formation of smaller capsules. Finally the leakage is (partly) reduced by the oily
environment in which the wax capsules reside. This preparation technique is referred to as
‘liquid preparation technique’. The use of this technique is described in this section.
Results of variation of temperature and turrax speed whilst preparing carnauba wax
particles using this technique is given in Table I.
In Table II it can be seen that particle sizes obtained are larger than desired from a
mouthfeel perspective <50 mm). Apparently the turrax is not sufficient to break up the wax
particles sufficiently quickly before the wax solidifies.
Especially by introducing higher stirrer speed, the average size is reduced. Unfortunately
leakage increases with decreasing particle size: the most stable particles are still too large
(350 mm).
In Table II it can be seen that the head with the finest split width gives smallest particles.
The best conditions give particle sizes of �100 mm. However, leakage of indigo carmine of
such particles is considerable: almost a quarter has left the capsules within 4 h. Note that in
many cases the leakage does not continue to increase at the same pace. The reason for this is
probably that the break-up of the wax is not optimal: colourant particles represent the most
important structural inhomogeneities in the wax and the structure is most likely to break up
on these places. This leads to wax particles with a major portion of the colourant on the
outside of the particles, i.e. not encapsulated.
This study has shown that the liquid preparation technique allows the formation of much
smaller capsules. It is believed this technique could be optimized giving particle sizes well
below the in-mouth detection limit or giving softer particles.
0
20
40
60
80
100
120
0 5 10 15 20
Storage time (weeks)
Lea
kag
e (%
) 1 mm 1.5 mm 2 mm 3 mm 4 mm 5 mm 6 mm 7 mm 9,10 mm
Figure 7. Results of leakage tests of wax capsules prepared by solid technique: percentage ofmaximum citric acid leaked out is shown as a function of time, for various capsule sizes.
Wax encapsulation of water-soluble compounds for application in foods 737
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Conclusions
Water-soluble ingredients have been successfully encapsulated in wax using two preparation
techniques. The first technique (‘solid preparation’) leads to relatively large wax particles.
The second technique (‘liquid preparation’) leads to relatively small wax particles immersed
in vegetable oil.
On the first technique: stable encapsulation of water-soluble colourants (dissolved at low
concentration in water) has been achieved making use of beeswax and PGPR. The leakage
from the capsules, for instance of size 2mm, is �30% after 16 weeks storage in water
at room temperature. To form such capsules a minimum wax mass of 40% relative to the
total mass is needed. High amounts of salt or acids at the inside water phase causes more
leaking, probably because of the osmotic pressure difference. Osmotic matching of inner and
outer phase can lead to a dramatic reduction in leakage. Fat capsules are less suitable to
incorporate water soluble colourants. The reason could be a difference in crystal structure
(fat is less ductile and more brittle).
On the second technique: stable encapsulation of water-soluble colourants (encapsulated
in solid wax particles) has been achieved making use of carnauba wax. The leakage from the
capsules, for instance of size 250mm, is �40% after 1 week storage in water at room
temperature.
Table I. Results of the leakage tests and particle sizes for different temperatures and stirring speeds during
preparation of carnauba capsules with indigo carmine. Turrax head split width was 2mm. Stirring was applied
for 60 s.
Process conditions Leakage (%) Particles size (mm)
Experiment
Temp
(�C)
Stirring speed
turrax (rpm) 88hrs 112 hrs 448 hrs D4,3
Overall
range
1 20 11000 0 3 3 350 70–680
2 20 24000 45 43 35 220 40–450
3 35 16000 22 22 24 300 30–610
4 50 11000 7 7 13 250 10–590
5 50 24000 28 20 20 170 20–340
Table II. Results of the leakage tests and particle sizes for different turrax heads and stirring speeds and times
during preparation of carnauba capsules with indigo carmine. Temperature was 20�C.
Leakage
Experiment
Turrax head
Split width (mm)
Turrax speed
[rpm]
Turrax time
[sec] 4 hrs 28 hrs 124 hrs
Particle size
D4,3
6 2.0 24000 180 17 37 39 161
7 1.0 16000 120 23 41 36 107
8 1.0 16000 60 28 40 41 190
9 1.0 16000 60 24 35 37 150
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Acknowledgements
J. P. T. Geurtsen and D. A. M. Taal-den Boon are thanked for assisting in some of the
experiments. A. Bot is thanked for reading the manuscript.
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