determination of droplet surface temperature and drying kinetics of ...

DETERMINATION OF DROPLET SURFACE
TEMPERATURE AND DRYING KINETICS
OF PROTEIN SOLUTIONS
USING AN ULTRASONIC LEVITATOR
Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades
vorgelegt von
Eva Cornelia Wulsten
aus Delmenhorst

Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät
der Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung:
Vorsitzender der Promotionskommission:
Erstberichterstatter:
Zweitberichterstatter:
Prof. Dr. Eberhard Bänsch
Prof. Dr. Geoffrey Lee
Prof. Dr. Achim Göpferich

Für meine Eltern
und Martin

Danksagung
Die vorliegende Arbeit wurde von Oktober 2005 bis Februar 2009 am Lehrstuhl für
Pharmazeutische Technologie der Friedrich-Alexander-Universität Erlangen-Nürnberg
angefertigt.
Zuerst möchte ich Prof. Dr. Geoffrey Lee für die Möglichkeit danken, in seinem
Arbeitskreis an einem sehr interessanten und innovativen Thema arbeiten zu dürfen. Für
seine Unterstützung und Diskussionsbereitschaft bei wissenschaftlichen Fragestellungen
danke ich ihm genauso, wie für den Freiraum für eigene Ideen und selbständiges Arbeiten
und das sehr angenehme Arbeitsklima am Lehrstuhl.
Ferner danke ich der Deutschen Forschungsgemeinschaft für die finanzielle
Unterstützung der Projekte (Le 626/8-1 und Le 626/8-2).
Prof. Dr. Achim Göpferich danke ich für die Übernahme des Zweitgutachtens.
Ganz besonders danke ich für alle Unterstützung, die ich am Lehrstuhl für
Pharmazeutische Technologie für meine Arbeit bekommen habe:
• Dr. Stefan Seyferth für seine geduldige Hilfe bei Computerproblemen
• Josef Hubert für seinen großartigen Einsatz beim Umbau des Levitators, außerdem
für seine Kreativität und Hilfe bei technischen Fragestellungen
• Luise Schedl für die rasche und geduldige Anfertigung unzähliger SEM-
Aufnahmen der Partikel und Pulver
• Christiane Blaha für die schnelle Beschaffung aller Substanzen und Materialien
• Petra Neubarth für ihre Hilfe bei allen Verwaltungsangelegenheiten und die
angenehme Zusammenarbeit bei der Organisation des Studentenpraktikums
• Dr. Heiko Schiffter für die umfangreiche Einführung in mein Forschungsthema und
seine Bereitschaft, fortwährend für meine Fragen zur Verfügung zu stehen
• Dr. Alexander Mauerer für die freundliche Aufnahme zu Beginn meiner
Doktorandenzeit in seinem Labor und die umfangreiche Hilfe bei Softwarefragen
• Dr. Joanna Manegold für die freundliche Aufnahme, die mir den Einstieg am
Lehrstuhl leicht gemacht hat, und den fortwährenden freundschaftlichen Kontakt

• Dr. Andreas Ziegler und Dr. Peter Lassner für die Einführung in das Praktikum
Liquida / Dermatika und für viele gute Gespräche im „Labor Harmonie“
• Harald Pudritz, Anke Saß und Georg Straller für die gute Zusammenarbeit bei der
Studentenbetreuung, für ihre Unterstützung in verschiedenen Bereichen meiner
Arbeit und außerdem für die vielen schönen Abende in Erlangen, Nürnberg und
Umgebung und bei den gemeinsam besuchten Weiterbildungsseminaren
• den Pharmaziestudentinnen Silvia Meffert und Adriane Giraud für ihren Einsatz bei
meiner Arbeit mit den Proteinen
Auch bei meinen Kolleginnen und Kollegen während meiner Doktorandenzeit Jakob
Beirowski, Dr. Jürgen Bögelein, Dr. Henning Gieseler, Silja von Graberg, Eva Meister,
Simone Reismann, Susanne Rutzinger, Stefan Schneid, Sebastian Vonhoff und Dr.
Henning Wegner möchte ich mich für ihre Unterstützung meiner Arbeit und das
angenehme Arbeitsklima am Lehrstuhl bedanken.
Allen meinen Freunden danke ich sehr für ihre Begleitung. Ganz besonderer Dank
an Dr. Kerstin und Dirk Schöttler für das Korrekturlesen dieser Arbeit.
Meinen Eltern gilt mein besonderer Dank für ihre liebevolle Unterstützung. Ohne
ihre persönliche sowie finanzielle Unterstützung wäre mir dieser Weg nicht möglich
gewesen. Meinem Bruder Jörg und Christine danke ich für die Unterstützung während
meines Studiums und in meiner Doktorandenzeit - nicht nur bei diversen Umzügen - und
für das Korrekturlesen dieser Arbeit.
Ganz besonders danke ich meinem Mann Martin, der mir mit seiner Liebe und
Unterstützung während der Doktorandenzeit eine sehr große Hilfe war. Angefangen bei
seiner Bereitschaft, mit mir nach Nürnberg zu gehen, hat er mich mit sehr viel Motivation
unterstützt. Für sein Interesse an meinen Versuchen, seine Hilfe bei Softwareproblemen
und für das Korrekturlesen dieser Arbeit bin ich ihm sehr dankbar.

Parts of this thesis have already been presented or published:
I. E. Wulsten and G. Lee (2008): “Drying behavior of levitated trehalose
microdroplets: surface temperature measurements and drying kinetics” 6 th World
Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology in
Barcelona (Spain)
II.
E. Wulsten and G. Lee (2008): “Surface temperature of acoustically levitated water
microdroplets measured using infra-red thermography” Chemical Engineering
Science 2008, 63 (22), 5420-5424
III.
E. Wulsten and G. Lee (2008): “Residual activity of carbonic anhydrase depending
on drying time using an ultrasonic levitator” DPhG Annual Meeting in Bonn
(Germany)
VI.
E. Wulsten, F. Kiekens, F. van Dycke, J. Voorspoels and G. Lee: “Applications of
levitated single droplet drying in process and formulation development. Case study
with itraconazole dried in binary organic solvent mixtures” International Journal of
Pharmaceutics 2009, in press

Contents I
Table of contents
1 Introduction ................................................................................................................ 1
2 Single droplet drying .................................................................................................. 4
2.1 A short history of single droplet drying ................................................................. 4
2.2 Heat transfer ........................................................................................................... 6
2.2.1 Conductive heat transfer .................................................................................. 6
2.2.2 Convective heat transfer ................................................................................... 6
2.2.3 Radiative heat transfer ...................................................................................... 7
2.3 Diffusion ............................................................................................................... 11
2.3.1 Fick’s first law of diffusion ............................................................................ 12
2.3.2 Diffusion coefficients for gases ..................................................................... 12
2.3.3 Convective mass transfer ............................................................................... 13
2.4 Mass transfer theories ........................................................................................... 13
2.5 Evaporation of pure solvent droplets.................................................................... 14
2.5.1 Evaporation of a single droplet ...................................................................... 14
2.5.2 The d²-law ...................................................................................................... 15
2.5.3 Diffusion-controlled evaporation of single droplets ...................................... 16
2.5.4 Evaporation of droplets containing solvent mixtures ..................................... 16
2.6 Evaporation of solution and suspension droplets ................................................. 17
2.6.1 Drying stages of solution and suspension droplets ........................................ 17
2.6.2 Particle formation ........................................................................................... 20
2.6.3 Models for the drying of single droplets containing solids ............................ 22
3 Acoustic levitation .................................................................................................... 24
3.1 History and application of acoustic levitation ...................................................... 24
3.2 A short introduction to acoustics .......................................................................... 25
3.3 Levitation forces ................................................................................................... 27
3.3.1 Standing acoustic wave .................................................................................. 27
3.3.2 Interaction of ultrasonic wave and droplet ..................................................... 28
3.4 Acoustic streaming ............................................................................................... 29
3.4.1 Inner acoustic streaming ................................................................................ 29

Contents II
3.4.2 Outer acoustic streaming ................................................................................ 30
3.5 Vertical position of the levitated droplet .............................................................. 31
3.6 Temperature of the levitation system ................................................................... 32
3.7 Single droplet drying using an acoustic levitator ................................................. 32
3.7.1 Pure solvent droplets ...................................................................................... 32
3.7.2 Solution and suspension droplets ................................................................... 35
4 Materials and Methods ............................................................................................ 37
4.1 Materials ............................................................................................................... 37
4.1.1 Proteins ........................................................................................................... 37
4.1.1.1 Carbonic anhydrase .................................................................................. 37
4.1.1.2 L-Lactic dehydrogenase ........................................................................... 39
4.1.1.3 Trypsinogen and Trypsin ......................................................................... 40
4.1.2 Itraconazole .................................................................................................... 41
4.1.3 Excipients and reagents .................................................................................. 42
4.2 Methods ................................................................................................................ 44
4.2.1 Acoustic levitation ......................................................................................... 44
4.2.2 Microdispensing system ................................................................................. 48
4.2.3 Droplet size measurements ............................................................................ 51
4.2.4 Droplet surface temperature measurements ................................................... 51
4.2.5 Spray drying ................................................................................................... 52
4.2.6 Scanning electron microscopy ....................................................................... 53
4.2.7 Enzyme activity assay of carbonic anhydrase ................................................ 53
4.2.8 Enzyme activity assay of L-lactic dehydrogenase ......................................... 54
4.2.9 Enzyme activity assay of trypsinogen ............................................................ 55
5 Results and Discussion ............................................................................................. 57
5.1 Preliminary experiments using the IR-camera ..................................................... 57
5.1.1 Heating-up of the levitation system ............................................................... 57
5.1.2 Influence of droplet size on the surface temperature measurement ............... 58
5.1.3 Determination of emissivities ........................................................................ 64
5.2 Evaporation of pure solvent droplets.................................................................... 67
5.2.1 Evaporation of pure water droplets ................................................................ 67

Contents III
5.2.1.1 Influences of the initial droplet size on the evaporation process ............. 67
5.2.1.2 Influence of the drying air stream on the evaporation process ................ 84
5.2.2 Evaporation of pure organic solvent droplets ................................................ 96
5.2.3 Evaporation of droplets containing solvent mixtures ................................... 103
5.3 Evaporation of excipient solution droplets ......................................................... 110
5.3.1 Trehalose solution droplets .......................................................................... 110
5.3.2 Mannitol solution droplets ........................................................................... 121
5.3.3 Sucrose solution droplets ............................................................................. 133
5.3.4 Copolyvidone and HPMC solution droplets ................................................ 136
5.4 Itraconazole formulation experiments ................................................................ 141
5.5 Drying of protein solutions ................................................................................ 148
5.5.1 Bovine carbonic anhydrase (bCA) droplets ................................................. 148
5.5.1.1 bCA-trehalose 10 % (w/v) total solids formulations ............................. 151
5.5.1.2 bCA 10 % (w/v) plus trehalose formulations ......................................... 157
5.5.2 L-Lactic dehydrogenase (LDH) droplets ..................................................... 161
5.5.2.1 LDH-trehalose 10 % (w/v) total solids formulations ............................. 163
5.5.2.2 LDH 10 % (w/v) plus trehalose formulations ........................................ 170
5.5.3 Trypsinogen droplets .................................................................................... 174
6 Summary and Conclusions .................................................................................... 177
7 Zusammenfassung .................................................................................................. 182
8 References ............................................................................................................... 188

Abbreviations IV
List of abbreviations
Capital letters
area
coefficient (Equation 3.16, Table 5.3)
, , liquid parameters (Equation 3.15, Table 3.2)
pressure amplitude at the source surface
effective pressure amplitude of the incident acoustic field
Spalding transfer number
binary diffusion coefficient
binary diffusion coefficient for water
horizontal diameter
vertical diameter
radial acoustic levitation force
axial acoustic levitation force
relative humidity
latent heat of evaporation
latent heat of evaporation for water
molar flux of component A
bulk modulus elasticity
constant of the acoustic field
sound pressure level
distance of the reflector from the transducer
molar mass
molar mass of a gas
molar mass of a liquid
molar mass of water
heat energy
heat flow
,
heat flux in the liquid / surrounding gas (Figure 2.4)
ideal gas constant

Abbreviations V
Reynolds number
surface area
Schmidt number
Sherwood number
temperature
experimentally-measured droplet surface temperature
droplet surface temperature calculated according to Yarin et al. [1999]
wet-bulb temperature
boiling point
critical temperature
temperature of the drying air
temperature of the bulk fluid
surface temperature
temperature at infinity
volume
emittance
emittance of a black body
emittance of a black body over the wavelength
mass fraction of the vapour
mass fraction of the vapour at infinity
characteristic acoustic impedance
Small letters
constants (Equation 3.12, Table 3.1)
concentration
, constants (Equation 2.5)
droplet diameter (calculated from a surface equivalent sphere)
frequency
gravitational acceleration
heat transfer coefficient
thermal conductivity
thermal conductivity of air

Abbreviations VI
wave number
mass flux of liquid
mass flux of the droplet material (Figure 2.4)
coefficient (Equation 3.16, Table 5.3)
pressure
0
∆
reference effective sound pressure value
partial vapour pressure at the surface
saturation vapour pressure
partial vapour pressure at infinity
sound pressure
effective sound pressure
heat flux
droplet radius (calculated from a surface equivalent sphere)
droplet radius at point of time t (calculated from a surface equivalent sphere)
initial droplet radius (calculated from a surface equivalent sphere)
measured horizontal droplet radius
measured vertical droplet radius
time
air velocity
particle velocity
sound velocity
length
distance centre of droplet mass to next upper pressure node
Small Greek letters
absorptivity
evaporation coefficient (calculated from droplet radius)
adiabatic index
thickness of layer / wall
diffusion boundary layer
acoustic boundary layer
emissivity

Abbreviations VII
λ
υ
diffusivity of drying gas
wavelength
wavelength with max. emittance of a black body
dynamic viscosity
particle displacement
reflectivity
density
density gas
density liquid
Stefan-Boltzmann constant
transmissivity
diffusion volume
angular frequency
Subscripts
A, B components
1, 2 numbering
Expressions
bSA bovine serum albumin
bCA bovine carbonic anhydrase
BAEE N α -benzoyl-L-arginine ethyl ester hydrochloride
CEM controlled evaporation mixer
CCD charged coupled device
Diff diffusion model
HF high frequency
HPMC hydroxypropylmethylcellulose
IR infrared
LDH L-lactic dehydrogenase
MCT mercury cadmium tellurium
MWCO molecular weight cut off
p.a. pro analysi (for analysis)

Abbreviations VIII
PCR
PNPA
RM
SPL
Treh
UV
VIS
polymerase chain reaction
p-nitrophenyl acetate
Ranz-Marshall model
sound pressure level
trehalose
ultraviolet
visible

Introduction 1
1 Introduction
The process of drying is an important step in the production of various pharmaceutical
dosage forms and has a wide influence on the quality and stability of the product. During
the drying process liquids can be thermally removed from a product by evaporation. In this
process heat transfer takes place via conduction, convection or radiation using various
types of dryers like drying ovens, infrared tunnel dryers, fluid bed dryers or spray dryers.
Spray drying has a special role in industrial drying technology, since it involves drying of
liquid feedstocks to powders with control over the final particle morphology and functional
powder properties [Masters 2002]. As a result spray drying is an appropriate method to
produce powders for various kinds of pharmaceutical applications.
An increasing number of peptides and proteins is now available for the therapy of
diseases like cancer, immune mediated or metabolic diseases and for carrying out
immunizations. One quarter of all drugs containing new active ingredients approved for
Germany in 2007 were biopharmaceuticals, and there are currently more than 350
biopharmaceuticals in clinical trials [BCG 2008]. Protein formulations are often developed
for parenteral administration. Due to the fact that proteins are often instable in aqueous
solution, it is not possible in most cases to achieve a sufficient shelf life stability. In this
case the protein is stored in a dry form and dissolved directly before use. Spray drying is a
potential alternative to lyophilisation for the production of parenteral powders for this use
[Mumenthaler et al. 1994]. Protein loaded powders are furthermore used for needle-free
powder injection. Vaccines for example can be efficiently delivered to the epidermis for
epidermal powder immunization [Dean and Chen 2004]. Inhalation is an appropriate way
to apply a protein not only for local treatment of respiratory diseases. An approved protein
formulation for pulmonary delivery as an aerosol with local effect is recombinant human
DNase (Pulmozyme ® ) for treatment of mucoviscidosis [Roche 2009]. For systemic
delivery of a protein by absorption via the lung inhalation is also useful as it is an
attractive, pain-free and self-administrable delivery method promoting a good compliance
by the patient. The lung, where some 300 million alveoli constitute a capillarized area of
around 100 m² [Almer et al. 2002], offers an enormous surface area with good blood
supply and a thin absorption mucosal membrane in the distal lung of 0.1 - 0.2 μm [Yu and
Chien 1997] for high absorption of the protein. Spray drying is an efficient method to

Introduction 2
produce powders loaded with various peptides and proteins for pulmonary delivery
containing particles in the size of 0.5 - 5 μm for optimal deep deposition in the respiratory
tract.
Spray drying offers various possibilities to influence the particle size and flow
properties via optimization of the drying conditions or the formulation ingredients. In
formulation development for a protein the risk of inactivation is often the main problem.
Chemical and physical instability of the protein can occur, the latter refers to a change in
the secondary, tertiary, or quaternary structure of a protein [Banga 1995]. Process stress
like thermal stress, shear stress associated with atomization and especially air-water
interfacial stress, has influence on protein stability during spray drying [Maa and
Prestrelski 2000]. Unfolding of the protein leads to adsorption, aggregation and
precipitation [Manning et al. 1989] and the activity of the protein decreases. This makes
the addition of stabilizing excipients like non-reducing sugars, amino acids or surfactants
in the spray drying formulation necessary [Adler and Lee 1999; Andya et al. 1999; Maury
et al. 2005b].
Single droplet drying experiments provide a possibility to analyze formulation
aspects with a very small amount of substance and are therefore very cost-efficient. It is
possible to investigate drying kinetics and particle formation during the whole drying
process of a sample droplet at different ambient conditions. During or after drying the
sample can be removed from the acoustic field for further analysis. In an ultrasonic
levitator a liquid or solid sample with a diameter of about 50 - 4000 μm [Kastner 2001] can
be levitated in the pressure node of a standing acoustic wave without heat transfer by a
thermal conducting holding device.
The aim of this thesis is the analysis of the drying kinetics, droplet surface
temperature and particle formation of acoustically-levitated sample droplets containing
proteins to determine the drying behaviour of the solution. The influence of the drying
process on the proteins is analyzed by enzymatic activity measurement of the protein
particles.
In the first part of this work, an infrared camera is established in an existing
levitation setup for simultaneous droplet surface temperature and size measurements. Pure
solvents and solvent mixtures are analyzed at different temperature and relative humidity
conditions. For water droplets the influence of different initial droplet diameter using a
micro-dispensing system and the influence of drying air rate is investigated. The suitability

Introduction 3
of droplet surface temperature measurements to detect the critical point and to monitor the
drying process in single droplet drying experiments is analyzed. In the second part
solvent / excipient formulations at different ambient conditions and solid contents are
examined. In the third part drying kinetics and droplet surface temperature of protein
solutions and protein / excipient formulations are examined and the enzymatic activity is
measured at the end of the drying process. Results from spray drying experiments are
compared to the results of the levitation experiments in terms of particle shape and protein
activity. The time-dependent protein activity during the drying process is analyzed.

Single droplet drying 4
2 Single droplet drying
2.1 A short history of single droplet drying
Since 1952 when Ranz and Marshall investigated the drying of a single droplet containing
dissolved and suspended solids, single droplet experiments have been used in the study of
particle morphology. This method has been criticized as being unrealistic for spray drying,
because the diameters are an order of magnitude larger than spray droplets and the droplets
are not allowed to rotate freely while drying. Furthermore the droplet is suspended on a
thermal conducting holding device, which is also a site for vapour bubble nucleation within
the droplet and may cause deformation of the droplet in later drying process. The method,
however, is still in use, because larger droplets are easier to handle and to observe [Lin and
Gentry 2003]. Table 2.1 gives a short review of single droplet drying experiments
containing particle formation and morphology studies [Lin and Gentry 2003; Schiffter
2006].
Charlesworth and Marshall [1960] investigated the drying rates of inorganic salts.
They analyzed the crust structure and appearance change in drying droplets and also
monitored droplet weight and temperature during drying process. Organic substances were
analyzed by El-Sayed et al. [1990]. Walton and Mumford [1999] carried out several
experiments with organic and inorganic substances as well as multicomponent mixtures.
They identified three distinct categories of particle morphology: crystalline, skin forming
and agglomerate. They found that although they are material-specific, each category is
evidence of a characteristic drying behaviour dependent on feed concentration, the degree
of feed aeration, and drying temperature. Lin and Gentry [2003] performed single droplet
drying studies with several inorganic salts and analyzed particle morphology. Low drying
temperature and a material with high latent heat of crystallization (for example
endothermic crystallization) were found to be favourable for small, dense, and regularlyshaped
particles. Materials that formed an elastic shell structure led to hollow particle
formation, whereas materials with high solubility led to small, dense and irregularlyshaped
particles. They found that a high initial solute concentration was favourable for the
formation of large dense particles.

Single droplet drying 5
Table 2.1: Selected single droplet drying studies [Lin and Gentry 2003; Schiffter
2006]
Investigators
Suspending method
Temperature
Test material and
Year
measurement
initial drop size
Ranz and Marshall
1952
Glass capillary
80 μm
Manganconstantan
thermocouple
Sodium chloride,
ammonium nitrate
0.6 - 11.0 mm
Charlesworth and
Marshall
1960
340 μm glass
filament, drop
formed from a
microburette
Thermoelement
formed from
manganin and
constantan wired
connected to a
recording
potentiometer
Sodium sulphate,
potassium sulphate,
copper sulphate,
ammonium nitrate
calcium chloride,
sodium acetate and
coffee extract
1.3 - 1.8 mm
El-Sayed, Wallack
and King
1990
Annealed glass with
a glass bead on the
tip
0.12 mm Type-Ethermocouple
Sucrose, maltodextrin,
coffee extract, skim
milk
0.38 - 1.5 mm
Walton and
Mumford
1999
Rotating glass
filament in a wind
tunnel
Thermocouple
Different inorganic
and organic salts,
gelatine, semi-instant
skimmed milk, codried
egg and
skimmed milk
powder, anionic
detergents
1.0 - 2.0 mm
Lin and Gentry
2003
Capillary filament
inside a drop holder
Thermocouple
Calcium acetate,
sodium acetate,
potassium carbonate,
sodium chloride,
ammonium chloride,
lithium manganous
nitrate, barium
aliumino boro silicate
1.2 - 1.3 mm

Single droplet drying 6
2.2 Heat transfer
A temperature gradient in a substance leads to heat flow from the part at higher
temperature to that at lower temperature. This process takes place in solid materials as well
as in liquids or gases [Eckert 1966]. Heat transfer can be divided in substance-bound heat
transfer via conduction and convection, and substance-unbound heat transfer via thermal
radiation.
2.2.1 Conductive heat transfer
Heat transfer via conduction occurs upon interaction of atoms and molecules, where
energy is transferred from the more energetic to the less energetic molecules. In liquids and
gases the faster molecules of the warmer regions will be decelerated and the molecules of
the colder regions will be accelerated, which leads to temperature equalization over all
parts of the liquid or gas. Heat transfer in solids that are poor conductors takes place via
longitudinal oscillations of the atoms, whereas in metallic solids thermal conduction results
from the motion of free electrons [Bosnjakovic 1997]. The basic equation used to express
heat flow by conduction is Fourier’s law:
Equation 2.1
· ·
·
with the heat energy , the thermal conductivity (specific for the material) , the heat
transfer area , the temperature difference across the material , the material
thickness , and the time . Considering the expressions for heat flow ⁄ and for
heat flux ⁄ , Equation 2.1 becomes:
Equation 2.2
·
·
The material thickness is and the negative algebraic sign recognizes that heat flow takes
place from warmer to colder regions.
2.2.2 Convective heat transfer
In liquids and gases thermal energy can be transported from one region to another by fluid
(liquid or gas) flow. Parts of the fluid move between regions of different temperature in the

Single droplet drying 7
substance and in this process they carry thermal energy. Convective heat transfer between
a surface and a moving fluid at a different temperature is a combination of diffusion and
bulk motion of molecules. Near the surface the fluid velocity is low and diffusion
dominates. Away from the surface bulk motion increases the influence and dominates
[Engineering Toolbox 2009]. Convection is closely associated with fluid mechanics.
Convective heat transfer takes the form of either natural (also known as free) or
forced (also known as assisted) convection, depending on the forces used to create
convection currents. In natural convection parts of the fluid heat up at the warmer surface
and the density of the liquid in this region decreases. The fluid will rise and be replaced by
cooler fluid that will also heat and rise generating natural convection. If the currents are set
in motion by the action of a mechanical device such as a pump or agitator, the flow is
independent of density gradients and is called forced convection [Bosnjakovic 1997;
McCabe et al. 2005].
The relation describing heat transfer by convection is Newton’s law of cooling:
Equation 2.3
·
which expresses how the heat flux is proportional to the temperature difference between
the surface and the bulk fluid . The proportionality coefficient is the heat transfer
coefficient , that is dependent on the type of media, its flow properties velocity or
viscosity, and also other flow and temperature dependent properties [Engineering Toolbox
2009]. Convective heat transfer is dependent on the intensity of bulk fluid motion, and thus
fluid mechanics play a decisive role for convective heat calculations [Bosnjakovic 1997].
2.2.3 Radiative heat transfer
Heat transfer through radiation takes place in the form of electromagnetic waves emitted
by an object and differs from conduction and convection, which both are substance-bound.
The radiation emitted by a body results from the thermal agitation of its molecules and can
spread even in vacuum due to its substance independence. In contrast to conductive and
convective heat transfer, thermal radiation is able to pass regions of lower temperature for
heat transfer between objects [Bosnjakovic 1997]. When the electromagnetic waves reach
an object, they will be transmitted, reflected or absorbed, whereby only the absorbed
energy appears quantitatively as heat.

Single droplet drying 8
Electromagnetic radiation covers a wide band of wavelengths, from short cosmic rays
having wavelengths of about 10 -11 cm to longwave broadcasting waves having lengths of
1000 m or more. The portion of the electromagnetic spectrum that is important for heat
flow by radiation is in the wavelength range between 0.1 and 100 μm. At temperatures
above about 500 °C heat radiation in the visible spectrum becomes substantial [McCabe et
al. 2005]. The infrared spectrum is attached to the visible spectrum and includes
wavelength from 780 nm up to 1 mm. For technical temperature measurement the
wavelength range from 0.8 - 20 μm is used. It can be divided (based on the response of
various detectors) into short-wave infrared (SWIR) from 1 - 3 μm, mid-wave infrared
(MWIR) from 3 - 5 μm, and long-wave infrared (LWIR) from 8 - 12 μm.
A black body is an ideal emitter, which has the maximum attainable emissive
power at any given temperature and absorbs all incoming radiation. The basic relationship
for black body radiation is the Stefan-Boltzmann law, which states that the emittance (rate
of radiant energy emission per unit area, equal to heat flux ) of a black body
is proportional to the fourth power of the absolute temperature:
Equation 2.4
·
using the Stefan-Boltzmann constant = 5.669·10 -12 W·cm -2·K -4 [McCabe et al. 2005;
Schuster and Kolobrodov 2004].
The distribution of energy in the spectrum of a black body is given by Planck’s law,
where the emittance of a black body over the wavelength λ ⁄ λ is:
Equation 2.5
λ
λ ·
1
⁄ · 1
with the constants = 3.74·10 -16 W·m 2 and = 1.44·10 -2 K·m, the wavelength of radiation
and the absolute temperature . Planck’s law at temperatures of 300, 800, 2500 and
5500 K is shown graphically in Figure 2.1 by curves of the same form without intersection.
Note that the chart is double logarithmic and therefore the real proportions are not clearly
visible. In the wavelength range of 8 - 14 μm for example the relation of the emittance of
the sun (5500 K curve) to that of a black body at 300 K is 470:1 [Schuster and Kolobrodov
2004].

Single droplet drying 9
Figure 2.1: Planck’s law according to Schuster and Kolobrodov [2004]
The curves in Figure 2.1 also show that the wavelength at which a black body has its
maximum emittance changes with the temperature of the body. The location of the
maximum can be calculated by Wien’s displacement law:
Equation 2.6
λ
2898 · μ
The lower the temperature of the object, the higher is the wavelength for the maximum
emittance [Schuster and Kolobrodov 2004].
Black bodies do not exist in nature, though its characteristics are approximated by a
hole in a box filled with highly absorptive material. For a real body the incident radiation is
partly reflected, absorbed or transmitted [Engineering Toolbox 2009]. The fraction of the
radiation falling on a body that is reflected is called the reflectivity , the fraction that is
absorbed is called absorptivity and the fraction that is transmitted is called the
transmissivity [McCabe et al. 2005]. All parts are wavelength-dependent, and the sum of
all fractions must be unity:

Single droplet drying 10
Equation 2.7
αλ λτλ 1
According to Kirchhoff’s law for a body in thermal equilibrium the absorptivity is equal to
the emissivity and the equation can be written as:
Equation 2.8
ελ λτλ 1
Solids and liquids are usually not transparent for infrared radiation, so that τ 0 and the
focus is on the emissivity and the reflectivity of the object.
The Stefan-Boltzmann law for real bodies uses the emissivity of the object:
Equation 2.9
ε · ·
The emissivity is defined as the emittance of an object in comparison to the emittance of
a black body:
Equation 2.10
ε
with ε 1 for a real body. A black body absorbs all radiation incident upon it, so that
α ε 1. The emissivity of an object must be determined experimentally. It depends on
the material, the wavelength, the temperature of the object, the surface texture, and the
direction of the emitted beam. For a grey body the emissivity is unique for all wavelengths.
If the emissivity is wavelength dependent, the object is called a selective radiator. Figure
2.2 shows the idealized course of the emissivity. For a black body 1 for all
wavelengths (curve 1). If 0 and 1, the substance is an ideal mirror, because it
does not absorb radiation but reflects all of it (curve 2). Curve 2 is also characteristic for an
ideal window in the case of 0 and 1, where all incident radiation is transmitted. A
grey body has a constant emissivity at all wavelength const (curve 3), and for a
selective radiator the emissivity depends on the wavelength of the measurement (curve 4).
The emissivity for various substances can be found in the literature, keeping in
mind that the emissivity is dependent on many parameters. For a few substances the
emissivity is shown in Table 2.2.

Single droplet drying 11
Figure 2.2: Emissivity depending on wavelength [Schuster and Kolobrodov 2004]
Table 2.2: Emissivity of selected substances (20 °C, , angle of
radiation ± 20°) [Schuster and Kolobrodov 2004]
Material
Emissivity ε
Iron (unmachined) 0.74
Iron (rusty) 0.69
Wooden board 0.96
Water 0.96
Snow 0.85
Human skin 0.98
2.3 Diffusion
Diffusion is the movement, under the influence of a physical stimulus, of an individual
component through a mixture. The most common cause of diffusion is a concentration
gradient of the diffusing component, which tends to move the component in such a
direction as to equalize concentrations and remove the gradient. In many mass-transfer
operations the steady-state flux of the diffusing component takes place, where the gradient
is maintained by constantly supplying the diffusing component to the high-concentration
end of the gradient and removing it at the low-concentration end [McCabe et al. 2005].

Single droplet drying 12
2.3.1 Fick’s first law of diffusion
For one-dimensional steady-state diffusion the following general equation, Fick’s first law
of diffusion, for the molar flux of component A, (similar to heat flux ), is given by:
Equation 2.11
·
using the binary diffusion coefficient and the concentration gradient ⁄ for a
binary mixture [McCabe et al. 2005].
2.3.2 Diffusion coefficients for gases
Diffusivities are best estimated by experimental measurements, but such information is not
always available for the system of interest. In this case the values can be estimated from
published correlations. Binary gas-phase diffusion coefficients in cm²/s at a given
temperature in °C and pressure in atm can be estimated by the method described by
Fuller et al. in 1966:
Equation 2.12
1.00 · 10 . · ·1⁄
1⁄
·∑ ⁄
⁄
∑
⁄
and are the molar masses in g/mol of the substances and ∑ and ∑ are the
summed atomic diffusion volumes given in Table 2.3 [Fuller et al. 1966].
Table 2.3: Special atomic diffusion volumes [Fuller et al. 1966]
Atom / substance
Diffusion volume
C 16.5
H 1.98
O 5.48
N 2 17.9
Air 20.1
H 2 O 12.7

Single droplet drying 13
2.3.3 Convective mass transfer
Mass transfer from the surface to a moving fluid or between immiscible fluids depends on
the material property of the components and the flow properties. As in heat transfer by
convection, convective mass transfer in a current can be caused by density differences due
to a gradient in temperature or concentration leading to free convection. It can also be
caused by external influences by a pump for example leading to forced convection [Baehr
and Stephan 2004]. Equimolar diffusion (both components move) and unimolar diffusion
(where component A diffuses through a non-diffusing component B) have to be
differentiated. Figure 2.3 gives a comparison of the concentration gradient for equimolar
and unimolar diffusion. In Figure 2.3 (a) components A and B are diffusing at same molar
rates in opposite directions. In Figure 2.3 (b) component A is diffusing and component B is
stationary with respect to interface. For a broad summary of calculations for different
convective mass transfer settings see Schiffter [2006], McCabe et al. [2005] or Baehr and
Stephan [2004].
(a)
Figure 2.3: Concentration gradient for (a) equimolar and (b) unimolar diffusion
[McCabe et al. 2005]
2.4 Mass transfer theories
Several mass transfer theories are used to describe mass transfer and to calculate mass
transfer coefficients. The film theory is based on a study by Lewis and Whitman in 1924.
(b)

Single droplet drying 14
Mass transfer from a surface to a moving fluid takes place in a very thin fluid film close to
the surface. Concentrations and velocities are allowed to change in the direction
perpendicular to the surface only, not with time or in the other two dimensions of space
[Baehr and Stephan 2004]. The film theory is often used as a basis for complex problems
of multicomponent diffusion or diffusion plus chemical reaction. The boundary layer
theory is based on the assumption that mass transfer takes place in a thin boundary layer
near a surface where the fluid is in laminar flow [McCabe et al. 2005]. In contrast to film
theory, concentrations and velocities are allowed to change in three dimensions in space. In
film theory and boundary layer theory a steady-state mass transfer is assumed. Both
theories are not applicable if concentrations change with time [Baehr and Stephan 2004].
The penetration theory makes use of the expression for the transient rate of
diffusion into a relatively thick mass of fluid with a constant concentration at the surface.
The theory was first used by Higbie in 1935 for the situation of gas absorption in a liquid,
showing that diffusing molecules will not reach the other side of a thin layer if the contact
time is short [McCabe et al. 2005]. In 1951 Danckwerts developed the surface renewal
theory as an extension of the penetration theory. Higbie stated that all elements of the fluid
have the same contact time, whereas Danckwerts expected that fluid elements have
different contact times so that elements of fluid at a transfer surface are randomly replaced
after some time with fresh fluid from the bulk stream [Baehr and Stephan 2004; McCabe et
al. 2005].
The two film theory, proposed by Whitman in 1923, recognizes that in many
separation processes material must diffuse from one phase into another, and the rates of
diffusion in both phases affect the overall rate of mass transfer. In the two film theory
equilibrium is assumed at the interface, and the resistances to mass transfer in the two
phases are added to give an overall resistance [McCabe et al. 2005].
2.5 Evaporation of pure solvent droplets
2.5.1 Evaporation of a single droplet
A single droplet with radius and temperature is brought into a gas phase with
temperature and mass fraction of the vapour of the droplet material. Figure 2.4
shows two possibilities for mass flux, heat flux and temperature profile in the evaporating
droplet. In the first example (Figure 2.4 (a)) the temperature of the droplet and the

Single droplet drying 15
ambiance are initially the same. An evaporation process starts and a mass flux of
droplet material will leave the droplet. The latent heat of evaporation is taken from the
inner energy of the droplet liquid, so that the temperature of the droplet will decrease and a
temperature gradient will be established in the droplet and in the surrounding gas. The
temperature inside the droplet is not constant.
and
represent the heat flux in the
droplet and in the surrounding gas towards the droplet surface. In Figure 2.4 (b) the
ambient temperature is higher than the boiling point of the droplet liquid. Here the latent
heat of evaporation is delivered by the heat flux
, which is directed towards the
droplet. The droplet is heated until the heat that is delivered to the surface is completely
needed for the latent heat of evaporation. Here the droplet stabilizes at a temperature below
the boiling point [Frohn and Roth 2000].
(a)
(b)
Figure 2.4: Mass flux, heat fluxes and radial temperature profiles in evaporating
droplets, (a) temperature of the droplet and the ambience are initially the same, (b)
ambient temperature is higher than the boiling point of the droplet liquid [Frohn and
Roth 2000]
2.5.2 The d²-law
Droplet evaporation at the wet-bulb temperature shows a linear decrease of its squared
radius with time as described by the d²-law:
Equation 2.13 0 ·
where 0 is the initial droplet radius, the time and is the evaporation coefficient
given by:

Single droplet drying 16
Equation 2.14
2· ·
·ln1
The density of the droplet liquid is , the density of the gas at the droplet surface is ,
the diffusion coefficient is and the Spalding transfer number is [Frohn and Roth
2000].
2.5.3 Diffusion-controlled evaporation of single droplets
In the case that a droplet is surrounded by an atmosphere that has approximately the same
temperature as the droplet liquid, and further this temperature is low in comparison with
the boiling point of the droplet liquid, the vaporization process is dominated by diffusion
processes in the vapour phase [Frohn and Roth 2000]. This situation can be described by:
Equation 2.15
0 2· ·
·
·
·
with the molar mass of the liquid , the ideal gas constant and the partial vapour
pressure at the droplet surface and at infinity . The evaporation coefficient is
given by:
Equation 2.16
2· ·
·
·
2.5.4 Evaporation of droplets containing solvent mixtures
For the evaporation of a single droplet containing different solvents, the decrease in the
radius squared with time is not linear but depends on the mass flux of the components. For
a binary solvent mixture, for example, the component with the higher mass flux will
predominate the evaporation in the first drying stage. As soon as most of the component
has evaporated, the second component will predominate the evaporation process and the
graph will show a slower decrease of the squared radius in the second stage [Yarin et al.
2002]. The droplet surface temperature changes during the drying process, when the
second component starts to dominate evaporation [Tuckermann et al. 2005].

Single droplet drying 17
2.6 Evaporation of solution and suspension droplets
2.6.1 Drying stages of solution and suspension droplets
The drying process of a solution or suspension droplet can be divided into two stages for
non-hygroscopic materials and three stages for hygroscopic materials. When the droplet
comes in contact with the hot drying air, there is a very short phase for heating-up of the
droplet. Then in the first drying stage, solvent evaporation takes place from the
completely wetted droplet surface comparable to the evaporation of pure solvent droplets.
The limiting factor for evaporation is the mass transfer from the droplet surface to the
drying air, and therefore the change in droplet diameter can be calculated using the d²-law
as for pure solvent droplets [Grassmann et al. 1997; Kastner 2001]. Diffusion of moisture
in liquid form from inside the droplet maintains saturated surface conditions. Capillary and
diffusional mechanisms are involved. Their dominance depends upon the nature of the
solid in the droplet as a solution or suspension. As long as the saturated surface conditions
last, evaporation takes place at a constant rate. For this reason the first drying stage is also
called the constant rate period. In this stage the evaporation rate is the highest during
droplet drying. Figure 2.5 shows the evaporation rate depending on drying time. The first
drying stage is represented by curve A-B. The droplet surface temperature approximates
the wet-bulb temperature and remains constant in the first drying stage. Solvent
evaporation leads to a decrease in droplet diameter, and the mass fraction of the dissolved
or suspended solid increases [Grassmann et al. 1997; Masters 2002].
When the moisture content becomes too low to maintain saturated surface
conditions, the critical point (critical moisture content) is reached and a dry layer starts to
form at the droplet surface. The surface is no longer completely wetted [Masters 2002].
The critical point is the transition point from the first drying stage to the second drying
stage. The evaporation rate decreases and the evaporation curve shows a sharp inflexion
point (Figure 2.5, point B). It is important to know when the critical point appears, because
the evaporation principles between the first and the second drying stage differ [Grassmann
et al. 1997].
The second drying stage or falling rate period starts at the critical point. For a
solution droplet the limiting factor for evaporation is the diffusion of moisture from inside
the droplet through the dried surface layer, which presents a formidable resistance to mass
transfer. The thickness of this layer increases with time as the moisture boundary retracts

Single droplet drying 18
toward the centre of the droplet and the evaporation rate decreases (Figure 2.5, curve B-E
for non-hygroscopic substances, curve B-C for hygroscopic substances). For a suspension
droplet the limiting factor for evaporation is the capillary moisture flow in the pores
forming in the particle [Kastner 2001; Masters 2002]. Due to decreasing evaporation rate,
the droplet or particle surface temperature starts to increase after the critical point close to
the ambient temperature.
Figure 2.5: Evaporation rate with time (continuous line: hygroscopic substance,
dashed line: non-hygroscopic substance) according to Grassmann et al. [1997]
The drying mechanisms for solution and suspension droplets differ in the second drying
stage [Masters 2002]. A solution droplet first evaporates at a constant rate, and the surface
temperature can be equated to that of a saturated solution. When the droplet moisture
content falls to a critical value, solid phase appears at the surface and the constant rate
ends. The rate of moisture migration to the surface decreases due to the increasing
resistance to mass transfer through the solid phase. The droplet begins to heat up, because
the rate of heat transfer exceeds that of mass transfer. If the heat transfer is high enough,
sub-surface evaporation can occur and pressure can be built up in the droplet. If the crust is
porous then the vapour can leak from the droplet. If it is a non-porous film then the droplet
may expand, collapse, rupture or even disintegrate. The movement of volatiles in drying of
solution droplets is first controlled by a liquid diffusional mechanism and then by a vapour
diffusional mechanism. Each product shows different drying characteristics in the second

Single droplet drying 19
drying stage, which are influenced by the solid surface layer. In a droplet with a porous
surface, the vapour migrates easily through the crust and the drying rate is high. For a nonporous
crust the drying rate will fall and evaporation time increases. A suspension droplet
evaporates also at a constant rate, as long as all suspended particles are completely wetted.
Moisture loss brings the insoluble solids closer together, until capillary mechanisms are
insufficient to maintain completely wet surfaces. The falling rate period with particles in a
funicular state continues until the pores are no longer filled with liquid and capillary flow
ends. In the following pendular state conditions a vapour diffusion mechanism
predominates until drying of the particle is completed [Masters 2002].
For hygroscopic materials a third drying stage occurs and a second inflexion point
in the evaporation curve appears (Figure 2.5, point C). The third drying stage begins as
soon as the highest hygroscopic moisture content is reached. From here on the evaporation
rate decreases faster than in the second drying stage (Figure 2.5, curve C-D) [Grassmann et
al. 1997].
The droplet temporal temperature curve together with the crust formation process
of a spherical droplet of liquid containing solid is described in Figure 2.6 [Farid 2003].
First the droplet is heated by the air until its surface reaches the air wet-bulb temperature
without substantial evaporation (Figure 2.6, curve A-B). In the following constant-rate
period droplet shrinkage occurs, with the droplet temperature remaining at the wet-bulb
temperature (Figure 2.6, curve B-C). The droplet does not heat up in this period and it can
be assumed that all heat transferred to the droplet is used for evaporation. The shrinkage
will continue until the solid starts crust formation. After the period of shrinkage,
evaporation continues with the formation of a moving interface. This is assumed to move
inward and separate the droplet into two regions, the dry crust and the inner wet core. The
temperature of the interface is assumed to remain at the wet-bulb temperature, whereas the
dry crust heats up. Farid [2003] calculated the average droplet temperature rising close to
the temperature of the drying air (Figure 2.6, curve C-E). After drying is completed, the
particle is heated up to the dry-bulb temperature of the air (Figure 2.6, curve E-F).

Single droplet drying 20
Figure 2.6: Drying stages of a liquid droplet containing solids [Farid 2003]
2.6.2 Particle formation
During the drying process a droplet undergoes both size and shape changes, the extent of
which is specific to the given product. In several studies the influence of process and
formulation parameters in spray drying on the particle shape has been analyzed. Elversson
et al. [2003] analyzed droplet and particle size relationship during spray drying. Increasing
droplet size increased the end particle size, but the effect was also influenced by feed
concentration whereby particles from low concentrated solutions were smaller than those
from higher concentrations. In a further study they demonstrated that larger particles could

Single droplet drying 21
be also obtained by decreasing the solubility of the solute. The apparent particle density
was inversely correlated to the feed concentration [Elversson and Millqvist-F. 2005].
Maa et al. [1997] investigated the shape and morphology of various spray dried
protein powders as a function of spray drying conditions and also protein formulation.
They observed a substantial change in particle shape from irregular to spherical with
decreasing outlet temperature. In formulations with sugars, the spray dried particles
exhibited a smooth surface when containing lactose. A rough surface appeared for
formulations containing mannitol at more than 30 % of total solids due to recrystallization.
The presence of surfactant resulted in noticeable smoother, more spherical particles. For
bovine serum albumin (bSA) / trehalose / polysorbate 80 formulations Adler et al. [2000]
confirmed that with rising amount of surfactant the surface shape becomes smooth,
whereas bSA / trehalose particle are more wrinkled.
Particle shapes formed during the spray drying process are given in Figure 2.7.
After the atomized droplet contacts the hot drying air (phase 1) a dry surface forms and its
properties control the ease of volatiles’ release and eventual particle shape and structure
(phase 2). Phase 3 shows various shapes and structures of the dried particles, some tend to
expand, other collapse, form blow holes or agglomerate. Very thin-shelled particles can be
so fragile that they disintegrate on mechanical handling [Masters 2002].
Single droplet drying experiments are a useful tool to examine particle formation of
solution and suspension droplets of inorganic and organic solids; some examples were
already given in Table 2.1. Charlesworth and Marshall [1960] gave a summary of crust
structure and consequential particle shape for suspended droplets of an inorganic salt in
aqueous solution. They found that the completion of the crust differed depending on the
nature of the solute and its crust structure, and also the surrounding air temperature after
the critical point. At a temperature of the surrounding air below the boiling point and with
a rigid and porous crust structure, the particle shape does not change. Less porous crust
structure can lead to fracture of the droplet and a crystal “fur” growing. Droplets with a
pliable and impervious skin will dimple or wrinkle. If the drying air temperature is above
the boiling point, the particle heats up after the critical point due to slowing of the mass
transfer rate by the crust and vapour formed inside the particle. If the crust is rigid and
porous, liquid is forced through the pores and evaporates quickly. Droplets with a nonporous
crust, which is not sufficiently permeable to the liquid, can fracture if the crust is
rigid, or inflate if the crust is plastic [Charlesworth and Marshall 1960].

Single droplet drying 22
Figure 2.7: Particle shapes formed during the spray drying process: 1: solid,
spherical; 2: shrivelled, misshapen; 3: hollow, spherical; 4: cenospherical; 5:
disintegrated [Masters 2002]
In recent studies Kastner [2001] analyzed the drying of suspension droplets in an ultrasonic
levitator using glass particles suspended in a water droplet as a model. In later experiments
he used a catalyst suspension. He developed a model for the computation of the drying
process. Schiffter and Lee [2007a; 2007b] investigated the drying behaviour of
formulations containing proteins, sugars and surfactants and compared the particle shape to
spray dried powders, obtaining good agreement.
2.6.3 Models for the drying of single droplets containing solids
Several models for heat and mass transfer during the drying of a single droplet containing
solids have been developed and described in the literature. Especially the changes in heat
and mass transfer in the second drying stage have to be taken into account, in contrast to
the evaporation of a pure solvent droplet. Several single droplet drying models are
summarised in Table 2.4.

Single droplet drying 23
Table 2.4: Single droplet drying models [Adhikari et al. 2000; Farid 2003]
Investigator
Wijlhuizen, Kerkhof and Bruin
[Wijlhuizen et al. 1979]
Sano and Keey
[Sano and Keey 1982]
Nesic and Vodnic
[Nesic and Vodnik 1991]
Cheong, Jeffreys and Mumford
[Cheong et al. 1986]
Farid
[Farid 2003]
Model conditions
Droplet is at a uniform temperature, water diffuses
through the solid and evaporates at the surface of the
droplet
Droplet is at a uniform temperature, model is based
on the formation of crust with a receding crust-bulk
interface, crust sensible heat was not included for
droplet temperature calculations
Model is based on a receding interface model with a
droplet core temperature different from that of the
surface
Moving boundary model considering a temperature
distribution inside the droplet and droplet shrinkage

Acoustic levitation 24
3 Acoustic levitation
3.1 History and application of acoustic levitation
In an acoustic levitator a droplet or particle can be levitated in a pressure node of a
standing acoustic wave generated by a piezoelectric crystal in the transducer. Acoustic
levitation provides the opportunity to perform single droplet drying experiments without
contact to a thermal-conducting holding device like a thermocouple or wire. The first
theoretical background was described by King in 1934 and Gorkov in 1962. Further
investigations were performed in the early 70s by the space agencies NASA and ESA for
the development of a tool for “containerless processing” under microgravity conditions. In
addition to acoustic levitation other levitation principles were also used, like electrostatic,
electromagnetic and aerodynamic levitation. Today acoustic levitation is readily performed
in terrestrial laboratories and is used for experiments in various fields of research [Lierke
1996a; Lierke 2002].
Lierke [1996a] described application fields for acoustic levitation experiments in
exactly defined experimental conditions. These are controlled evaporation experiments,
concentration of samples for trace analysis, single crystal growth, heat and mass transfer
between single droplets and their ambiance, measurements at isolated biological samples,
or stability studies of suspensions and emulsions. A summary of experimental research
using an acoustic levitation system for the application of density, surface tension and
viscosity measurements, micro and trace analysis, gas-solid reactions and determination of
enzyme kinetics for example is given by Schiffter [2006].
Single droplet drying studies using an ultrasonic levitator were used to describe
evaporation processes in several studies. The influence of the acoustic field and acoustic
streaming on the evaporation process was examined [Burdukov et al. 1963]. Seaver et al.
[1989] worked with an acoustic levitator in a free-jet wind tunnel and analyzed evaporation
of liquid droplets using different initial droplet sizes and the condensation of vapour on
evaporating droplets. In more recent studies Yarin et al. [1998] developed a method for
calculation of the acoustic radiation pressure and gave a relationship between droplet
shape, droplet displacement and sound pressure in an acoustic levitator. In later studies
they developed further calculations for the influence of the acoustic streaming on both
droplet drying and droplet surface temperature [Yarin et al. 1999]. The drying process of

Acoustic levitation 25
solution and suspension droplets was investigated by Kastner [2001] using an acoustic tube
levitator, who also developed a model for the evaporation process of droplets. The
influence of droplet surface oscillations for different solvents, solvent mixtures and
suspensions on the evaporation was studied by Rensink [2004]. For droplet surface
temperature measurement Tuckermann et al. [2005] used an infrared camera and carried
out experiments with different solvent droplets. They also analyzed the formation of
octadecanol monolayers on a liquid droplet using the temperature change as a detection
method [Tuckermann et al. 2007]. For pharmaceutical applications Schiffter and Lee
[2007a; 2007b] analyzed excipients and model proteins in different formulations and under
different experimental conditions. Drying kinetics, particle formation and protein activity
in comparison to spray dried powders were examined.
3.2 A short introduction to acoustics
Acoustic waves are mechanical waves that spread in gases, liquids, and solids due to their
elastic properties. Mechanical oscillations occur when molecules of the substance are
moved out of their position of equilibrium by external forces. Due to elastic and inertial
behaviour they start to oscillate periodically around their rest position. In acoustic waves
the molecules do not move forwards, rather just the sound energy is transported. The
appearance of sound depends on the existence of material, and therefore acoustic waves do
not spread in a vacuum [Günther et al. 1978]. In liquids and gases acoustic waves occur as
longitudinal waves. The human ear is able to hear frequencies from 16 Hz to 20 kHz.
Ultrasonic waves have a frequency of > 20 kHz, and acoustic waves having a frequency of
< 16 Hz are called infrasound [Stroppe 2008].
The sound velocity in a liquid can be calculated using the bulk modulus
elasticity and the liquid density [Stroppe 2008]:
Equation 3.1
For an ideal gas the sound velocity is calculated using the adiabatic index , the ideal gas
constant , the absolute temperature and the molar mass of the gas [Stroppe 2008]:
Equation 3.2
··

Acoustic levitation 26
The particle velocity and the sound pressure are used to describe the sound field and to
calculate the sound pressure level. The particle velocity is the alternate velocity of a
particle in a medium that transmits a longitudinal wave of pressure. It is calculated for
sinusoidal movement using the particle displacement and the angular frequency ω
[Günther et al. 1978]:
Equation 3.3
··2··
The sound pressure is the force of sound on a surface area perpendicular to the
direction of sound. is an alternating quantity, that means it is a function of time
which can be expressed as a peak value, arithmetic mean value, instantaneous value or as
root-mean-square-value. In most cases the root-mean-square-value, the effective sound
pressure , is used [Günther et al. 1978]. For sinusoidal waves the relation is
[Hering et al. 2007]:
Equation 3.4
√2
The relation of sound pressure and particle velocity in a plane wave is constant at any point
and any time, so points with maximum particle velocity have also maximum sound
pressure values. The proportionality factor is characteristic for the transmitting medium
and is called the characteristic acoustic impedance . It is dependent on the density of
the medium and the sound velocity [Stroppe 2008]:
Equation 3.5
·
Using this relation can be calculated by [Stroppe 2008]:
Equation 3.6
· ·

Acoustic levitation 27
The sound pressure level (SPL or ) characterizes the acoustic field. Using the effective
sound pressure values in the equation for it is defined as the logarithmic value of the
sound pressure compared to a reference sound pressure [Hering et al. 2007]:
Equation 3.7
20·log
The reference sound pressure value is = 2·10 -5 Pa (human auditory threshold) and
has the nondimensional unit decibel (dB) expressing the ratio of two values. A different
definition for the SPL that is used for standing acoustic waves by Yarin et al. [1998] is
introduced in Equation 3.24.
A standing acoustic wave is generated by reflection of an acoustic wave. At the
surface of reflection there is 0 and 0, whereas doubles. In the standing
acoustic wave sound pressure and particle velocity nodes and antinodes are phase-shifted
by 90° [Günther et al. 1978].
3.3 Levitation forces
3.3.1 Standing acoustic wave
In an acoustic levitator an acoustic wave generated by a piezoelectric crystal in the
transducer is reflected creating a standing acoustic wave when the distance between
transducer and reflector is an integral multiple of the half wavelength. In the standing
acoustic wave pressure nodes and anti-nodes appear at fixed points separated by a distance
of λ⁄ 2 (Figure 3.1). Small liquid or solid samples can be levitated in the vicinity of the
pressure nodes. Under gravitational conditions the sphere is levitated just below the
pressure node, dependent on its volume and density [Tuckermann 2002]. Calculations of
the position of the pressure nodes and anti-nodes and the levitation forces are given by
Yarin et al. [1998].

Acoustic levitation 28
Transducer
Sound pressure
Sample
Sound particle
velocity
Reflector
Figure 3.1: Sound pressure and sound particle velocity in a standing acoustic wave
[Schiffter 2006; Tuckermann 2002]
3.3.2 Interaction of ultrasonic wave and droplet
The levitation forces have influence on the droplet
shape, because axial and radial forces are unequal.
Calculations of the forces are given by Lierke
[1996b]. The axial forces are mainly responsible for
compensating the gravitational forces, whereas the
radial forces (Bernoulli forces) hold the sample
horizontally in the pressure node, if there is radial
displacement of the sample. For a deformable
sample the axial and radial levitation forces lead to a
deformation of the sample to the appearance of a
spheroid (Figure 3.2). With increasing sound
pressure the deformation increases; the sample can
even disintegrate [Tuckermann 2002]. The ratio
F r
F z
F z
F r
Figure 3.2: Radial (F r ) and axial
(F z ) levitation forces according to
Tuckermann [2002]

Acoustic levitation 29
between the horizontal and vertical diameter is the aspect ratio of the droplet. It depends
not only on the SPL, but also on the surface tension and the droplet size.
If a sample is levitated in a pressure node, then some reflection of the ultrasonic
wave occurs that is dependent on the sample size. As a result the SPL decreases. At the
beginning of drying the droplet has a known volume and a definite SPL exists sufficient to
levitate it. During the drying process droplet shrinkage leads to an increase in SPL due to a
decreasing reflection area. The calculative link between droplet shape / droplet displacement
and SPL is given by Yarin et al. [1998]. Kastner [2001] showed that the calculation
model by Yarin corresponds with experimental values for the SPL. Rensink [2004]
experimentally verified an increasing SPL with decreasing droplet volume for several pure
solvents.
3.4 Acoustic streaming
3.4.1 Inner acoustic streaming
The acoustic field induces streaming in the gas around a levitated droplet. This acoustical
streaming can be divided into inner acoustic streaming that is located directly on the
surface of the levitated droplet forming an acoustic boundary layer, and outer acoustic
streaming which forms two toroidal vortices above and below the droplet. A sketch of the
inner acoustic streaming field is given in Figure 3.3 taken from Yarin et al. [1999]. The
four closed loops (AB, CD, EF, GH) inside the acoustic boundary layer represent the
structure of the steady state flow with the acoustic and diffusion boundary layers and
. The acoustic boundary layer is very thin in comparison to the diffusion boundary
layer. The inner acoustic streaming has to be taken into account for the droplet evaporation
process, because it induces an additional convective influence that can increase mass
transfer.

Acoustic levitation 30
Figure 3.3: Inner acoustic streaming according to Yarin et al. [1999]
3.4.2 Outer acoustic streaming
Figure 3.4 shows the outer acoustical streaming field [Yarin et al. 1999]. The streaming of
the outer vortex on the left side above the droplet is left-handed and the one below the
droplet is right-handed. The surrounding air streams therefore towards the droplet
equatorially and leaves the droplet at the poles [Kastner 2001]. In the outer acoustic
streaming field vapour of the droplet liquid can accumulate. This has a wide influence on
droplet evaporation because the evaporation rate can be slowed down. Using an axial
ventilation airstream the vapour can, however, be blown out of the vortices, causing the
concentration of vapour to decrease and the evaporation rate to increase. The ventilation
airstream has to be adjusted so that the amount of vapour blown out of the vortices is equal
to the amount of vapour accumulating in the vortices from the droplet. If the ventilation
airstream is too strong, so that it has influence not only on the outer acoustic streaming but
also on the droplet surface, a further convectional mass transfer is induced together with a
rising evaporation rate [Rensink 2004].

Acoustic levitation 31
Figure 3.4: Outer acoustic streaming according to Yarin et al. [1999]
3.5 Vertical position of the levitated droplet
The position of the sample in an ultrasonic levitator under gravitational forces lies slightly
below the pressure node due to mass of the droplet, as explained in 3.3. This displacement
also depends on the SPL. A higher SPL as well as mass loss from the sample leads to a
higher droplet position. During droplet evaporation there is mass loss of the droplet, and
this droplet shrinkage leads to an increase in the SPL. The droplet rises in the ultrasonic
field towards the next upper pressure node during the drying process.
Kastner [2001] performed calculations to analyze the influence of volume decrease
(at constant density) and density decrease (at constant volume). A decrease in density has a
much higher influence on the sample position than does the droplet volume [Kastner
2001].

Acoustic levitation 32
3.6 Temperature of the levitation system
As soon as the levitator is put in operation the levitator transducer and the levitation
chamber start to heat up due to oscillations of the transducer. Kastner found a temperature
increase of about 5 °C at an ambient temperature of 20 °C without additional ventilation in
the levitation system [Kastner 2001]. For experiments at room temperature this
temperature increase has to be taken into account, because the temperature increase
influences the evaporation rate. Experiments at higher temperature need much external
heat supply, so that here the temperature increase from the levitator can be neglected.
3.7 Single droplet drying using an acoustic levitator
3.7.1 Pure solvent droplets
For the calculation of droplet drying kinetics in an ultrasonic levitator the influence of the
ultrasonic wave on the evaporation rate has to be included. The evaporation coefficient
in the equation for diffusion-controlled evaporation of a pure solvent droplet (Equation
2.16) is now modified to include the influence of inner acoustic streaming to mass transfer
via the Sherwood number [Tuckermann et al. 2002]:
Equation 3.8
2· ·
·
·
· 2
The droplet surface temperature of the levitated droplet can be calculated using the
model described by Yarin et al. [1999]:
Equation 3.9
⁄
· ⁄
· ·
·
·
Equation 3.10
1 2 · ⁄
· · ·
·
· ·
is the diffusivity of the ambient gas ( 0.208 cm 2·s -1 for air), is the thermal
conductivity of the ambient gas, is the latent heat of evaporation, is the relative
humidity and and are the vapour pressure and temperature of the gas. The properties
of the substance are dependent on the surface temperature, so that the equation has to be
solved iteratively. For the evaporation of water droplets and . The

Acoustic levitation 33
thermal conductivity of air in cal·cm -1·°C -1·s -1 with in degrees Celsius is calculated
by [Seaver et al. 1989]:
Equation 3.11
5.8·10 1.6·10 ·
For water vapour at atmospheric pressure the saturation vapour pressure in mbar is
given by [Seaver et al. 1989]:
Equation 3.12
· ·
· · · ·
is taken in degrees Celsius and the values for the constants are given in Table
3.1.
Table 3.1: Parameters for the calculation of the saturation vapour pressure of water
[Seaver et al. 1989]
6.107799961
4.436518521∙10 -1
1.428945805∙10 -2
2.650648731∙10 -4
3.031240396∙10 -6
2.034080948∙10 -8
6.136820929∙10 -11
The diffusion coefficient for water in cm 2·s -1 and the latent heat of evaporation for
water in cal·g -1 for water vapour at atmospheric pressure are calculated by [Seaver et
al. 1989]:
Equation 3.13 0.211 · 273.15
273.15 .

Acoustic levitation 34
Equation 3.14
595.3 · 0.483 ·1.2·10
For solution droplets other than water the saturation vapour pressure in bar is
calculated using the Antoine equation [Reid et al. 1987]:
Equation 3.15 0.001333224 ·
Here is taken in degrees Kelvin and some values for the liquid parameters A-C are given
in Table 3.2.
Table 3.2: Liquid parameters for calculation of the saturation pressure of the vapour
[Reid et al. 1987]
Liquid A B C
Acetone 16.6513 2940.46 -35.93
2-Butanone 16.5986 3150.42 -36.65
Dichloromethane 16.3029 2622.44 -41.70
Ethanol 18.9919 3803.98 -41.68
Ethyl acetate 16.1516 2790.50 -57.15
Methanol 18.5875 3626.55 -34.29
2-Propanol 18.6929 3640.20 -53.54
Tetrahydrofuran 16.1069 2768.38 -46.90
For solvent droplets other than water the binary gas diffusion coefficient has to be
calculated using the method by Fuller described in 2.3.2. The values for the latent heat of
evaporation can be found for each solvent in tables, where the values for are usually
given for 25 °C or for the boiling point of the substance. An online-handbook containing
values for numerous organic substances is “Yaws’ Handbook of Thermodynamic and
Physical Properties of Chemical Compounds” [Yaws 2003]. To calculate in kJ·mol -1 for
a special temperature in Kelvin the conversion is given by:

Acoustic levitation 35
Equation 3.16
· 1
For each substance the values for the coefficients , , and the molar mass for
conversion of to kJ·kg -1 are also given in Yaws [2003]. For the calculation of droplets
containing binary solvent mixtures see Yarin et al. [2002].
3.7.2 Solution and suspension droplets
The drying process of solution and suspension droplets also has to be divided into two
parts. In the first drying stage (constant rate) the droplet’s behaviour is like for pure solvent
droplets and can be calculated using the equations in 3.7.1. The vapour pressure lowering
effect of the dissolved solids has to be taken into account. The mass flux in the first
drying stage can be calculated directly from the volume decrease ∆ per time ∆ and the
density of the solvent liquid [Kastner 2001]:
Equation 3.17
· ∆
∆
With the assumption of a volume decrease the negative algebraic sign leads to positive
values for the mass flux. After the critical point the droplet volume remains constant, but
mass and density of the levitated droplet decrease due to further evaporation through the
crust from the liquid centre. The droplet rises in the ultrasonic field, and by using the
distance to the pressure node the mass flux can be calculated [Kastner 2001]:
Equation 3.18
∆
· sin2 · ·∆ sin2 · ·∆
The wave number is ⁄ and the distance from the mass centre of the droplet to its
next upper pressure node is ∆ . The constant for the intensity of the ultrasonic field, ,
is given by [Kastner 2001]:
Equation 3.19
·
·
· · Ω
·

Acoustic levitation 36
The function Ω with Ω · is given by [Kastner 2001]:
Equation 3.20
Ω 1
Ω ·
· Ω
2
Ω ·
·Ω 3·1
1 1 ·
Ω ·
· Ω · 2
using for , and the spherical Bessel- and Neumann-functions Ω and Ω:
Equation 3.21
Ω Ω · Ω
Ω · Ω
Ω Ω · Ω
Ω · Ω
Ω Ω Ω ⁄
For values at Ω 1 Lierke [1996a] gives a ready approximation:
Equation 3.22 Ω 5 6 ·Ω· 3
2Ω · sin2Ω 2Ω
cos2Ω
The effective pressure amplitude of the incident acoustic field is defined by Yarin et
al. [1998] in relation to the pressure amplitude at the source surface using the distance
of the reflector from the transducer , the angular frequency and the sound velocity :
Equation 3.23
cos · ⁄
The sound pressure level SPL is defined using the effective amplitude in dyn·cm -2 by
Yarin et al. [1998] and for a given SPL can be calculated using this equation:
Equation 3.24
20 · log 74
The SPL can be calculated using the aspect ratio ( ⁄ ) of the levitated droplet in a
numerical solution according to Yarin et al. [1998], because droplet deformation is
dependent on the SPL.

Materials and Methods 37
4 Materials and Methods
4.1 Materials
4.1.1 Proteins
Table 4.1 gives a summary of the proteins used in this work. Carbonic anhydrase from
bovine erythrocytes, L-lactic dehydrogenase type II from rabbit muscle and trypsinogen
from bovine pancreas were used as model proteins in the levitation experiments. Trypsin
from bovine pancreas was used as a reagent in the enzyme assay of trypsinogen.
Table 4.1: Enzymes with lot number and supplier used in this work
Substance Lot Number Supplied by (Product Number)
Carbonic anhydrase from bovine
erythrocytes
L-Lactic dehydrogenase solution
Type II from rabbit muscle
057K1277
127K1564
028K1625
086K2026
107K7405
036K7400
Sigma-Aldrich, Germany
(C 3934)
Sigma-Aldrich, Germany
(L 2500)
Trypsin from bovine pancreas 097K7025 Sigma-Aldrich, Germany
(T 9201)
Trypsinogen from bovine pancreas 077K7004 Sigma-Aldrich, Germany
(T 1143)
4.1.1.1 Carbonic anhydrase
Carbonic anhydrase facilitates the hydration of carbon dioxide to hydrogen carbonate and a
proton.
CO 2
+ H 2
O
carbonic anhydrase
⎯⎯⎯⎯⎯⎯⎯⎯→ HCO - 3 + H+

Materials and Methods 38
It is found in all animals and photosynthesizing organisms examined, as well as in some
nonphotosynthetic bacteria. Carbon dioxide is the starting point for photosynthesis and the
end point of substrate oxidation, therefore carbonic anhydrase is an ubiquitous enzyme
[Cox and Phillips 2008]. Carbonic anhydrase is one of the fastest of all known enzymes.
One enzyme is able to hydrolyse 10 6 molecules of CO 2 per second [Berg et al. 2007].
Carbonic anhydrase can be found in different locations. In erythrocytes it is included in the
transport of carbonic dioxide, in the kidneys in the reabsorption of hydrogen carbonate, in
the stomach in the secretion of gastric acid, and in the eye in the production of aqueous
humor. Carbonic anhydrase is a mostly spherical metalloenzyme containing zinc in the
active site located in a cavity that almost reaches the centre of the molecule. The zinc ion is
coordinated by three nitrogen atoms from histidin components in a tetrahedral geometry
completed by water (or hydroxide) [Cox and Phillips 2008]. There are more than 120
single-crystal X-ray structures of the native enzyme, its mutants, isozymes and various
metal-substituted forms [Cronin et al. 2001]. Figure 4.1 shows the structure of human
carbonic anhydrase.
Figure 4.1: Structure of human carbonic anhydrase II and the active site with zinc
according to [Berg et al. 2007; PDB 2009]
In the reaction mechanism of carbonic anhydrase the enzyme uses the zinc-bond hydroxide
ion to hydratase carbon dioxide as shown in Figure 4.2. First the water molecule is
deprotonated (1) and carbon dioxide is bonded (2). Then a nucleophilic attack of the
hydroxide ion to the carbon dioxide takes place and forms a hydrogen carbonate ion (3)
that is set free and replaced by a new water molecule (4) [Berg et al. 2007]. In this thesis

Materials and Methods 39
carbonic anhydrase from bovine erythrocytes (bCA) is used. The product is a mixture of
isoforms with the typically used molecular weight of 30 kDa [Sigma-Aldrich 2009].
Figure 4.2: Reaction mechanism of carbonic anhydrase [Berg et al. 2007]
4.1.1.2 L-Lactic dehydrogenase
Lactic dehydrogenase is an enzyme in glycolysis that catalyzes the reduction of pyruvate to
lactate. It is found in nearly all kinds of cells. Coenzyme is NADH+H + , which delivers the
hydrogen for the reaction. L-Lactic dehydrogenase is specific for L-lactate.
Pyruvate + NADH + H +
lactic dehydrogenase
⎯⎯⎯⎯⎯⎯⎯⎯→ lactate + NAD +
Lactic dehydrogenase is composed of four subunits with a total molecular weight of
140 kDa. The subunits are either H- or M-subunits, which are similar in molecular weight,
but differ in their amino acid composition [Sigma-Aldrich 2009]. The human polypeptide
chains of the subunits differ by 25 % [Berg et al. 2007]. With this two types of subunits
five isoenzymes, which are predominantly found in different organs, are built: H 4 (heart),
MH 3 , M 2 H 2 , M 3 H and M 4 (skeletal muscles). Figure 4.3 shows the active site of the protein
and the substrate and coenzyme binding. The reaction is stereospecific for the C4-
hydrogens of NADH due to the positioning of the molecules [Cox and Phillips 2008]. In
this thesis L-lactic dehydrogenase Type II from rabbit muscle (LDH) as crystalline
suspension in 3.2 M (NH 4 ) 2 SO 4 solution pH 6.0 is used.

Materials and Methods 40
Figure 4.3: The lactate dehydrogenase reaction [Cox and Phillips 2008]
4.1.1.3 Trypsinogen and Trypsin
Trypsinogen is an enzyme located in the pancreatic juice. It is the proenzyme of the
pancreatic enzyme trypsin, which is a member of the serine protease family. The activation
takes place after the proenzyme reaches the lumen of the small intestine by
enteropeptidase. A hexapeptide is splitted off the NH 2 -terminus, which leads to a change in
conformation to reduce the distance from side chains in the active site of the protein. This
way of using proenzymes prevents the pancreas from self-digestion. Trypsin itself is also
able to activate trypsinogen to trypsin by autocatalysis. The physiological relevance of
trypsin is to cut peptide bonds by hydrolysis for the decomposition of amino acids in
digestion [Karlson 1988; Sigma-Aldrich 2009]. Trypsinogen from bovine pancreas is used
in this thesis. Bovine trypsinogen consists of a single polypeptide chain of 229 amino acids
and is cross linked by six disulfide bridges (Figure 4.4). It has a molecular weight of
23.7 kDa [Karlson 1988; Sigma-Aldrich 2009].

Materials and Methods 41
Figure 4.4: Structure of bovine trypsinogen [PDB 2009]
4.1.2 Itraconazole
Itraconazole (C 35 H 38 Cl 2 N 8 O 4 ) is a synthetic broad-spectrum triazole antifungal agent. It
inhibits the synthesis of ergosterol, a vital cell membrane component in fungi. The molar
mass is 705.63 g/mol [Sigma-Aldrich 2009]. The itraconazole used in this work was
supplied by Janssen Pharmaceutica, Belgium (Lot Nr. 60000142). The chemical structure
of itraconazole is given in Figure 4.5 and shows that the molecule is highly lipophilic.
Itraconazole is applicable for oral and parenteral use [Mutschler et al. 2001].
Figure 4.5: Chemical structure of itraconazole [Mutschler et al. 2001]

Materials and Methods 42
4.1.3 Excipients and reagents
The following tables give a summary of all substances used in this work as solvents (Table
4.2), sugars (Table 4.3) or reagents (Table 4.4). The water for buffers and reagent solutions
was prepared by double distillation in an all-glass apparatus and filtration using filter
membranes with 0.2 μm pores.
Table 4.2: Solvents with lot number and supplier used in this work
Substance Lot Number Supplied by (Product Number)
Acetone pure 03676547 Roth, Germany (CP40.1)
2-Butanone p.a. 031K14530709 Merck, Germany (9708)
Dichloromethane pure 15678452 Roth, Germany (CP45.1)
Ethanol pure 19679127 Roth, Germany (P075.1)
Ethyl acetate pure 03676549 Roth, Germany (CP42.1)
Methanol pure 03676545 Roth, Germany (CP43.1)
2-Propanol pure 39681217 Roth, Germany (CP41.1)
Tetrahydrofuran p.a. 210K17393531 Merck, Germany (9731)
Water p.a.
OC683183
HC803830
Merck, Germany (1.16754.5000)
Table 4.3: Sugars with lot number and supplier used in this work
Substance Lot Number Supplied by (Product Number)
D-Mannitol, SigmaUltra 066K00411 Sigma-Aldrich, Germany (M 9546)
Sucrose (min. 99.5 %) 096K0026 Sigma-Aldrich, Germany (S 9378)
D-(+)-Trehalose dihydrate
124K3793
018K3791-1
Sigma-Aldrich, Germany (T 5251)

Materials and Methods 43
Table 4.4: Reagents with lot number and supplier used in this work
Substance Lot Number Supplied by (Product Number)
N α -Benzoyl-L-arginine
ethyl ester hydrochloride
035K0767
033K0991
Sigma-Aldrich, Germany (B 4500)
Calcium chloride dihydrate 31K1251 Sigma-Aldrich, Germany (C 3881)
Copolyvidone
(Kollidon® VA 64)
59933416KO BASF, Germany (-)
Hydrochloric acid, conc. 15786449 Roth, Germany (4625.1)
Hydroxypropylmethylcellulose
(Pharmacoat 615)
20022509 Dow Chemicals, USA (-)
β - Nicotinamide adenine
dinucleotide, reduced
disodium salt hydrate
126K7025
097K7016
Sigma-Aldrich, Germany (N8129)
p-Nitrophenyl acetate 1329140
35007229
Sigma-Aldrich, Germany (N 8130)
Phosphoric acid, conc. K23985682 724 Merck, Germany (1.59382.0050)
Potassium di-hydrogen
phosphate
di-Potassium hydrogen
phosphate anhydrous
Potassium hydroxide
solution 1 M
18887607 Roth, Germany (P018.1)
07358012 Roth, Germany (T875.2)
20420 Sigma-Aldrich, Germany (35113)
Sodium pyruvate 117K0662 Sigma-Aldrich, Germany (P 2256)
Trizma base 064K5405 Sigma-Aldrich, Germany (T 1503)
Trizma HCl
(reagent grade)
086K5461
057K5418
Sigma-Aldrich, Germany (T 3253)

Materials and Methods 44
4.2 Methods
4.2.1 Acoustic levitation
A 58 kHz levitator (tec5 AG, Oberursel, Germany) was used. According to tec5 AG the
wavelength of the standing acoustic wave is 5.71 mm and the high frequency (HF)-power
(continuously variable) can be adjusted from 0.65 to 5 Watt. The particle diameter range
for levitation experiments is about 15 μm to 2.5 mm. The levitator can be used in a
temperature range from 0 - 70 °C and a relative humidity (not condensating) of 10 - 90 %.
In Figure 4.6 the transducer and reflector of the levitation system is shown. The transducer
with the piezo-crystal is located at the top and connected to a power supply unit. The metal
reflector at the base is replaceable. A
planar reflector is an integral part of
the system on which the concave
reflector can be placed as shown in
the picture. All experiments in this
Transducer
work were performed using the
concave reflector. The distance from
transducer and reflector can be
adjusted by a micrometer screw to a
distance of a multiple of halfwavelengths.
By two additional
Reflector
micrometer screws the horizontal
position of the reflector can be
adjusted. The hole in the reflector can
be used for a drying air stream that is
directed to the droplet. The
dimensions of the transducer and the Figure 4.6: Transducer and reflector
reflector are given in Figure 4.7.

Materials and Methods 45
40.0 mm
16.0 mm
Transducer
15.0 mm
12.0 mm
0.8 mm
6.1 mm
2.0 mm
Concave
reflector
5.0 mm
9.0 mm
26.2 mm
3.8 mm
11.2 mm
Planar
reflector
8.0 mm
25.0 mm
Figure 4.7: Dimensions of the transducer and the concave and planar (fixed) reflector

Materials and Methods 46
The transducer and the reflector are
located in an acrylic glass chamber.
The chamber has a sample input
window at the front that can be
closed during measurement (Figure
4.8 Nr. 1). Liquid samples are
brought in the pressure node by a
Hamilton 7105N 5 μl microliter
syringe (Hamilton Company Europe,
Bonaduz, Switzerland) or by a
micro-dispensing system described
in 4.2.2. Solid samples are placed in
by tweezers or spoon net. The spoon
net is also used to remove the dried
3
particles out of the acoustic field. If
liquid samples have to be removed,
they are caught in 0.2 ml polymerase Figure 4.8: Inner acrylic glass box with
levitator
chain reaction (PCR) tubes
(Eppendorf AG, Hamburg, Germany) where they can be dissolved for further analysis
(Figure 4.9).
1
2
Figure 4.9: Spoon net and PCR tube holder
A Testo 605-H1 dew point hygrometer (Testo AG, Lenzkirch, Germany) is built in the
chamber for humidity and temperature measurements (Figure 4.8 Nr. 2). A flexible tube
(Figure 4.8 Nr. 3) connects the levitation chamber with the controlled evaporation mixer
(CEM). The CEM-system consists of a flow meter for the liquid (L1-FAC-33-0, flow
range 0.2 - 10.0 g/h H 2 O), a flow controller for the drying gas (F201C-FAC-33-V, flow

Materials and Methods 47
range 0.04 - 2.0 ln/min N 2 ) and a temperature controlled evaporation unit (W202-330-P,
range up to 200 °C) (Bronkhorst, Ruurlo, the Netherlands, supplied by Wagner Mess- und
Regeltechnik, Offenbach / Main, Germany). The feeding pressure was 1 bar and the system
was used with water and dry air or nitrogen. The inner acrylic glass chamber is contained
in an outer acrylic glass chamber which contains a Leister air heater Type 8D1 3000W
(Leister, Kägiswill, Switzerland) and has a front plate with two holes. The front plate has
to be changed if the microdispensing system is used. The levitation system was
equilibrated for 1 h (temperature and relative humidity) before starting an experiment. The
conditions during the experiments were constantly monitored by the Testo dew point
hygrometer. The setup of the levitation system is given in Figure 4.10 (without
microdispensing system).
Germanium
window
IR-Camera
with
macrolens
External
heating
Controlled
evaporation mixer
CCD-Camera with
macrolens
Light source
Transducer /
reflector
Inner and outer acrylic
glass chamber
Sample input
Figure 4.10: Setup of the levitation system
Figure 4.11 shows a picture of the complete setup of the levitation system including
computer, control units and water supply: 1) CEM (three units), 2) charge coupled device
(CCD) camera (left side of the chamber) and infrared (IR) camera (back side of the
chamber), 3) light source, 4) inner and outer acrylic glass chamber with levitator, 5)
external heating, 6) computer and control units for the levitator and CEM, 7) water supply.

Materials and Methods 48
2
1
4
3
7
6
5
Figure 4.11: Picture of the levitation setup
4.2.2 Microdispensing system
For contactless dispensing of very small droplet
sizes into the pressure nodes of the levitator a
microdispensing system was used (microdrop
Technologies GmbH, Norderstedt, Germany). A
dispenser head (MD-K-130-012) was used with an
inner nozzle diameter of 70 μm corresponding to a
droplet volume of about 180 pl (Figure 4.12),
dependent on the fluid, together with a control unit
(MD-E-201-H). The dispenser head is suitable for
liquids with a viscosity up to about 15 mPa·s. The
droplets have a velocity of about 2 m/s and the
maximum droplet rate is 2000 Hz (depending on
the fluid used).
The dispenser head is based on piezodriven
inkjet printing technology. The integrated
piezo activator induces a shock-wave into the fluid
contained in the head which causes a droplet to be
emitted from the nozzle [microdrop 2009]. A
series of droplets is repeatedly shot into the
Figure 4.12: Microdispensing
system with rail (front view)

Materials and Methods 49
pressure node of the standing acoustic wave of the levitator, so that the initial volume of
the levitated droplet is achieved by the number of single droplets.
The dispenser head has to be brought out of the levitation chamber, especially at
experiments at high temperature, because of the temperature influence on the sample in the
reservoir. For this reason a rail was constructed, on which the dispenser head and the
reservoir are fixed on a mobile unit (Figure 4.12). The position of the rail can be adjusted
in height and gradient to optimum position to the pressure node by screws fixed in the
perpendicular bars (Figure 4.13). The total length of the rail minus stopper is 58 cm. The
rail is fixed on a wooden plate that can be adjusted via four screws.
Figure 4.13: Microdispensing system with rail (side view)
For the fixation of the dispenser head the standard holder (MD-H-711-07) had to be
modified (Figure 4.14) so that it can be moved through the small window into the inner
levitation chamber close to the pressure node. The reservoir is outside the inner acrylic
glass chamber while dispensing.
Figure 4.15 shows the levitation setup for experiments using the microdispensing
system. The rail is fixed directly on the front side of the outer acrylic glass box and an
alternative front plate was used. There is one rectangular hole for the microdispensing head
and one round hole for a hand. By several screws the rail can be adjusted for the best
dispensing position.

Materials and Methods 50
C
A
B
Figure 4.14: Dispenser head holder: (A) dispenser head, (B) reservoir and (c) air filter
Figure 4.15: Setup of the levitation system containing the microdispensing system

Materials and Methods 51
4.2.3 Droplet size measurements
For the droplet size measurements a JAI CV-M4 2/3’’ monochrome CCD-camera (JAI-
AG, Copenhagen, Denmark) was used together with a Nikon Micro 60 mm objective 2.8
diaphragm and a bellow. The CCD-camera is connected to a computer by a PcDIG LVDS
frame grabber card. The pictures were taken using back-light illumination by a Schott KL
1500 cold light source and recorded and analyzed by Image Pro Plus Software 4.51
(MediaCybernetics, Bethesda, USA). The complete imaging system was supplied by Weiss
Imaging and Solutions GmbH, Günding, Germany. The camera system was calibrated for
each setting of the macro lens and bellows using a micrometer scale on a microscope slide
that was placed in the levitator. For verification of the calibration a polypropylene sphere
(Spherotech, Fulda, Germany) was levitated and the diameter was measured and compared
to the given value of 1.85 ± 0.03 mm.
During droplet drying pictures were taken in different intervals depending on the
evaporation rate of the sample. The horizontal and vertical diameter and the aspect ratio
were measured together with the position of the centre of mass directly in the picture and
saved in a data collector.
4.2.4 Droplet surface temperature measurements
Droplet surface temperature measurements were performed using the VARIOSCAN 3021
ST high resolution camera (InfraTec, Dresden, Germany). The camera has a single
mercury cadmium tellurium (MCT) detector for scanning measurements in the temperature
range from -40 to 1200 °C. A Stirling cooler is used as a cooling system for the detector.
The camera has a thermal resolution of +/- 0.03 K and a spectral sensitivity in the range
from 8 to 12 μm. The geometric resolution is 1.5 mrad and the field of view is 30° (H) x
20° (V). Up to five infrared pictures can be taken per second dependent on the chosen
camera settings with a sixfold electro optical zoom. The camera was used together with a
trifold 25 μm macro lens [InfraTec 2009]. Since IR-measurements cannot be performed
through acrylic glass, a round germanium window (100 mm x 5 mm) is located in the back
wall of the acrylic glass box. From inside the levitation chamber the germanium window is
covered by a thin plastic film for protection from the sample solvents. The IR-camera is
connected to a computer via Ethernet and IRBIS®control software (InfraTec, Dresden,
Germany). For data analysis the IRBIS®professional 2.2 software (InfraTec, Dresden,

Materials and Methods 52
Germany) was used. The droplet surface temperature was measured at different time
intervals during the drying process. The emissivity for the droplet substances was analyzed
by comparison of the measured temperature by the IR-camera to the temperature of an
Omega Type T thermocouple (OMEGA Engineering, Stamford, USA) with 0.13 mm
diameter of the wire, on which the sample droplet was suspended according to
Tuckermann [2002]. The temperature data were collected by an OM-CP-QUADTEMP
data logger using the software OMEGA engineering 2.00 (OMEGA Engineering,
Stamford, USA).
4.2.5 Spray drying
For spray drying experiments a
Büchi Mini Spray Dryer B-191 was
used (Büchi AG, Flawil,
Switzerland) using a high efficiency
cyclone. The sample was collected
directly in a 50 ml Sarstedt tube. The
drying temperature was higher than
in the levitation experiments, with
130 °C inlet temperature and 80 °C
outlet temperature. The atomization
flow rate was 700 l/h and the sample
volume was 0.4 - 5 ml, depending on
the substance, with a liquid feed of
2 ml/min. The aspirator was set to
Figure 4.16: Sketch of a Büchi Mini Spray
90 % of the maximum rate of
Dryer B-191 [Büchi 1999]
60 m³/h. Figure 4.16 shows a Büchi
B-191 Mini Spray Dryer and the path of the drying air: 1) air inlet (air filter is connected
upstream), 2) heating, 3) entry in drying chamber, 4) cyclone, in this work replaced by an
improved cyclone for maximum powder recovery [Maury et al. 2005a], 5) aspirator, 6)
temperature sensor air inlet, 7) temperature sensor air outlet, 8) product container, in this
work replaced by a Sarstedt tube [Büchi 1999].

Materials and Methods 53
4.2.6 Scanning electron microscopy
Scanning electron microscopic pictures were taken by an Amray 1810T scanning electron
microscope (Amray, Bedford, USA) for particle size and morphology analysis. The
samples were sputtered with gold for 1.5 min (Hummer JR Technics, Munich, Germany)
after fixing on aluminium sample stubs G301 (Plano GmbH, Wetzlar, Germany).
4.2.7 Enzyme activity assay of carbonic anhydrase
The enzymatic activity measurement of bovine carbonic anhydrase (bCA) is based on
assay procedures by Sigma-Aldrich [2009] and by Verpoorte et al. [1967] and was used in
a modified form. The principle of the assay is the reaction of p-nitrophenyl acetate (PNPA)
and water to p-nitrophenol and acetate catalyzed by bCA:
p - nitrophenyl acetate + H 2
O
bCA
⎯⎯⎯→ p - nitrophenol + acetate + H +
The samples were dissolved in 50 mM trizma buffer pH 7.5 (at 25 °C) and stored on ice.
The buffer was prepared using trizma base, trizma hydrochloride and double-distilled
water. The pH was adjusted to 7.5 at 25 °C using hydrochloric acid and potassium
hydroxide solution. In the case of no inactivation, the final activity of the sample solution
should be 100 - 200 units/ml. The levitation samples could be dissolved directly in the
PCR tubes using 250 μl trizma buffer. Spray dried samples were dissolved in Sarstedt
tubes. A reference sample using untreated bCA was also analyzed, the untreated sample
solution being used as reference. A droplet was brought in the ultrasonic field for droplet
volume determination using the CCD-camera, immediately removed, diluted and stored on
ice until measurement. A 3 mM PNPA-solution was prepared freshly daily by dissolving
the PNPA in acetone and filling up the volume with double-distilled water. It was stored
protected from light on ice. For the autozero the trizma buffer was used. The increase in
absorption at 348 nm at 25 °C was measured by UV-spectroscopy using a Lambda 25
UV/VIS-spectrometer connected to a PC with PerkinElmer UV WinLab V 5.0 software
(PerkinElmer LAS GmbH, Rodgau, Germany) in a 10 mm quartz cuvette. For sample
measurements the cuvette contained 1.90 ml trizma buffer, 1.00 ml PNPA-solution and
0.10 ml of the enzyme solution. Blank measurements were performed using 2.00 ml trizma
buffer and 1.00 ml PNPA-solution. The increase in absorption was recorded for 3 minutes
after an initial 3 minutes’ equilibration time in the cuvette holder connected to a water bath

Materials and Methods 54
Julabo Type MP-5 (Julabo Labortechnik GmbH, Seelbach, Germany) at 25 °C. The slopes
of the sample measurements were compared to the slope of the reference sample (set to
100 % activity) and the residual activity of the sample was thus calculated. All
measurements could be performed only twice due to the small amount of sample solution
in the PCR tubes.
4.2.8 Enzyme activity assay of L-lactic dehydrogenase
The L-lactic dehydrogenase (LDH) suspension was dialysed using Spectra/Por ® Dialysis
Membrane with a molecular weight cut off (MWCO) of 12 - 14,000 (Spectrum
Laboratories, Rancho Domingues, USA) according to Adler [1999]. The dialysis tubing
was placed in a hundredfold volume of a 100 mM potassium phosphate buffer pH 7.0 (at
25 °C) on ice on a magnetic stirrer for 3 hours. The potassium phosphate buffer was
prepared using potassium di-hydrogene phosphate and double-distilled water. The pH was
adjusted using 1 M potassium hydroxide solution. After renewal of the dialysis medium the
dialysis was continued for 14 hours. To achieve a high protein concentration the solution
was concentrated using a centrifugal filter device Amicon Ultra-15 with a MWCO of
30,000 (Millipore Corporation, Billerica, USA) in the centrifugal rotor Minifuge RF
(Heraeus Sepatech GmbH, Osterode, Germany). The concentration was first measured at
280 nm after equilibration on 25 °C for 1 minute in a 10 mm quartz cuvette and then the
sample was diluted to a suitable concentration.
The enzymatic assay for LDH is based on the instruction by Sigma-Aldrich [2009]
and the information given by Adler and Lee [1999]. LDH catalyzes the oxidation of
pyruvate and β-NADH to L-lactate and β-NAD + (see 4.1.1.2). The decrease of absorption
results from the decomposition of β-NADH. The sample was dissolved in 100 mM
potassium phosphate buffer pH 7.0 (at 25 °C) in PCR tubes or in Sarstedt tubes and diluted
to an appropriate activity for the measurement with potassium phosphate buffer. In the case
of no inactivation, the final activity of the solution should be about 1.3 units/ml. For
autozero the potassium phosphate buffer was used. The decrease in absorption at 340 nm at
25 °C is measured in a 10 mm quartz cuvette by UV-spectroscopy using a Lambda 25
UV/VIS-spectrometer connected to a computer with PerkinElmer UV WinLab V 5.0
software (PerkinElmer LAS GmbH, Rodgau, Germany). For sample measurements 2.80 ml
of a 0.13 mM β-NADH solution and 0.10 ml of a 69 mM sodium pyruvate solution were

Materials and Methods 55
pipetted into the quartz cuvette. The 0.13 mM β-NADH solution and the 69 mM sodium
pyryvate solution were prepared using potassium phosphate buffer as solvent. The cuvette
was first equilibrated in the cuvette holder connected to a water bath (Julabo Labortechnik
GmbH, Seelbach, Germany) at 25 °C for one minute. Then 0.10 ml of the sample solution
was added and the decrease in absorption at 340 nm was measured for 2 minutes. Blank
measurements were performed by using 0.10 ml potassium phosphate buffer instead of the
protein solution. The slopes of the sample measurements were compared to the slope of a
reference substance (set to 100 % activity, as described in 4.2.7) and the residual activity
was calculated.
4.2.9 Enzyme activity assay of trypsinogen
In the enzyme activity assay of trypsinogen first the trypsinogen is activated to trypsin
before the UV-detectable reaction takes place according to Sigma-Aldrich [2009] and
Faizyme Laboratories [2007].
Trypsinogen
trypsin
⎯⎯⎯→ trypsin + hexapeptide
Trypsin is able to catalyze the hydrolysis of esther and amide linkages of synthetic
derivates of amino acids as in N α -benzoyl-L-arginine ethyl ester hydrochloride (BAEE). It
is decomposed to N α -benzoyl-L-arginine and ethanol [Sigma-Aldrich 2009].
BAEE + H 2
O
trypsin
⎯⎯⎯⎯→ N α -benzoyl-L-arginine + ethanol
For activation of trypsinogen the sample was dissolved in 1 mM hydrochloric acid.
Levitation samples were dissolved in Eppendorf tubes and spray dried samples in Sarstedt
tubes. The trypsinogen solution was added to an activation mixture containing 1 M CaCl 2 -
solution, 400 mM trizma buffer pH 8.4 (at 25 °C) and 0.02 % (w/v) trypsin enzyme
solution (1:19:1) and incubated at 4 - 8 °C for 24 hours. The CaCl 2 -solution and the trizma
buffer were prepared using double-distilled water as a solvent. Trypsin was dissolved in
1 M hydrochloric acid and freshly prepared daily. To stop the activation process the
solution was diluted with 1 mM HCl. In the case of no inactivation, the final activity of the
solution should be 500 units/ml. For autozero a potassium phosphate buffer was used. For
activity measurement 3.00 ml of a 0.25 mM BAEE solution, prepared using a 67 mM
potassium phosphate buffer pH 7.6 (at 25 °C) as solvent, and 0.20 ml of the sample

Materials and Methods 56
solution were pipetted in a 10 mm quartz cuvette. The increase in absorption at 253 nm at
25 °C was recorded for 1.5 minutes by using a Lambda 25 UV/VIS- spectrometer
connected to a computer with PerkinElmer UV WinLab V 5.0 software (PerkinElmer LAS
GmbH, Rodgau, Germany). The slope of the sample measurement was compared to the
slope of a reference sample set to 100 % activity (as described in 4.2.7) and the residual
activity was calculated.

Results and Discussion 57
5 Results and Discussion
5.1 Preliminary experiments using the IR-camera
5.1.1 Heating-up of the levitation system
The transducer heats up due to its oscillations after switch-on of the levitator [Kastner
2001]. In a preliminary experiment the infrared camera was used to depict this behaviour
and to give a quantitative impression on the temperature increase for the levitation system.
Figure 5.1 shows infrared pictures of the levitator (a) before power-on, (b) after 5 min and
(c) after 10 min from power-on at an ambient temperature of about 20 °C. The temperature
scale is set constant in all pictures and the increasing red colour shows higher temperature.
(a)
(b)
(c)
Figure 5.1: IR-pictures of the transducer (a) before power-on, (b) 5 minutes after
power-on, (c) 10 minutes after power-on
The heating-up of the transducer unit can be clearly seen. The temperature increase is
about 2.6 °C after 10 minutes. The air temperature in the inner acrylic glass chamber also
increases. Thus the consequence for the following experiments is that the minimum drying
air temperature must be set to 25 °C. Experiments at 20 °C are therefore not possible using

Results and Discussion 58
this levitation set-up, because there is no cooling system and lower temperatures cannot be
kept constant. For a constant temperature and relative humidity of the drying air it is
necessary to equilibrate the chamber for one hour before an experiment is started. During
the experiment the temperature and relative humidity are controlled continuously by a
thermometer and a dew point hygrometer. For measurements at higher temperature, the
temperature increase has little influence because of the higher external heat supply that can
be adjusted to the correct temperature measured directly in the inner levitator chamber.
The temperature distribution in the standing ultrasonic field of a 20 kHz levitator at
21 °C, 43 % relative humidity and a SPL of about 168 dB was measured by Tuckermann
[2002]. Tuckermann found that the maximum temperature in the levitation axis is located
at the pressure antinodes where the particle velocity is maximal (2 - 3 °C temperature
increase). But also the other axial parts are heated up by a lower value of 1 - 1.5 °C. By use
of different settings Tuckermann found that with rising SPL the temperature difference
increases. At the maximum SPL of about 175 dB the temperature increase was about 8 °C
in the pressure antinodes.
5.1.2 Influence of droplet size on the surface temperature measurement
Droplet drying experiments will be performed using different initial droplet sizes. Before
starting the experiments the surface temperature measurement of droplets and particles
with different initial sizes was analyzed. At first the surface temperature of plastic spheres
was analyzed at 25 °C and 40 °C at 1 % relative humidity using spheres having a diameter
of 0.62 mm, 0.98 mm and 1.23 mm. Then the temperature profiles of water droplets at
three different initial droplet sizes of about 500 μm, 800 μm, and 1200 μm horizontal
diameter are examined at 25 °C, 40 °C, and 60 °C at 1 % relative humidity. The
temperature profile was taken at the maximum diameter of the droplet or sphere. The size
of the droplets or spheres was measured using the CCD-camera.
In comparison to the temperature of the ambient air the temperature profiles for the
large sphere show a higher surface temperature for measurements at an ambient
temperature of 25 °C (Figure 5.2 (a)) and lower surface temperature at 40 °C (Figure
5.2 (b)). The smaller spheres showed similar behaviour. Note that there is no emissivity
correction included for the material of these spheres, the focus lies on the profile seen by

Results and Discussion 59
the IR-camera and the area of minimum surface temperature. In the sphere profiles for
clarity a circle shows the size of the sphere.
(a)
Figure 5.2: Surface temperature profile of a levitated sphere (sphere diameter: 1.23
mm) at (a) 25 °C and (b) 40 °C ambient temperature and 1% relative humidity
The heating-up of the solid sphere placed in the ultrasonic levitator is shown in Figure
5.3 (a). The maximum heating appears at the poles, the equatorial part is cooler. This effect
was already described by Tuckermann [2002]. Tuckermann found that the heating of the
poles is induced by the energy supplied by the ultrasonic field, whereas the cooling of the
equatorial part is a consequence of the radial streaming air to the levitated sample due to
acoustic convection. This phenomenon is not found for higher air temperatures, for
example at 40 °C in Figure 5.3 (b).
(b)
(a)
Figure 5.3: IR-pictures of a 1.23 mm plastic sphere at ambient conditions of (a) 25 °C
and (b) 40 °C and 1% relative humidity
(b)

Results and Discussion 60
The temperature distribution is now overpowered by the high external heat supply from the
ambient air. The temperature of the sphere appears cooler than the ambience because the
air temperature cannot be measured by the IR-camera. This might also explain the decrease
of the ambient temperature to the boundary of the temperature profile.
For water droplets of all three initial sizes there is a decrease in the droplet surface
temperature due to the cooling effect of the evaporating solvent (Figure 5.4). This can be
found for all drying air temperatures. The temperature of the water droplets is assumed to
be constant over the droplet surface, but the IR-camera does not show a uniform
temperature at the droplet boundary to the ambience on the IR-picture. The temperature at
the border of the droplet does not change immediately, but there is rather a transition
region on the droplet surface where the temperature decreases. This effect was also seen
for the plastics spheres at 40 °C.
(a)
(b)
(c)
Figure 5.4: IR-pictures of water droplets at 25 °C and 1 % relative humidity, droplet
size: (a) 0.51 x 0.52 mm, (b) 0.77 x 0.97 mm and (c) 1.03 x 1.08 mm
The IR-camera cannot be focused on the whole droplet surface area because of the
sphericity of the droplet, and it also cannot be in a perpendicular direction of measurement
to the droplet surface for all positions on the surface. Furthermore, the amount of measured

Results and Discussion 61
background heat radiation, from the levitation chamber walls for example, by the camera
on the droplet surface is higher at the border in comparison to the middle of the droplet or
particle. This transition area is expected to have a higher influence on droplet surface
temperature measurement of small droplets than for larger droplets. In this case it would
have to be taken into account that for small droplets the measured surface temperature
(even for measurement in the middle of the droplet surface) may lead to higher values than
the true droplet surface temperature would be.
The comparison of the temperature profiles of the 500 μm droplets (Figure 5.5)
shows that with increasing drying air temperature (T da ) the profile of the droplet results in a
smaller constant minimum temperature part.
Figure 5.5: Surface temperature profile of levitated water droplets at droplet
diameters about 0.5 mm at 25 °C (droplet size 0.51 x 0.52 mm), 40 °C (droplet size
0.57 x 0.58 mm) and 60 °C (droplet size 0.49 x 0.51 mm) and 1 % relative humidity
In the comparison for different droplet sizes there is no visible dependence of the minimum
droplet temperature on the droplet size (Figure 5.6, Figure 5.7, Figure 5.8). The minimum
droplet temperatures change due to small differences in the drying air conditions.

Results and Discussion 62
Figure 5.6: Surface temperature profiles of levitated water droplets at 25 °C and 1 %
relative humidity (droplet size: 0.51 x 0.52 mm, 0.77 x 0.79 mm and 1.03 x 1.08 mm)
Figure 5.7: Surface temperature profiles of levitated water droplets at 40 °C and 1 %
relative humidity (droplet size: 0.57 x 0.58 mm, 0.83 x 0.84 mm and 1.13 x 1.03 mm)

Results and Discussion 63
Figure 5.8: Surface temperature profiles of levitated water droplets at 60 °C and 1 %
relative humidity (droplet size: 0.49 x 0.51 mm, 0.75 x 0.90 mm and 1.35 x 1.13 mm)
Note that the drying air temperature cannot be determined from the profile, because the
temperature of gas is not measureable for the IR-camera. Therefore the drying air
temperature must be continuously measured by the thermometer in the dew point
hygrometer during the experiments.
There is no indication of an influence of the initial horizontal droplet diameter of
500, 800 and 1200 μm on the surface temperature measurement. For this reason for all
three initial droplet sizes the minimum droplet surface temperature can be used for
analysis. For very small droplet sizes at the end of the drying process (about 200 - 300 μm)
there is a large temperature increase measurable before the droplet disappears in the
acoustic field (see data plots in 5.2). The temperature increase can be caused by
measurement inaccuracy and by non-volatile impurities contained in the solvent or from
the drying air [Tuckermann 2002]. For pure solvents the droplet surface temperature at the
beginning of the measurement (after the surface temperature equilibration) is taken for
comparison. For solution droplets the droplet has a finite size after the critical point due to
its solid content. Therefore the small size, where the increasing error due to incorrectly
measured surface temperature has to be taken into account, will not be reached.

Results and Discussion 64
5.1.3 Determination of emissivities
For the droplet surface temperature measurements the emissivity ε of the measured
substance has to be known. Values of ε for several substances are given in the literature.
The emissivity has, however, to be determined experimentally because it depends on many
factors like the wavelength range of the camera and the ambient temperature. The
emissivities used for data analysis in this work were determined by measuring the
temperature of a droplet suspended on a thermocouple by using both the IR-camera and the
thermocouple. The temperature value of the thermocouple was used as a reference for the
temperature measured by the IR-camera that was adjusted by variation of the emissivity. It
is assumed that the droplet surface temperature measured by the IR-camera and the mean
droplet temperature measured by the thermocouple correspond. This assumption is
justified by the small size and the rapid internal convection of acoustically levitated drops
[Yarin et al. 1999] which lead to rapid temperature equilibration [Tuckermann et al. 2005].
The transmissivity of the germanium window was assumed to be 0.95 (according to
the transmission spectrum of the window). Influences on the temperature measurement
inside the levitation chamber were included in the value for the emissivity.
This method was described by Tuckermann [2002]. Tuckermann determined
experimentally emissivities for water and several organic solvents and found that during
the drying process the temperature determined by the camera and the thermocouple
diverges towards the end of measurement. Tuckermann attributed this to decreasing
wetting of the thermocouple during the measurement. In this work the emissivity was
determined using the temperature values as soon as a constant droplet temperature
appeared. The experiments were each performed three times. This method had to be
expanded for solution droplets because the surface characteristic of the droplet changes
after the critical point. An example for the measurement of the temperature curve on a
droplet containing mannitol 15 % (w/w) in water p.a. at 40 °C and 1 % relative humidity is
given in Figure 5.9. Here the temperature curve measured by the thermocouple and the
curve measured by the IR-camera using the best-fit emissivity in the first drying stage are
compared. It is not possible to fit the whole curve with just one setting of the emissivity.
For this reason the analysis of the temperature measurement has to be divided into two
parts, before and after the critical point, each with a different setting for data analysis. A

Results and Discussion 65
summary of the experimentally determined emissivities for pure water droplets in the
levitation system is given in Table 5.1.
Figure 5.9: Droplet surface temperature curves of a mannitol 15 % (w/w) in water
p.a. solution droplet suspended on a thermocouple, (continuous line: droplet surface
temperature measured by the IR-camera using best fitting emissivity for the droplet,
dashed line: temperature measured by the thermocouple)
The published value for the emissivity of water (20 °C) is 0.96 [Schuster and Kolobrodov
2004]. Note that the values determined in the experiments include the properties of the
levitation chamber and are determined by assuming a transmissivity of the germanium
window of 0.95.
Table 5.1: Summary of the emissivities ε for pure water droplets
1 % rel. humidity 20 % rel. humidity 40 % rel. humidity 60 % rel. humidity
25 °C 0.96 0.99 1.00 1.00
40 °C 0.98 1.00 1.00 1.00
60 °C 0.96 1.00 1.00 1.00

Results and Discussion 66
The emissivities were determined again using a 0.91 m/s drying air stream directed
towards the droplet to make sure ventilation does not disturb the measurement. The result
is that single values differ in comparison to the measurements without ventilation
airstream, but the change is in both directions and stays in the range of 0.96 to 1.00. No
clear dependency can be found for the influence of a direct drying air stream. Even in
experiments with different drying air velocity settings of 0.75 m/s to 3.02 m/s, the
emissivities remain in the range of 0.95 to 1.00.
For organic solvents the emissivities were also examined. The problem was the
temperature of 50 °C at which the evaporation experiments should be performed and
therefore the emissivities had to be analyzed. At a drying air temperature of 50 °C the
temperature difference to the boiling point of the solvents (that lie between 39.0 °C and
82.4 °C) is low. This low temperature difference to the boiling point leads for some
solvents to a very fast evaporation of the droplets and a rapidly decreasing wetting of the
thermocouple, so that the values for the emissivity were not reproducible. The published
values for emissivities of organic solvents (especially hydrocarbons) are in a range of 0.9
to 1.0 [Daif et al. 1999]. Therefore it was decided to use 0.90 as value for the emissivity for
data analysis of the measurements of all organic solvents in this thesis, even though some
experimentally determinate values were lower.
The emissivities of solutions containing mannitol and trehalose 15 % (w/w) were
also analyzed at different drying air temperature and relative humidity settings. Here the
emissivities of the droplet and additionally of the particle forming after the critical point
need to be determined. The measurement of the wet droplet leads to similar results as in
the measurements for pure water. After the critical point the measurement started to be
difficult. Mannitol did not form a particle, but rather started to crystallize at the wire of the
thermocouple forming fur-like crystals. Trehalose climbed the wire and filled the space
between the two thermocouple wires. The emissivities were determined by variation of the
values for the transmissivity and the emissivity in the second drying stage. The values for
mannitol and trehalose solutions differ slightly, so that for the protein solutions and
protein / sugar formulations the averaged values for emissivity and transmissivity are used
without performing new experiments. For data analysis the course of the temperature
increase at the critical point is more important than the exact particle temperature that is
assumed to be close to the temperature of the drying air. Therefore a small inaccuracy due

Results and Discussion 67
to the emissivities in the second drying stage does not influence the interpretation of the
data curves.
For quantitative analysis note that there are inaccuracies due to the measuring error
of the thermocouple and the thermometer of the dew point hygrometer. Together with
temperature variation of the drying air during the measurement and the inaccuracy of the
emissivities for data analysis, a variation in temperature of 1 - 2 °C has to be accepted.
5.2 Evaporation of pure solvent droplets
5.2.1 Evaporation of pure water droplets
5.2.1.1 Influences of the initial droplet size on the evaporation process
The drying behaviour of pure solvents was analyzed using the radius squared curves, the
aspect ratio curves and the droplet surface temperature curves. The horizontal and vertical
droplet radii are unequal due to the flattening by the levitation forces and are converted to
the radius of a surface equivalent sphere. The droplet is assumed to be an oblate spheroid,
so that the surface area of the droplet can be calculated using the measured horizontal
radius and vertical radius :
Equation 5.1
2··
·
·ln1
1
where is given by:
Equation 5.2
1
Via the formula for the surface of a sphere, the surface equivalent radius is calculated by
solving the equation for the radius using the calculated droplet surface area value of the
spheroid. To have comparable curves for different droplet sizes, the value of the radius
squared and also for the drying time is divided by the initial radius squared of the droplet.
This correction provides a comparison of the experimental data of variations in initial
droplet size. If equal initial droplet sizes are required this correction is also needed,
because even using a syringe without dead volume or the microdispensing system it is not
possible to obtain absolutely identical initial droplet sizes. Using this correction the
equation for the graph is given by Equation 2.13 [Frohn and Roth 2000]:

Results and Discussion 68
Equation 5.3
²
0² 1 ·
0²
The evaporation coefficient given in Equation 2.16 is included in the equation by:
Equation 5.4
²
12· ·
·
·
0² ·
0²
with the surface-equivalent droplet radius squared as a function of time ², the initial
surface equivalent droplet radius squared 0 , the drying time , the binary diffusion
coefficient calculated using the method by Fuller [1966], the molar mass , the
density of water , the ideal gas constant , the partial vapour pressure at the droplet
surface and at infinity , the droplet surface temperature , and the drying air
temperature . The curves show a linear decrease in the radius squared if the evaporation
process can be described by the d²-law as a model for diffusional evaporation processes of
pure, spherical solvent droplets at a fixed surface temperature and a fixed drying air
temperature [Frohn and Roth 2000].
The evaporation coefficient is directly given as the negative slope of the
² ⁄ 0² versus ⁄ r0² plot and characterises the evaporation process. It is dependent
on the thermodynamic properties of the droplet liquid and on conditions of the ambience
like the temperature. The aspect ratio curve is given by the relation of the horizontal and
vertical diameter, measured in the shadow image taken by the CCD-camera using
ImageProPlus software. The droplet surface temperature curve is measured by the IRcamera.
The minimum droplet surface temperature values are corrected using the measured
emissivity of the solvent at given conditions. For comparison of the droplet surface
temperatures at different conditions the droplet surface temperature was taken from the
linear part of the temperature curve after equilibration time and before the temperature
increases at the end of the measurement. All experiments were performed six times and the
graphs presented are the averaged curves.
Three different initial droplet sizes for water p.a. of approx. 500 μm, 800 μm and
1200 μm initial horizontal diameter were analyzed at a drying air temperature of 25 °C,
40 °C and 60 °C and a relative humidity of 1 %, 20 %, 40 % and 60 % without a direct
drying air stream towards the droplet. The water p.a. was filtered freshly before starting the
experiments using a filtration unit with 0.2 μm pore diameter (Whatman®, FP 30/0.2,

Results and Discussion 69
Schleicher & Schuell, Dassel, Germany). The droplets were inserted in the acoustic field
using the microdispenser system. Figure 5.10 shows the curves for 1200 μm water droplets
at 25 °C and 60 °C at 1 % relative humidity as an example for the curve progression.
(a)
(b)
Figure 5.10: Drying behaviour of a 1200 μm water droplet at (a) 25 °C and (b) 60 °C
at 1 % relative humidity

Results and Discussion 70
The ² ⁄ 0² -graphs show a linear decrease of the radius squared. Only the last part of
the curve is a little flattened. For this reason the evaporation coefficient is calculated using
the linear part of the curve. The aspect ratio is nearly constant at the beginning of the
measurement and shows fluctuations at the end of the evaporation process due to droplet
oscillations. For higher temperatures a higher SPL is necessary to provide stable levitation
of the droplet and to minimize oscillations that can influence the droplet size and surface
temperature measurement by the cameras. In this example the SPL at 25 °C was 161 dB
and at 60 °C 165 dB. A higher SPL leads to further flattening of the droplet and therefore
the aspect ratio is increased at 60 °C. After a short equilibration time the droplet surface
temperature curves show a constant temperature that is lower than the drying air
temperature due to the evaporation of water. At the end of the measurement the
temperature increases.
Figure 5.11, Figure 5.12 and Figure 5.13 show the evaporation behaviour of water
droplets at 25 °C and 1 %, 20 %, 40 % and 60 % relative humidity in comparison of the
different initial droplet sizes. With rising relative humidity the drying time increases and
the evaporation coefficient decreases. The droplet surface temperature increases with
increasing relative humidity for all initial droplet sizes. The droplet surface temperature
values correspond for all initial droplet sizes.
Figure 5.11: Drying behaviour of water droplets at 25 °C and 1 %, 20 %, 40 % and
60 % relative humidity with an initial droplet diameter of 500 μm (SPL = 160 dB)

Results and Discussion 71
Figure 5.12: Drying behaviour of water droplets at 25 °C and 1 %, 20 %, 40 % and
60 % relative humidity with an initial droplet diameter of 800 μm (SPL = 160 dB)
Figure 5.13: Drying behaviour of water droplets at 25 °C and 1 %, 20 %, 40 % and
60 % relative humidity with an initial droplet diameters of 1200 μm (SPL = 161 dB)
The aspect ratio curves show that with the shrinking of the droplet more droplet
oscillations occur leading to an unsteady graph. For the aspect ratio no dependence on the
relative humidity can be seen in these measurements. The aspect ratio increases with

Results and Discussion 72
increasing initial droplet diameter, because droplets having a higher mass need a stronger
SPL for stable levitation that flattens the droplet. The initial SPL in the experiments was
1 dB higher for the 1200 μm droplets (161 dB) than for the smaller ones (160 dB).
Figure 5.11 for the initial droplet size of 500 μm shows an aspect ratio value < 1
leading to a prolate spheroid, whereas the droplet sizes of 1200 μm show mostly as
assumed an oblate spheroid with an aspect ratio > 1. The droplets having an initial droplet
size of 800 μm show an aspect ratio around 1. A conversion from an oblate to a prolate
spheroid can sometimes also be seen for shrinking droplets of higher initial diameters at
the end of the evaporation process. In the case of an aspect ratio < 1 the calculation of the
surface equivalent diameter has to be adapted using the formula for a prolate spheroid:
Equation 5.5
2··
2·· ·
·sin
Equation 5.6
1
The same evaporation behaviour in dependence of the relative humidity given for a drying
air temperature of 25 °C was found at higher drying air temperatures of 40 °C and 60 °C.
The evaporation behaviour of water has previously been analyzed by Tuckermann
[2002] and Schiffter and Lee [2007a]. Tuckermann published a measurement of a water
droplet at 20 °C and 28 % relative humidity. For data analysis Tuckermann used the
droplet surface area decrease which is also linear. The droplet surface temperature was also
monitored and was also almost constant at about 12.1 °C. The measurements for water
were performed without variations of the drying air conditions. Schiffter and Lee
performed measurements of evaporating water droplets at different ambient conditions
using a CCD-camera. They also found an almost linear decrease of the radius squared at
different drying air temperatures and relative humidity.
Figure 5.14 is a summary of the experimental evaporation coefficients (a)
depending on the drying air temperature and (b) on the relative humidity for 1200 μm
initial diameter droplets. The values of the evaporation coefficients were compared to the
experiments performed by Schiffter [2006]. The experimental values in Schiffter’s thesis
show the same tendency as shown for the present experiments in Figure 5.14.

Results and Discussion 73
(a)
Figure 5.14: Evaporation coefficients of water droplets with an initial horizontal
droplet diameter of 1200 μm dependent on (a) the drying air temperature and (b) the
relative humidity
The evaporation coefficient increases with increasing drying air temperature and
decreasing relative humidity. The absolute values for 25 °C and 40 °C in the experiments
correspond with the values by Schiffter, but the evaporation coefficients at 60 °C show
lower values. This might be due to the way of bringing the droplet in the pressure node. By
using the microdispensing system, the droplet has to be built up by several injections of a
droplet stream into the pressure node. The droplets can be redirected by streaming near the
pressure node, so that this procedure needs more time and another setting of the SPL than
with injection using a microsyringe. This may lead to temperature and relative humidity
variations that are more noticeable at higher drying air temperature. Additionally the
different SPL settings can lead to differences in the evaporation process.
The predicted droplet surface temperature according to Yarin et al. [1999]
(T(Yarin)) was calculated according to Equation 3.9 for all experimental conditions. The
surface temperature values were then used to calculate an evaporation coefficient.
Additionally, the wet-bulb temperature of water (T(wb)) at the respective conditions was
taken from a psychrometric calculator (www.linric.com/webpsysi.htm, Linric Company,
Bedford, USA) and a psychrometric chart (Nautical dehumidifiers Inc., Huntington, USA).
The evaporation coefficients using the wet-bulb temperature were also calculated for the
comparison of the predicted (using T(Yarin) and T(wb)) and experimental droplet surface
temperatures and evaporation coefficients. The comparison of the evaporation coefficients
and the droplet surface temperatures is given in Figure 5.15. The initial SPL was about
(b)

Results and Discussion 74
161 dB for experiments at 25 °C and 162 - 165 dB for experiments at 40 °C and 60 °C
depending on the droplet size.
(a)
(b)
(c)
(d)
(e)
Figure 5.15: Comparison of the experimental and calculated values for the
evaporation coefficients and for the droplet surface temperatures at (a, b) 25 °C (c, d)
40 °C and (e, f) 60 °C drying air temperature dependent on the relative humidity
(f)

Results and Discussion 75
Generally the evaporation coefficient increases with increasing drying air temperature and
decreasing relative humidity and the droplet surface temperature increases with increasing
drying air temperature and increasing relative humidity. The comparisons of the
evaporation coefficients show for all drying air temperatures a decrease with smaller initial
droplet diameter that is most clearly seen at a relative humidity of 1 % (Figure 5.15). The
calculated value using the wet-bulb temperature was lower than all measured evaporation
coefficients. The evaporation coefficient calculated using the method described by Yarin et
al. [1999] leads to lower values than using T(wb). With increasing relative humidity this
order is constant for 25 °C, but the deviation between the values decrease. In the graphs for
the 40 °C and the 60 °C experiments the values are closer together with rising relative
humidity and at 60 °C and 40 % / 60 % relative humidity the calculated evaporation
coefficients are now higher than the measured ones.
The droplet surface temperature values also show a deviation between the
experimental and the calculative values. The experimental surface temperature values are
higher than the wet-bulb temperature and the calculated surface temperature using the
method by Yarin et al. [1999]. In the surface temperature the experimental values show no
substantial deviation for the three droplet sizes. As for the evaporation coefficient, with
increasing relative humidity the theoretical and experimental values for the droplet surface
temperature become closer and are almost equal for high temperature and high relative
humidity. As mentioned before, a temperature deviation of 1 - 2 °C may be due to
measurement inaccuracy.
Figure 5.16, Figure 5.17 and Figure 5.18 show this phenomenon in the
experimental evaporation curves at 25 °C, 40 °C and 60 °C given at 1 % and 60 % relative
humidity. Additionally, the calculated evaporation curves using the wet-bulb temperature
for the droplet surface temperature and a horizontal line depicting the wet-bulb temperature
are displayed. The diagrams for 25 °C clearly show that the wet-bulb temperature lies
below the experimental droplet surface temperatures for low as well as for high relative
humidity. At higher drying air temperature of 40 °C and 60 °C the wet-bulb temperature is
at 1 % relative humidity lower than the experimental values, but at 60 % relative humidity
the values are almost equal. The experimental ² ⁄ 0² -graphs for 25 °C drying air
temperature have a steeper decrease than the calculated graph using the wet-bulb
temperature values. For higher drying air temperature combined with high relative
humidity this relation changes and the calculated evaporation coefficient is higher.

Results and Discussion 76
(a)
(b)
Figure 5.16: Comparison of the experimental r(t)²/r(0)²-graphs, droplet surface
temperature curves and aspect ratio curves and the calculated evaporation curve
using the wet-bulb temperature for water droplets at (a) 25 °C and 1 % relative
humidity and (b) 25 °C and 60 % relative humidity

Results and Discussion 77
(a)
(b)
Figure 5.17: Comparison of the experimental r(t)²/r(0)²-graphs, droplet surface
temperature curves and aspect ratio curves and the calculated evaporation curve
using the wet-bulb temperature for water droplets at (a) 40 °C and 1 % relative
humidity and (b) 40 °C and 60 % relative humidity

Results and Discussion 78
(a)
(b)
Figure 5.18: Comparison of the experimental r(t)²/r(0)²-graphs, droplet surface
temperature curves and aspect ratio curves and the calculated evaporation curve
using the wet-bulb temperature for water droplets at (a) 60 °C and 1 % relative
humidity and (b) 60 °C and 60 % relative humidity

Results and Discussion 79
If the evaporation process of the three droplet sizes would correspond to the d²-law, then
the curve progression should be independent of the initial droplet size. The reason for the
increasing evaporation coefficient at a higher initial droplet radius can be found in the SPL
used for levitation of the droplet. The aspect ratio is a parameter that shows the influence
of the acoustic field. A higher SPL leads to a more flattened droplet and therefore to a
higher aspect ratio. For stable levitation of the droplets of 1200 μm initial diameter a
higher SPL has to be used that can be seen at the higher aspect ratio > 1 in comparison to
the smaller initial droplet sizes. The 800 μm and 500 μm droplets have a lower aspect ratio
of about 1 or even < 1. For the 25 °C and 40 °C experiments the values are similar, but in
the 60 °C experiments a clear dependency on the droplet size can be seen. The higher SPL
leads to a higher influence of primary acoustic streaming which enhances the evaporation
process and leads to higher evaporation coefficients for droplets having a higher initial
droplet size. An additional effect is given with increasing drying air temperature, where a
higher SPL has to be used to provide stable levitation for the experiments.
The deviation of experimental and calculated graphs was already observed by
Schiffter and Lee [2007a] for water and ethanol in comparison to diffusion controlled
model. Yarin et al. [1999] gave as an explanation for the deviating evaporation coefficient
the constitution of the inner and outer acoustic streaming which both increases and
decreases evaporation (see 3.4). The deviation in experimentally-measured droplet surface
temperature and the wet-bulb temperature or the surface temperature predicted by Yarin et
al. [1999] can be explained by influences of the standing acoustic wave. This gap expands
for increasing drying air temperature and decreasing relative humidity.
Tuckermann [2002] found for a levitated water droplet at 20 °C and 28 % relative
humidity at 167 dB a droplet surface temperature of about 12.2 °C that is also above the
wet-bulb temperature of 10.5 °C. In further experiments Tuckermann found for a levitated
water droplet at 21 °C drying air temperature, a relative humidity of 35 - 40 % and a SPL
of 160 dB in the pressure nodes a droplet surface temperature of about 13 °C that is similar
to the wet-bulb temperature of 12.3 - 13.1 °C [Tuckermann et al. 2005]. In the calculation
model for the droplet surface temperature of acoustically levitated droplets Yarin et al.
[1999] take into account the influence of the inner acoustic streaming. The calculated
values using this relation (3.7.1) are located further below the values of the wet-bulb
temperature that is due to the additional convective driven evaporation induced by the
inner acoustic streaming. The experiments presented in this chapter were not conducted

Results and Discussion 80
with a drying air stream that was directed on the levitated droplet, so the outer acoustic
streaming was not eliminated to fit the premises for the calculations by Yarin et al. [1999].
The deviations between the experimental droplet surface temperatures and the values for
the wet-bulb temperature are assumed to be due to the additional energy supply to the
levitated droplet by the acoustic field. The temperature increase in the levitation chamber
after power-on of the levitator (without CEM or external heating influence) due to the
acoustic wave could clearly be seen and was in the range of up to 4 °C in this levitation
set-up. The explanation of a heat input to the droplet by the acoustic wave seems therefore
possible. This temperature increase leads to a faster evaporation of the water at higher
temperature and therefore to the higher evaporation coefficients.
To describe the deviation from the diffusion controlled evaporation process the
Sherwood number is calculated using Equation 3.8 introduced in Equation 5.3 by:
Equation 5.7
²
12· ·
·
·
0² · 2 ·
0²
For either the experimental droplet surface temperature or the wet-bulb temperature can
be used. By fitting this equation to the experimental values for ² ⁄ 0² versus ⁄
0²
the values for the Sherwood number are calculated during the evaporation process (Figure
5.19, Figure 5.20 and Figure 5.21). A value for the Sherwood number of 2 shows close
agreement with the d²-law. Values > 2 are a sign for higher drying rates than predicted by
the d²-law. The values calculated using the wet-bulb temperature are > 2 for most drying
air conditions and also higher than the values calculated using the experimental droplet
surface temperature which are close to 2. This shows good agreement using the
experimental surface temperature values for the d²-law for experiments without direct
drying air stream to the droplet. Regarding the d²-law, a higher droplet surface temperature
in the calculation will increase the binary diffusion coefficient and has also an influence on
the saturation vapour pressure at T s . The increasing evaporation coefficients are in this case
not sufficiently explained by the influence of inner acoustic streaming. The experimental
droplet surface temperatures lead to close agreement with the d²-law and the reason for the
higher evaporation coefficients can be given by the higher droplet surface temperature.

Results and Discussion 81
(a)
(b)
Figure 5.19: Comparison of the Sherwood numbers of water droplets calculated using
the experimental surface temperature T(exp) and the wet-bulb temperature T(wb) at
(a) 25 °C and 1 % relative humidity and (b) 25 °C and 60 % relative humidity

Results and Discussion 82
(a)
(b)
Figure 5.20: Comparison of the Sherwood numbers of water droplets calculated using
the experimental surface temperature T(exp) and the wet-bulb temperature T(wb) at
(a) 40 °C and 1 % relative humidity and (b) 40 °C and 60 % relative humidity

Results and Discussion 83
(a)
(b)
Figure 5.21: Comparison of the Sherwood numbers of water droplets calculated using
the experimental surface temperature T(exp) and the wet-bulb temperature T(wb) at
(a) 60 °C and 1 % relative humidity and (b) 60 °C and 40 % relative humidity

Results and Discussion 84
The Sherwood number also shows that droplets with a larger initial size have a higher
divergence from the d²-law than the 500 μm and 800 μm droplets. That shows a higher
drying rate with a higher initial droplet size. Here the influence of acoustic streaming on
droplet evaporation can be seen, because larger droplets need a higher SPL for levitation
that was already shown for the aspect ratio. With rising relative humidity the deviation of
the Sherwood number calculated using the different surface temperatures decreases.
The deviation between the experimental and calculated values for the evaporation
coefficient and the droplet surface temperature decreases for higher relative humidity of
the drying air. The initial SPL does not change noticeably, so this decrease cannot be
sufficiently explained by less application of energy by the acoustic field to the levitated
droplet. The decreasing deviation of the evaporation coefficients appears to be due to
accumulation of water vapour in the outer acoustic streaming that decreases the
evaporation process. This seems to be more important at higher relative humidity. But this
does not explain the disappearance of the surface temperature deviation, because with
increasing relative humidity around the droplet the surface temperature should increase. A
possible explanation is that with increasing relative humidity the drying air temperature
close to the droplet decreases in comparison to the drying air temperature in the chamber
due to the shielding effect of outer acoustic streaming. Especially for high drying air
temperatures this effect could lead to a drying air temperature deviation. This idea could
not be verified experimentally by temperature measurements in the levitation setup.
5.2.1.2 Influence of the drying air stream on the evaporation process
Evaporation experiments were performed using a drying air stream directed towards the
droplet at different settings of the drying air velocity in order to minimize the influence of
outer acoustic streaming on the evaporation process of the droplets. The effect on the
droplet surface temperature was also analyzed. In these experiments the air streamed
directly towards the droplets via the hole in the reflector. To exclude the influence of the
airstream on the emissivities used for the surface temperature analysis, the emissivities of
water at different conditions were determined again using different air stream settings. No
dependency with the airstream velocity was detectable, the values are similar to the ones
without airstream.
In a first set of experiments the influence of different drying air velocities of 0.75,
1.51, 2.26 and 3.02 m/s was analyzed at 25 °C and 1 % relative humidity and at 40 °C and

Results and Discussion 85
20 % relative humidity. The droplets of initial volume 1 μl were inserted into the ultrasonic
field using the microsyringe. The SPL for experiments with and without ventilation
airstream at 25 °C and 1 % relative humidity is 164.0 - 165.4 dB and at 40 °C and 20 %
relative humidity 163.4 - 164.8 dB. Experiments at a drying air velocity of 3.02 m/s require
a slightly higher SPL to avoid oscillations. The comparison of the /0²-graphs,
droplet surface temperatures and aspect ratios is given in Figure 5.22 and Figure 5.23. The
evaporation coefficient increases as expected with increasing drying air velocity for both
drying air conditions due to reduction of the slowing influence of outer acoustic streaming.
The aspect ratio is not influenced by the drying air stream. For all airstream settings it is
about 1.1 - 1.2. The droplet surface temperature seems to increase for increasing drying air
velocity at 40 °C.
The temperature curves in Figure 5.23 show fluctuating lines, which means that the
temperature measurement is subject to error by oscillations of the droplets that increase
with higher drying air velocity. The temperature fluctuations lead to measurement
inaccuracies because of changes in the droplet distance to the IR-camera. The surface
temperature is slightly higher for increasing drying air velocity, but this variation is with
1 - 2 °C close to the measurement inaccuracy.
Figure 5.22: Drying behaviour of levitated water p.a. droplets at 25 °C and 1 %
relative humidity using different drying air stream settings

Results and Discussion 86
Figure 5.23: Drying behaviour of levitated water p.a. droplets at 40 °C and 20 %
relative humidity using different drying air stream settings
Rensink [2004] investigated the evaporation of water and organic solvent droplets at
different drying air velocities and found 1 m/s as the optimum airstream velocity in his
experimental setup. At this setting the airflow was able to remove humidity accumulation
in the outer acoustic streaming, whereas additional convective influence on the evaporation
process was prevented. Schiffter [2006] found for his experimental setup the optimum
drying air velocity of 0.82 m/s. In the following experiments the drying air velocity is set
to 0.91 m/s at several drying air conditions. The droplets of initial volume of 1 μl were
inserted using the microsyringe. To ensure equal SPL settings the experiments without
direct airstream were repeated using the microsyringe. The SPL for experiments with and
without a direct drying air stream is 163.6 dB at 25 °C, 164.5 dB at 40 °C and 166.6 dB at
60 °C.
Figure 5.24, Figure 5.25 and Figure 5.26 show the comparison of the evaporation
behaviour with and without a drying air stream directed towards the droplet at different
drying air conditions. The evaporation coefficient is as expected clearly higher for the
measurements with a direct drying air stream at all drying air settings, and the drying time
decreases. The aspect ratio does not show a dependency on the drying air stream, though
droplet oscillations at high drying air velocities occur.

Results and Discussion 87
(a)
(b)
Figure 5.24: Evaporation of water droplets using 0.0 and 0.91 m/s drying air velocity
at (a) 25 °C and 1 % relative humidity and (b) 25 °C and 40 % relative humidity

Results and Discussion 88
(a)
(b)
Figure 5.25: Evaporation of water droplets using 0.0 and 0.91 m/s drying air velocity
at (a) 40 °C and 1 % relative humidity and (b) 40 °C and 40 % relative humidity

Results and Discussion 89
(a)
(b)
Figure 5.26: Evaporation of water droplets using 0.0 and 0.91 m/s drying air velocity
at (a) 60 °C and 1 % relative humidity and (b) 60 °C and 40 % relative humidity
The droplet surface temperature for the measurements without a direct air stream in the
experiments at a drying air temperature of 25 °C is higher than the surface temperature
with a direct air stream to the droplet. With increasing drying air velocity the droplet
surface is cooled by the faster evaporation, so that the droplet surface temperature

Results and Discussion 90
decreases. At a drying air temperature of 40 °C the surface temperatures are similar,
whereas at 60 °C the droplet surface temperature with a direct drying air stream is higher.
This might be due to the faster supply of fresh hot drying air to the droplet at higher drying
air velocity that overcomes the effect of increasing evaporation.
Figure 5.27 shows a comparison of the evaporation coefficients and the droplet
surface temperatures of the experiments with and without a direct drying air stream
towards the droplet. Additionally the calculated evaporation coefficients (using T(wb)) and
the wet-bulb temperatures are depicted. The evaporation coefficients for 0.91 m/s drying
air velocity lie at all ambient conditions above the values for the measurements without a
direct drying air stream and also above the values calculated using the wet-bulb
temperature in the diffusion model. This is due to the reduction of the influence of the
outer acoustic streaming via the drying air stream. The deviation becomes less with rising
relative humidity. In the experiments without additional ventilation, the increase in
accumulation of water vapour in the outer vortices might have less influence on the
evaporation process at high relative humidity, because there is a higher drying air humidity
in the whole levitation chamber.
The relation of the droplet surface temperatures changes with increasing drying air
temperature as already shown in Figure 5.24, Figure 5.25 and Figure 5.26. At low drying
air temperatures the droplet surface temperature is less higher in the experiments without
ventilation than in the experiments with 0.91 m/s drying air velocity. At 60 °C drying air
temperature this relation changes and the droplet surface temperature of the measurements
using a direct drying air stream is higher. The clearest deviation is given in the experiments
at 60 °C, where the droplet surface temperature using a direct drying air stream is about
5 °C higher than in the experiments without ventilation. A temperature deviation of about
1 °C can be due to measurement errors.
The increase of the evaporation coefficient with increasing drying air velocity
corresponds with the results by Schiffter [2006]. The absolute values are slightly higher
than the values given here due to the higher SPL at Schiffter’s experiments. Kastner [2001]
also measured faster evaporation using a direct drying air stream.

Results and Discussion 91
(a)
(b)
(c)
(d)
(e)
Figure 5.27: Experimental values for the evaporation coefficient at (a) 25 °C (c) 40 °C
and (e) 60 °C and the droplet surface temperature at (b) 25 °C (d) 40 °C and (f) 60 °C
for 0.0 and 0.91 m/s drying air velocity in comparison to the calculated evaporation
coefficient (using the wet-bulb temperature) and T(wb)
(f)

Results and Discussion 92
To quantify the deviation from diffusion controlled evaporation in the experiments the
Sherwood number is calculated. It is calculated using the diffusion model (Diff). For the
experiments with a direct drying air stream the Ranz-Marshall (RM) model for forced
convection is also used [Ranz and Marshall 1952a; Ranz and Marshall 1952b]:
Equation 5.8
2 0.6 · ⁄ · ⁄
The Reynolds number is calculated by [Baehr and Stephan 2004]:
Equation 5.9
··
using the droplet diameter (of a surface equivalent sphere) , the density of the air , the
air velocity and the dynamic viscosity of the air . The Schmidt number is
calculated using the dynamic viscosity of air , the binary diffusion coefficient and the
density of the air by [Baehr and Stephan 2004]:
Equation 5.10
·
Schiffter [2006] performed experiments using various drying air velocities and found that
the Ranz-Marshall model is accurate for an air velocity of 1 m/s or more. The air velocity
setting of 0.91 m/s in the present experiments is close to this setting, so that the comparison
using the Ranz-Marshall model is assumed to be applicable in this case.
The Sherwood number curves are given in Figure 5.28, Figure 5.29 and Figure
5.30. The experiments without a direct drying air stream using the microsyringe show a
Sherwood number at about 2, as well as in the experiments using the microdispenser head
in 5.2.1.1. The experiments with a direct drying air stream have initial Sherwood numbers
of about 4 - 6 calculated using the diffusion model, and about 6 calculated using the Ranz-
Marshall model for forced convection. This shows the increasing evaporation rate due to
the drying air stream by reducing vapour accumulation around the droplet in the outer
acoustic streaming. The calculation using the diffusion model shows decreasing Sherwood
numbers with increasing temperature and relative humidity. This means that the
evaporation approximates diffusional rates. For measurements with drying air stream
directed to the droplet the Ranz-Marshall model for forced convection is more applicable.

Results and Discussion 93
(a)
(b)
Figure 5.28: Comparison of the Sherwood numbers of water droplets calculated using
the experimental droplet surface temperature at (a) 25 °C and 1 % relative humidity
and (b) 25 °C and 40 % relative humidity (Diff = diffusion model; RM = Ranz-
Marshall model)

Results and Discussion 94
(a)
(b)
Figure 5.29: Comparison of the Sherwood number of water droplets calculated using
the experimental droplet surface temperature at (a) 40 °C and 1 % relative humidity
and (b) 40 °C and 40 % relative humidity (Diff = diffusion model; RM = Ranz-
Marshall model)

Results and Discussion 95
(a)
(b)
Figure 5.30: Comparison of the Sherwood number of water droplets calculated using
the experimental droplet surface temperature at (a) 60 °C and 1 % relative humidity
and (b) 60 °C and 40 % relative humidity (Diff = diffusion model; RM = Ranz-
Marshall model)

Results and Discussion 96
5.2.2 Evaporation of pure organic solvent droplets
The experiments were performed at just one single drying air temperature, 50 °C, and 1 %
relative humidity. The droplet surface temperature for each organic solvent was
additionally calculated using the method by Yarin et al. [1999]. The material properties
and the coefficients for the formulas are given in Table 5.2 and Table 5.3. The calculation
steps were already described in 3.7.1.
The droplets of 1 μl initial volume were inserted into the acoustic field using the
microsyringe. Due to the fast evaporation of acetone, 2-butanone, ethyl acetate and
tetrahydrofuran the initial droplet volume for these solvents was set to 2 μl to have a
sufficient test duration. The SPL was adjusted to the lowest possible setting and was
between 162.8 - 165.5 dB dependent on the organic solvent. All experiments were
performed without a direct drying air stream.
Table 5.2: Properties of the organic solvents [Engineering Toolbox 2009; Fuller et al.
1966; Yaws 2003]
Substance name Molecular
Molecular
Boiling
Density at
Diffusion
formula
weight
point T b
20 °C
volume
[g/mol]
[K]
[kg/m³]
(Fuller)
Acetone C 3 H 6 O 58.08 329.44 791 66.86
2-Butanone C 4 H 8 O 72.11 352.79 805 87.32
Dichloromethane CH 2 Cl 2 84.93 312.90 1326 59.46
Ethanol C 2 H 6 O 46.07 351.44 789 50.36
Ethyl acetate C 4 H 8 O 2 88.11 350.21 901 92.80
Methanol CH 4 O 32.04 337.85 791 29.90
2-Propanol C 3 H 8 O 60.10 355.41 785 70.82
Tetrahydrofuran C 4 H 8 O 72.11 338.00 888 67.12
Water H 2 O 18.02 373.15 998 12.70

Results and Discussion 97
Table 5.3: Material properties and coefficients for the calculation of the enthalpy of
vaporization [Yaws 2003]
Substance name Enthalpy of
vaporization at
T b [kJ/mol]
Coefficients for the formula:
· ⁄
A T c [K] n
Acetone 29.79 49.24 508.2 0.481
2-Butanone 31.22 50.65 535.5 0.450
Dichloromethane 28.38 41.91 510.0 0.410
Ethanol 39.40 43.12 516.3 0.079
Ethyl acetate 32.23 49.35 523.3 0.385
Methanol 35.14 52.72 512.6 0.377
2-Propanol 39.87 58.98 508.3 0.326
Tetrahydrofuran 30.26 44.44 540.2 0.391
Experiments with pure dichloromethane could not be performed, because the droplets
explosively evaporated in the acoustic field, even when the levitator settings (variations of
the distance between transducer and reflector or different SPL settings) were changed. The
boiling point of the substance is 39.8 °C, lower than the drying air temperature. In general
it is possible to analyze the evaporation behaviour of droplets at a temperature higher than
their boiling point due to the cooling effect of the evaporation. Dichloromethane was
analyzed as a component in solvent mixtures.
The evaporation behaviour for each organic solvent averaged from three
measurements is given in Figure 5.31 (acetone and 2-butanone), Figure 5.32 (ethanol and
ethyl acetate), Figure 5.33 (methanol and 2-propanol) and Figure 5.34 (tetrahydrofuran and
water). The aspect ratio is higher than for pure water droplets and decreases towards
sphericity at the end of the measurement. This is due to the higher surface tension of water
in comparison to other solvents that leads to more spherical droplets for water. The
measured droplet surface temperature is, as in the experiments with water, higher than the
calculated values according to Yarin et al. [1999].

Results and Discussion 98
This deviation is assumed to be caused by the energy supply of the acoustic field. The
increase in droplet surface temperature at the end of the measurement can also be seen. The
measured evaporation coefficient is much higher than the calculated one using the
diffusion model due to the influence of inner acoustic streaming that increases the
evaporation rate. This behaviour was also seen for pure water droplets.
Experiments for the analysis of the evaporation behaviour of organic solvents using
a IR-camera were already performed by Tuckermann et al. [2002; 2005]. They used a
different drying air temperature of 21 °C and 35 - 40 % relative humidity. The organic
solvents were alkanes and alkanols, but also dichloromethane was examined successfully
at the lower temperature. The surface area decrease and the surface temperature were
monitored. Most of the organic solvents show a linear decrease in surface area as for the
radius in the present experiments at higher drying air temperature. However, some solvents
show a break in the surface area decrease. Tuckermann et al. [2002; 2005] explained this
effect by condensation of water vapour on the cold droplet surface at the experimental
conditions of 35 - 40 % relative humidity. This leads to an increase in the water fraction in
the droplet. The water now evaporates predominately in the second part of the curves with
a slower evaporation rate. The droplet surface temperature curves confirm this. After a
short surface temperature decrease in the equilibration time of the droplet, the droplet
surface temperature has an almost constant value and increases only at the end of the
measurement.
In the case of water condensation on the droplet the temperature increases, when
water predominately starts to evaporate and is constant for the water evaporation time. The
equilibration time at the beginning of the measurement described by Tuckermann cannot
be seen in the experiments at 50 °C. It might take place in the time of the adjustment of the
acoustic field before the measurement could be started. Water condensation may be absent
at a relative humidity of 1 % in the 50 °C experiments. Tuckermann et al. [2002; 2005] did
not present experiments at higher drying air temperatures or lower relative humidity,
therefore the absolute values cannot be compared.

Results and Discussion 99
(a)
(b)
Figure 5.31: Evaporation behaviour of (a) acetone and (b) 2-butanone droplets,
drying air conditions: 50 °C and 1 % relative humidity

Results and Discussion 100
(a)
(b)
Figure 5.32: Evaporation behaviour of (a) ethanol and (b) ethyl acetate droplets,
drying air conditions: 50 °C and 1 % relative humidity

Results and Discussion 101
(a)
(b)
Figure 5.33: Evaporation behaviour of (a) methanol and (b) 2-propanol droplets,
drying air conditions: 50 °C and 1 % relative humidity

Results and Discussion 102
(a)
(b)
Figure 5.34: Drying behaviour of (a) tetrahydrofuran and (b) water droplets, drying
air conditions: 50 °C and 1 % relative humidity

Results and Discussion 103
Figure 5.35 shows the comparison of the evaporation coefficients and the experimental and
predicted droplet surface temperature versus the boiling point of the substances. The
evaporation coefficient has no clear dependence on the boiling point, but the tendency is
that for a higher boiling point the evaporation coefficient decreases. This can be due to
different influence of inner acoustic streaming and to droplet oscillations. The solvents
needed different adjustments of the SPL to provide less oscillations and an acceptable
sphericity. The droplet surface temperature increases clearly with increasing boiling point
and the calculated temperature values are always lower than the experimental droplet
surface temperatures.
(a)
Figure 5.35: (a) Evaporation coefficient and (b) droplet surface temperature of the
organic solvents ordered by the boiling point (squares: experimental values, triangles:
calculated values), drying air conditions: 50 °C and 1 % relative humidity
5.2.3 Evaporation of droplets containing solvent mixtures
The evaporation of solvent mixtures was analyzed for the combination of
ethanol / dichloromethane (Figure 5.36), ethanol / ethyl acetate (Figure 5.37) and
ethanol / tetrahydrofuran (Figure 5.38) at 50 °C, 1 % relative humidity and an initial
droplet volume of 2 μl. The weight proportions of ethanol-second solvent of 10:90, 25:75,
50:50, 75:25 and 90:10 were analyzed at a SPL of about 165.8 dB for
ethanol / dichloromethane and 163.3 dB for ethanol / ethyl acetate or tetrahydrofuran. For
all solvent mixtures with a high ethanol content droplet oscillations occur that can be seen
in the /0²-graphs.
(b)

Results and Discussion 104
(a)
(b)
Figure 5.36: (a) r(t)²/r(0)² and (b) droplet surface temperature curves of solvent
mixture droplets composed by ethanol and dichloromethane, drying air conditions:
50 °C and 1 % relative humidity

Results and Discussion 105
(a)
(b)
Figure 5.37: (a) r(t)²/r(0)² and (b) droplet surface temperature curves of solvent
mixture droplets composed by ethanol and ethyl acetate, drying air condition: 50 °C
and 1 % relative humidity

Results and Discussion 106
(a)
(b)
Figure 5.38: (a) r(t)²/r(0)² and (b) droplet surface temperature curves of solvent
mixture droplets composed by ethanol and tetrahydrofuran, drying air conditions:
50 °C and 1 % relative humidity

Results and Discussion 107
Dichloromethane has a very low boiling point in comparison to ethanol leading to very fast
evaporation. For very small amounts of dichloromethane its evaporation cannot be seen in
the graph, because the solvent has evaporated before the SPL could be finally adjusted and
the measurement started. For a higher dichloromethane content a break in the curve where
the ethanol starts to dominate the evaporation process can clearly be seen.
The graphs for ethanol / dichloromethane do not show a sequence dependent on the
mass content of the solvents. This might be due to the very fast evaporation of low
amounts of dichloromethane that cannot be exactly determined. The trend is that the
evaporation is faster at a higher dichloromethane content. The temperature curves for high
ethanol content do not show an increase at the beginning of the measurement due to
dichloromethane evaporation. This confirms the theory of measurement inaccuracy by fast
evaporation of small amounts of dichloromethane. For higher dichloromethane content a
decrease in the initial droplet surface temperature occurs as expected.
Ethyl acetate and ethanol neither show clear breaks in the /0²-curves nor a
clear dependency of the droplet surface temperature on the solvent ratio. A dependency of
the /0²-graph on the mixture ratio of this solvents can be seen. For higher ethanol
content the evaporation slows down, though ethyl acetate has only a slightly lower boiling
point. The curves for the ethanol / tetrahydrofuran mixtures also show breaks in the
/0²-graph with slower evaporation for higher ethanol content. Here the increase in
droplet surface temperature with increasing ethanol content can clearly be seen.
The aspect ratio curves in Figure 5.39, Figure 5.40 and Figure 5.41 show clearly the
increasing droplet oscillations with increasing ethanol content for all solvent mixtures.
Most flattened droplets occur for the ethanol / dichloromethane mixtures with an initial
aspect ratio of 1.5 - 2.0 (without considering the oscillations). Ethanol in combination with
ethyl acetate and tetrahydrofuran droplets are more spherical having an initial aspect ratio
of about 1.5. For all mixture samples the aspect ratio decreases towards the end of the
measurement.
Schiffter [2006] performed experiments for ethanol / water mixtures at 40 °C and
60 °C. For the evaporation coefficient he found an increase with decreasing water content.
Calculation of the evaporation coefficient in the first and last part of the measurement
shows that the terminal evaporation coefficient corresponds for all mixture ratios well with
the value for pure water. At the beginning the evaporation coefficient is higher with higher
ethanol content.

Results and Discussion 108
Figure 5.39: Aspect ratio curves of solvent mixture droplets composed by ethanol and
dichloromethane, drying air conditions: 50 °C and 1 % relative humidity
Figure 5.40: Aspect ratio curves of solvent mixture droplets composed by ethanol and
ethyl acetate, drying air conditions: 50 °C and 1 % relative humidity

Results and Discussion 109
Figure 5.41: Aspect ratio curves of solvent mixture droplets composed by ethanol and
tetrahydrofuran, drying air conditions: 50 °C and 1 % relative humidity
This phenomenon can be also seen in the present experiments, where the steeper curves
flatten to the end of the measurement. The relation for ethanol is inverse, because ethanol
is the solvent having the highest boiling point in comparison to the experiments with water
by Schiffter [2006].
Tuckermann et al. [2005] analyzed ideal and azeotropic solvent mixtures. For
water / methanol as an almost ideal mixture they found a decreasing surface temperature of
the evaporation droplet with increasing methanol content. Water / propanol mixtures show
a temperature minimum for middle mixture ratios and maximum surface temperature for
the pure solvents. This is due to the azeotropic mixture and corresponds closely with the
boiling points of the solvent mixture ratios. The mixture ethanol / dichloromethane has also
azeotropic behaviour. The azeotropic mixture ratio is 0.09:0.91. This cannot be determined
by the droplet surface temperature in the experiments at 50 °C, because the pure
dichloromethane that should consequently have a higher surface temperature as the
ethanol / dichloromethane 10:90 mixture was not measureable under this condition.

Results and Discussion 110
5.3 Evaporation of excipient solution droplets
5.3.1 Trehalose solution droplets
Trehalose (α,α-trehalose) is a disaccharide formed by two 1→1 linked α,α units of
glucopyranose [Gil et al. 1996]. The bond is via the reducing groups, so trehalose itself is a
non-reducing sugar. It is a natural substance that can be found in some insects, plants and
microorganisms [Richards et al. 2002]. In periods of desiccation trehalose has a protective
effect on the cells. Trehalose has the molecular formula C 12 H 22 O 11 and a molecular weight
of 342.31 g/mol. It is commonly used as a dihydrate with a molecular weight of
378.33 g/mol. The melting point of trehalose dihydrate is 97 °C, where the water
evaporates and the trehalose solidifies again at about 130 °C. The anhydrous trehalose
melts at 203 °C [Richards et al. 2002]. The maximum solubility in water at 20 °C is 68.9 g
in 100 g water [Higashiyama 2002]. Trehalose is used as an excipient in pharmaceutical
spray drying formulation due to its protective effect on proteins in aqueous solution and in
the dry product [Adler and Lee 1999]. In this chapter the drying behaviour of pure
trehalose solutions is analyzed before subsequently in 5.5 the protective effect on the
protein formulation is analyzed.
Trehalose has an amorphous modification that affects its drying behaviour. Walton
and Mumford [1999] defined in their study three distinct morphological types for powder
samples: agglomerate, skin-forming and crystalline structure. If classified in this way, then
trehalose is an example for skin-forming behaviour. Solutions of 15 % (w/w) trehalose in
water p.a. were prepared and filtered using filters of 0.2 μm pores (Schleicher and Schuell,
Dassel, Germany) before use. The drying conditions were 25 °C, 40 °C, 60 °C and 1 %,
20 %, 40 % relative humidity and the experiments were performed without a direct drying
air stream. The starting droplets had a volume of 1.5 μl and the initial SPL was between
164.4 - 166.4 dB. All experiments were performed six times and the average curves were
calculated. As in the water experiments, the /0²-curve, the aspect ratio and the
droplet surface temperature were used for analysis of the drying behaviour and here also
for the detection of the critical point.
The graphs for measurement at 25 °C and 1 % relative humidity are given in Figure
5.42 as an example. The /0²-graph shows a linear decrease of the radius squared
until the critical point appears where a crust forms. The transition is not a sharp break, but

Results and Discussion 111
changes over a long time forming a bend in the curve. The initial aspect ratio is about 1.2
and almost constant in this phase. At the critical point the droplet starts to flatten and
slowly the aspect ratio increases to about 2.0 at the end of the drying process. Therefore the
aspect ratio curve is useful to determine the critical point in the drying process. The droplet
surface temperature decreases at the beginning of measurement to a constant value in the
first drying stage. At the critical point it starts to increase and approaches the drying air
temperature. Also at low drying air temperature the droplet surface temperature curve is
rounded and shows no sharp transition. This is due to the amorphous character of trehalose.
When the water evaporates it starts to form a film on the surface of the droplet in a rubberlike
state which is deformable. Afterwards the solid glassy state is reached. This behaviour
avoids sharp transition points in the curves.
Figure 5.42: Drying behaviour of a trehalose 15 % (w/w) solution droplet at 25 °C
and 1 % relative humidity
The comparison of the curves for different drying air conditions in Figure 5.43, Figure 5.44
and Figure 5.45 shows that this behaviour can be found for all settings. The drying time
increases for increasing relative humidity due to a decrease in the water vapour pressure
gradient between the droplet surface and the surrounding air. The critical point appears
later in the /0²-graph as well as for the aspect ratio and the droplet surface

Results and Discussion 112
temperature curves. The final particle size is similar under all drying air conditions and
also the aspect ratio shows no clear dependence on the drying air conditions.
Figure 5.43: Evaporation behaviour of trehalose 15 % (w/w) solution droplets at
25 °C drying air temperature
Figure 5.44: Evaporation behaviour of trehalose 15 % (w/w) solution droplets at
40 °C drying air temperature

Results and Discussion 113
Figure 5.45: Evaporation behaviour of trehalose 15 % (w/w) solution droplets at
60 °C drying air temperature
Schiffter [2006] and Schiffter and Lee [2007b] performed experiments using trehalose
solutions without droplet surface temperature measurement. They found similar results for
the drying curves, but in the example for a solution of 100 mg/ml and 200 mg/ml trehalose
content at 60 °C and 0.1 % / 5 % relative humidity the aspect ratio increased only to 1.5.
Figure 5.46 gives a comparison of the evaporation coefficients and the droplet
surface temperatures in the first drying stage for all drying air conditions. As expected the
evaporation coefficient increases with increasing drying air temperature and decreasing
relative humidity. The droplet surface temperature increases with increasing drying air
temperature and increasing relative humidity.
In comparison to water (5.2.1.1) the values for the evaporation coefficient and the
droplet surface temperature are similar. The dissolved solid should lead to a depression of
the water vapour pressure and the droplet temperature should increase in comparison to the
pure solvent [Masters 1991]. The evaporation coefficients for water at 25 °C are even
lower than the values for the trehalose solution, but at higher drying air temperatures the
values approximate and no evident deviation due to the vapour pressure lowering effect of
trehalose can be seen.

Results and Discussion 114
(a)
Figure 5.46: (a) Evaporation coefficients and (b) droplet surface temperatures of a
15 % (w/w) trehalose solution droplet at different drying air conditions
The evaporation rate for solution droplets was calculated using the equations given in
3.7.2. In the first drying stage the droplet shrinkage was used for the calculations, whereas
in the second drying stage the vertical position of the mass centre of the droplet / particle to
the next upper pressure node was measured. The problem here is that small droplet
oscillations have a large influence on the vertical droplet position measurement. This leads
to fluctuations, so that at the end of the second drying stage slightly negative evaporation
rates are calculated.
In spite of these inaccuracies, the drying stages can be clearly seen in the graphs
given in Figure 5.47, Figure 5.48 and Figure 5.49 for different drying air temperature and
relative humidity. The first part of the curves shows a linear evaporation rate that increases
somewhat towards the critical point. At the critical point the evaporation starts to decrease
sharply because of the reduced evaporation due to formation of a surface layer on the
droplet. The fluctuations at the end of the measurement are caused by slight oscillations of
the particle around its vertical position. The trehalose 15 % (w/w) solution droplets show
the highest initial evaporation rate at drying air conditions of 60 °C and 1 % relative
humidity. The initial evaporation rate decreases for increasing relative humidity and
decreasing drying air temperature, as expected. Therefore, the lowest initial evaporation
rate can be seen in the experiments at drying air conditions of 25 °C and 40 % relative
humidity.
(b)

Results and Discussion 115
Figure 5.47: Evaporation rate of trehalose 15 % (w/w) solution droplets at 1 %
relative humidity
Figure 5.48: Evaporation rate of trehalose 15 % (w/w) solution droplets at 20 %
relative humidity

Results and Discussion 116
Figure 5.49: Evaporation rate of trehalose 15 % (w/w) solution droplets at 40 %
relative humidity
The dried particles were removed from the acoustic field using the spoon net, stored in
Eppendorf tubes, and analyzed by scanning electron microscopy. The SEM-pictures are
given in Figure 5.50. The particles for all drying air conditions are flattened, corresponding
to the aspect ratio curves. The surface is smooth and on the top side folded and wrinkled.
This corresponds with the surface of spray dried trehalose particles which also have
a smooth surface. The spray dried particles are spherical in contrast to the levitated ones. In
spray drying the droplets are allowed to rotate freely while drying. The levitated samples
are flattened due to the influence of the levitation forces that do not occur in the spray
drying process. Additionally in the levitation experiments the top side starts to collapse
downwards in the second drying stage.
This behaviour was also shown for particles produced from trehalose solutions of
100 mg/ml and 200 mg/ml at 60 °C and 5 % relative humidity [Schiffter 2006; Schiffter
and Lee 2007b]. In the present experiments for the 15 % (w/w) solution droplets no
influence of the drying air conditions on the droplet shape or surface smoothness can be
observed.

Results and Discussion 117
(a) 25 °C, 20 % rel. humidity (100x)
(b) 25 °C, 20 % rel. humidity (1000x)
(c) 40 °C, 1 % rel. humidity (100x)
(d) 40 °C, 1 % rel. humidity (1000x)
(e) 60 °C, 20 % rel. humidity (100x)
(f) spray dried at T in = 130 °C (3000x)
Figure 5.50: SEM-pictures of trehalose particles, (a-e) levitated and (f) spray dried
using a 15 % (w/w) trehalose solution at different drying air conditions

Results and Discussion 118
In additional experiments the influence of the trehalose content of the solution was
analyzed. The trehalose content was changed from 0 % to 40 % (w/w) and the solution
droplets were dried at 60 °C and 1 % relative humidity. The starting droplet volume was
1.5 μl and the initial SPL was between 165.9 - 166.7 dB. Figure 5.51 and Figure 5.52 give
the comparison of the resulting curves. The ²/0² -curve shows an earlier critical
point with increasing trehalose content, and the particles are larger. The droplet surface
temperature curves correspond with the critical point of the radius squared curves.
Figure 5.51: Drying behaviour of trehalose solution droplets at 60 °C and 1 %
relative humidity dependent on the trehalose content (r(t)²/r(0)² and droplet surface
temperature)
The temperature increase is sharper for a lower trehalose content. Droplets containing
30 % and 40 % trehalose start to form a surface layer immediately after the droplet is
brought into the acoustic field. Their droplet surface temperature increase is slower in
comparison to the less concentrated droplets at their critical point. The more concentrated
droplets did not dry, though their droplet surface temperature and aspect ratio did not
change further. They stuck to the tube wall so that the SEM pictures cannot show the
original shape. This may be due to fast formation of a surface layer that encloses moisture
inside the particle even for long drying times. The aspect ratio (Figure 5.52) has a tendency
to increase with increasing trehalose content. There are also more droplet oscillations at the
critical point especially with the more concentrated samples.

Results and Discussion 119
Figure 5.52: Drying behaviour of trehalose solution droplets at 60 °C and 1 %
relative humidity dependent on the trehalose content (aspect ratio)
The comparison of the evaporation coefficients in the first drying stage (Figure 5.53)
shows an initial increase for increasing solids content until the trehalose content produces
reduced evaporation due to rapid crust formation at 30 % and 40 % (w/w) trehalose
content.
Figure 5.53: Evaporation coefficients and droplet surface temperatures dependent on
the trehalose content of the solutions, drying air conditions: 60 °C and 1 % relative
humidity

Results and Discussion 120
These values have to be interpreted with circumspection because of droplet oscillations
even at the beginning of the drying process. The initial droplet surface temperature does
not change much. There is a tendency to a higher initial surface temperature at 30 % and
40 % trehalose content, but the difference of 1 °C is in the range of the measurement
inaccuracy. Masters [1991] predicts a vapour pressure lowering effect of the dissolved
solids. In experiments with calcium chloride solutions of different solids content an
increase in salt concentration led to a decrease in the evaporation rate [Charlesworth and
Marshall 1960]. Unfortunately, this relation cannot be shown for the complete
concentration range in these experiments.
The SEM-pictures for trehalose particles from 5 %, 10 %, 15 % and 20 % (w/w)
trehalose content are given in Figure 5.54.
(a) 5 % (w/w) trehalose (100x)
(b) 10 % (w/w) trehalose (100x)
(c) 15 % (w/w) trehalose (100x)
(d) 20 % (w/w) trehalose (100x)
Figure 5.54: SEM-pictures of trehalose particles dried using solutions of different
trehalose content, drying air conditions: 60 °C and 1 % relative humidity

Results and Discussion 121
For all initial trehalose concentrations the surface of the particle is smooth and the particle
flattened, they just differ in size due to their trehalose content. Schiffter [2006] performed
experiments at different trehalose content. Schiffter achieved similar evaporation rate
curves, which also show no clear first drying stage at high trehalose content (400 mg/ml).
With increasing trehalose content the evaporation rate in the first drying stage decreases
due to the amount of dissolved molecules and the related water vapour pressure depression
[Schiffter 2006]. Schiffter also calculated the evaporation coefficients for different
trehalose content and found a continuous decrease with increasing trehalose content. This
effect cannot clearly be seen in the evaporation coefficients of the present experiments.
However, here the evaporation coefficients increase slightly for low trehalose content, but
decrease for high trehalose content as expected. In summary, the trehalose experiments
show that for the determination of the critical point in the evaporation experiments it is
useful to measure the droplet surface temperature in addition to the droplet radius and the
aspect ratio. The results regarding particle shape and surface structure are comparable to
spray drying experiments.
5.3.2 Mannitol solution droplets
Mannitol is a sugar alcohol with the molecular formula C 6 H 14 O 6 and a molecular weight of
182.17 g/mol. The melting point of D-mannitol is 167 - 170 °C and its maximum solubility
in water at 20 °C is 182 g/l [Sigma-Aldrich 2009]. D-Mannitol is a common excipient in
freeze and spray drying. Its chief advantage is chemical stability [Yu et al. 1998]. D-
mannitol has a strong tendency to crystallize, but also exists in a fully or partially
amorphous state in certain formulations [Yu et al. 1998]. It is used in this work as a model
for the drying behaviour of a crystallizing substance according to the morphological types
given by Walton and Mumford [1999].
A mannitol 15 % (w/w) solution in water p.a. was prepared and filtered using filters
of 0.2 μm pores. The drying air conditions were 25 °C, 40 °C, 60 °C and 1 %, 20 % and
40 % relative humidity without a direct drying air stream. The droplets were inserted in the
pressure node using a microsyringe and had an initial droplet volume of 1.5 μl. The SPL
was set to the lowest level that gives sufficient droplet stability. In the experiments it was
between 162.2 - 167.9 dB depending on the drying air conditions. The experiments were
performed six times and the average curves were calculated. Figure 5.55 gives an example

Results and Discussion 122
for the curve progression of drying mannitol solution droplets at 25 °C and 1 % relative
humidity. In contrast to the trehalose experiments the transition from the first in the second
drying stage at the critical point is very steep and clearly visible in the ²/0² -graph
and the droplet surface temperature curve. The aspect ratio increases after the critical point
to 1.3. This increase is not as high as for the trehalose solution droplets that have a final
aspect ratio of up to 2.0. The mannitol particles are therefore more spherical than the
trehalose particles.
Figure 5.55: Drying behaviour of a mannitol 15 % (w/w) solution droplet at 25 °C
and 1 % relative humidity
A comparison of the curves for different drying air conditions in Figure 5.56, Figure 5.57
and Figure 5.58 shows that with increasing drying air temperature the transition to the
second drying stage at the critical point becomes sharper for the droplet surface
temperature and the /0²-curves. The aspect ratio is constant after the critical point
or decreases at high drying air temperature. Due to the crystallization of mannitol the
particle formation is faster than for amorphous substances like trehalose leading to the
sharp breaks seen in the mannitol curves. The determination of the critical point in the
drying curves is therefore easier for the crystallizing substance with sharp transition than
for trehalose with longer transition times. The droplet surface temperature is especially at

Results and Discussion 123
higher drying air temperatures a useful tool for the determination of the critical point due
to the fast increase in temperature.
Figure 5.56: Drying behaviour of mannitol 15 % (w/w) solution droplets at 25 °C
drying air temperature
Figure 5.57: Drying behaviour of mannitol 15 % (w/w) solution droplets at 40° drying
air temperature

Results and Discussion 124
Figure 5.58: Drying behaviour of mannitol 15 % (w/w) solution droplets at 60 °C
drying air temperature
The values for the evaporation coefficients and the droplet surface temperatures are given
in Figure 5.59. The evaporation coefficients at 40 °C / 60 °C and low relative humidity are
higher for mannitol in comparison to trehalose. At higher relative humidity and for the
25 °C-measurements the values correspond to the trehalose experiments. The droplet
surface temperature is similar for droplets of both solutions.
(a)
Figure 5.59: (a) Evaporation coefficients and (b) droplet surface temperatures of
mannitol 15 % (w/w) solution droplets at different drying air conditions
(b)

Results and Discussion 125
Due to the lower molecular weight of mannitol the 15 % (w/w) solution contains more
molecules than a trehalose 15 % (w/w) solution. This should lead to higher vapour pressure
depression for mannitol and lower evaporation coefficients. However, the results do not
correspond with this assumption, especially for high drying air temperatures. Schiffter
[2006] performed mannitol experiments with a 100 mg/ml mannitol solution. The results
were higher evaporation rates for mannitol than for trehalose or maltodextrin solutions.
The evaporation rate curves of mannitol 15 % (w/w) solutions at different drying
air conditions are given in Figure 5.60, Figure 5.61 and Figure 5.62. As for the trehalose
experiments the drying rate increases with increasing drying air temperature and
decreasing relative humidity. The problem that occurs in the calculation of the evaporation
rate was the precise measurement of the vertical droplet position. At the critical point the
droplets often started to oscillate and the determination of the vertical position of the
droplet or particle was imprecise or even impossible. The oscillations lead to fluctuations
in the evaporation rate curves, especially at the critical point and in the second drying
stage. This has to be considered for curve interpretation.
Figure 5.60: Evaporation rate of mannitol 15 % (w/w) solution droplets at 1 %
relative humidity

Results and Discussion 126
Figure 5.61: Evaporation rate of mannitol 15 % (w/w) solution droplets at 20 %
relative humidity
Figure 5.62: Evaporation rate of mannitol 15 % (w/w) solution droplets at 40 %
relative humidity

Results and Discussion 127
The initial evaporation rates are similar for the mannitol and the trehalose experiments.
The values of the initial evaporation rates are in the range of 0.2 - 1.4 μg/s/mm² depending
on the drying air conditions. The experiments at 40 °C and 1 % relative humidity are an
exception. Here the evaporation rate of the mannitol solution is slightly higher. The curve
progression for mannitol solutions at the critical point cannot be compared to the trehalose
curves, because droplet oscillations deform the curves. Especially at 40 % relative
humidity the graphs show strong fluctuations.
Schiffter [2006] found a sharp break at the critical point of the evaporation rate
curves for solutions of 100 mg/ml mannitol, but he also had a problem with oscillating
droplets at the critical point. The sharp breaks in the evaporation rates from his
experiments correspond with the /0²-graphs and droplet surface temperature
curves of the present measurements.
The particle shape was analyzed by scanning electron microscopy of the dried
particle. Several examples for different drying air conditions are presented in Figure 5.63.
The particles dried at low temperature are more spherical than the trehalose particles,
leading to a lower aspect ratio. The surface is rough due to the crystallizing properties of
mannitol, and the particles have “blow holes”. At higher temperature and relative humidity
the particles are also mostly spherical, but both rough and smoothed particle surfaces can
be found. In some experiments at 60 °C and 40 % relative humidity the droplets did not
form a closed spherical particle, but rather a “bowl” that is open at the top. The spray dried
particles of a mannitol 15 % (w/w) solution are more spherical and seem to have a
smoother surface than most of the levitated particles. Blow holes can also be seen in the
powder particles.
The SEM-pictures correspond with the pictures given by Schiffter [2006] and
Schiffter and Lee [2007b] for a lower concentrated solution of 100 mg/ml mannitol. The
surface shape is rough for low drying air temperature and smoother at higher drying air
temperature. The particle shape is also dependent on the relative humidity of the drying air.
At low relative humidity the particles were smoother and rougher for high relative
humidity. In the present experiments a clear dependency of the surface shape on the
relative humidity of the drying air could not be observed.

Results and Discussion 128
(a) 25°C, 1% rel. humidity (75x)
(b) 25°C, 1% rel. humidity (750x)
(c) 40 °C, 20 % rel. humidity (75x)
(d) 60 °C, 40 % rel. humidity (100x)
(e) 60 °C, 40 % rel. humidity (100x)
(f) spray dried T in = 130 °C (3000x)
Figure 5.63: SEM-pictures of (a-e) levitated and (f) spray dried mannitol 15 % (w/w)
solution particles at different drying air conditions

Results and Discussion 129
Experiments at different mannitol concentrations were performed. Due to the low
maximum solubility in water the concentration range of 0 - 15 % (w/w) mannitol content
was chosen. The inserted droplet volume was 1.0 μl and the initial SPL was between
163.1 - 167.6 dB depending on the mannitol content. The drying air temperature was 60 °C
and 1 % relative humidity. The curves for the radius squared and the droplet surface
temperature are given in Figure 5.64. For all mannitol contents the break at the critical
point is very sharp and corresponds for the /0²-graphs and the droplet surface
temperature curves. Due to the higher solids content the critical point appears earlier in the
curves for higher concentration. The initial droplet surface temperature is almost identical
at 30 °C for all mannitol concentrations.
Figure 5.64: Drying behaviour of mannitol solution droplets at 60 °C and 1 % relative
humidity dependent on the solids content (r(t)²/r(0)² and droplet surface temperature)
The aspect ratio curves (Figure 5.65) stay constant or even decrease at the critical point, so
that the aspect ratio graphs are not always useful for the detection of the critical point. A
comparison of the evaporation rates (Figure 5.66) for higher mannitol content shows a
decrease in the initial evaporation rate. This corresponds with the assumption of a slower
evaporation due to an increasing number of dissolved molecules. The critical point appears
earlier in the evaporation curves for the higher concentrated samples.

Results and Discussion 130
Figure 5.65: Drying behaviour of mannitol solution droplets at 60 °C and 1 % relative
humidity dependent on the solids content (aspect ratio)
Figure 5.66: Drying behaviour of mannitol solution droplets at 60 °C and 1 % relative
humidity dependent on the solids content (evaporation rate)
The unusual curves of the 5 % and especially for the 1 % (w/w) mannitol solution droplets
can be explained by droplet oscillations. For these concentrations oscillations occured even
at the beginning of the measurement leading to measurement inaccuracies.

Results and Discussion 131
The SEM-pictures of the particles produced using solutions of different mannitol content
are given in Figure 5.67 and Figure 5.68. The particles at low mannitol content have a
smooth surface and a relatively large hole in the middle of the particle. The picture at
larger magnification shows a star-like pattern on the surface that shows crystalline
influence during the drying process.
The particles are irregular, more so than the trehalose particles. At higher mannitol
content the particles start to become rougher on the surface. In the present experiments this
behaviour can be found at a concentration ≥ 12.5 % (w/w). Schiffter [2006] found rough
particles for a 100 mg/ml mannitol solution at 60 °C and 20 % relative humidity.
(a) 1 % (w/w) mannitol (100x)
(b) 5 % (w/w) mannitol (100x)
(c) 5 % (w/w) mannitol (1500x)
(d) 7.5 % (w/w) mannitol (100x)
Figure 5.67: SEM-pictures of mannitol particles dried using solutions of different
mannitol content, drying air conditions: 60 °C and 1 % relative humidity

Results and Discussion 132
(a) 10 % (w/w) mannitol (100x)
(b) 12.5 % (w/w) mannitol (100x)
(c) 15 % (w/w) mannitol (75x)
(d) 15 % (w/w) mannitol (1000x)
Figure 5.68: SEM-pictures of mannitol particles dried using solutions of different
mannitol content, drying air conditions: 60 °C and 1 % relative humidity
The comparison of spray dried samples of 5 % and 15 % (w/w) mannitol content solutions
(T in = 130 °C) is given in Figure 5.69. The SEM-pictures show little difference in particle
shape for the mannitol solutions, but the particles from the lower concentrated solution are
smaller. The spray dried particles show blow holes as seen with the levitated samples and
seem to have a smooth surface. The surface structure cannot clearly be seen, even in the
picture at the highest magnification from the 15 % (w/w) mannitol solution particles in
Figure 5.63 (f). Here at a magnification of 3000x the surface also seems to be smooth, and
the blow holes can be seen more clearly. For the spray dried mannitol particles no clear
dependence of the surface structure on the mannitol content, as found for the levitated
mannitol particles, could be observed in the present experiments.

Results and Discussion 133
(a) 5 % (w/w) mannitol (2000x)
(b) 15 % (w/w) mannitol (2000x)
Figure 5.69: SEM-pictures of spray dried mannitol using solutions of different
mannitol content, T in = 130 °C
5.3.3 Sucrose solution droplets
Sucrose was analyzed as a substance with high solubility in water in order to achieve a
high solids content in the sample solutions. The solubility of sucrose in water is ca.
200 g/100 ml at 20 °C and therefore higher than the solubility of trehalose or mannitol.
Sucrose is a disaccharide of D-fructose and D-glucose connected at their reducing groups.
Therefore sucrose is a non-reducing sugar with molecular formula C 12 H 22 O 11 and
molecular weight 342.30 g/mol. The melting point is 185 - 187 °C [Sigma-Aldrich 2009].
Solutions of 20 % - 60 % (w/w) sucrose content in water p.a. were prepared and
filtered as before. The droplets of 1.5 μl were inserted into the pressure node using the
microsyringe. The initial SPL was between 164.5 dB and 165.0 dB. The drying air
conditions were 60 °C and 1 % relative humidity without a direct drying air stream to the
droplet and the experiments for each concentration were performed three times. The
comparison of the /0 -curves, the droplet surface temperature curves and the
aspect ratio curves is given in Figure 5.70 and Figure 5.71. The curves show typical
amorphous behaviour without a sharp transition at the critical point. This behaviour has
already been explained for the trehalose solutions. For high concentrated sucrose solution
droplets the curve transition at the critical point is highly rounded. At sucrose
concentrations ≥ 40 % (w/w) the first drying stage and the critical point is not clearly
visible in the /0 -curves or in the droplet surface temperature curves.

Results and Discussion 134
Figure 5.70: Drying behaviour of sucrose solution droplets at 60 °C and 1 % relative
humidity dependent on the sucrose content (r(t)²/r(0)² and droplet surface
temperature)
Figure 5.71: Drying behaviour of sucrose solution droplets at 60 °C and 1 % relative
humidity dependent on the sucrose content (aspect ratio curves)
The evaporation coefficients (Figure 5.72) are almost equal, except at sucrose contents of
50 % and 60 % (w/w). Here the droplets formed a surface film immediately after insertion

Results and Discussion 135
in the acoustic field leading to reduced evaporation. The initial droplet surface temperature
is also equal for low concentrations, but increases for 50 % and 60 % (w/w) sucrose
content due to the fast formation of the surface film and therefore reduced evaporation
cooling of the droplet. In the radius squared and droplet surface temperature curves the
critical point appears later with decreasing sucrose content, as expected. The aspect ratio
curves show that the particles, especially the low concentration samples, become more
spherical during the drying time.
Figure 5.72: Evaporation coefficients and droplet surface temperatures of sucrose
solution droplets at 60 °C and 1 % relative humidity dependent on the sucrose
content
The problem in the drying of sucrose solution droplets was that for a high sucrose content
the particles did not dry completely. The particles of low concentration have a smooth
surface and were solid, whereas the high concentrated samples at 50 % and 60 % (w/w)
sucrose content stayed sticky and deformable even after long drying times. This can also
be seen in the SEM-pictures in Figure 5.73, where the 20 % (w/w) sucrose solution particle
shows a smooth surface. The 30 % (w/w) sucrose solution particle has a popcorn-like
shape because it burst during the preparation for the SEM analysis due to the residual
water content. This happened to all particles produced from higher concentrated sample
droplets and is a good indication of not completely dry particles.

Results and Discussion 136
(a) 20 % (w/w) sucrose (50x)
(b) 30 % (w/w) sucrose (35x)
Figure 5.73: SEM-pictures of sucrose particles dried using solutions of different
sucrose content, drying air conditions: 60 °C and 1 % relative humidity
Spray drying of pure sucrose solutions is difficult [Adler 1999], because the droplets stick
in the chamber and cyclone walls forming a glass-like layer. The glass transition
temperature of pure sucrose is 75 °C [Hancock and Zografi 1997]. Considering the residual
water content of the powder, the temperature of the cyclone wall is higher than the glass
transition temperature of the powder [Adler 1999]. For this reason the behaviour of high
concentrated sucrose solutions in the levitation experiments is not surprising. For high
water content in the particles it is possible that the glass transition temperature is lower
than the drying air conditions in the levitator. Even for the very wide concentration range
of the sucrose solutions it was not possible to detect a difference in the initial evaporation
rate or the initial droplet surface temperature (except for immediate film building at high
concentrations of 50 % and 60 % (w/w)). The experiments show again how important the
additional measurement of the droplet surface temperature for the detection of the critical
point is.
5.3.4 Copolyvidone and HPMC solution droplets
Preliminary experiments for the itraconazole measurements in Chapter 5.4 examine the
excipients copolyvidone and hydroxypropylmethylcellulose (HPMC) dissolved in a solvent
mixture containing different ratios of ethanol and dichloromethane. The drying
experiments were performed using nitrogen as drying gas at a drying gas temperature of
50 °C and 0.5 % relative humidity without a drying air stream directed towards the droplet.
The droplets with an initial volume of 2 μl were inserted in the pressure node using the

Results and Discussion 137
microsyringe. The SPL was set to the lowest possible value of 160.0 dB (copolyvidone)
and 160.4 dB (HPMC). The solvent ratios of dichloromethane-ethanol were 58.8:41.2
(start formulation), 70:30, 60:40, 50:50, 40:60 and 30:70. The polymer content for the pure
excipient solution was 4.9 % (w/w) for each polymer.
The drying behaviour of copolyvidone and HPMC in the start formulations is given
in Figure 5.74 and Figure 5.75. The experiments were performed for a longer time than
depicted, and the figure is a part of the drying behaviour in the first drying stage and the
critical point. The copolyvidone solution droplets show the common curve progression for
an amorphous substance that was already found for trehalose and sucrose. The droplets
form a crust at the critical point that leads to an immediate decrease in evaporation,
therefore the critical point is clearly visible in all three curves. In contrast to the
copolyvidone solution the HPMC solution does not show a clear first drying stage in the
droplet surface temperature and the aspect ratio curves. The droplet surface temperature
increases immediately after the droplet was placed into the pressure node. The aspect ratio
shows fluctuations at the beginning of the measurement and then it increases slightly. The
radius squared plot changes its slope, but the critical point cannot clearly be seen due to
oscillations at the beginning of the measurement. For both excipient solutions the known
drying behaviour of the pure binary solvent mixture cannot be seen in the graphs. The
initial droplet surface temperature is higher than the droplet surface temperature of the
binary solvent mixture. Here the influence of the fast dichloromethane evaporation that
leads to cooling to very low temperature of the droplet surface at the beginning of the
measurement is strongly reduced by addition of the polymer. The droplet surface
temperature of the polymer solutions is more similar to the ethanol surface temperature.
When placing the sample in the pressure node it could be observed for the HPMC
solutions that immediately after the solution leaves the syringe a film appears on the
droplet surface. This leads to the early increase in droplet surface temperature and the
differing drying behaviour in comparison to the copolyvidone solutions. In the first drying
stage strong droplet oscillations occur until the surface film is sufficiently solidified. This
leads to strong measurement inaccuracies in the /0²-graph that make it impossible
to determine the evaporation coefficients for the HPMC formulations. Neither different
SPL settings nor different levitator adjustments were able to stop the oscillations and give
more precise results.

Results and Discussion 138
Figure 5.74: Drying behaviour of a copolyvidone start formulation droplet at 50 °C
and 0.5 % relative humidity
Figure 5.75: Drying behaviour of a HPMC start formulation droplet at 50 °C and
0.5 % relative humidity

Results and Discussion 139
The comparison of the formulations at different solvent ratios is given in Figure 5.76 for
copolyvidone and Figure 5.77 for HPMC. The copolyvidone temperature graphs show a
tendency to a lower initial droplet surface temperature for higher dichloromethane content
due to its faster evaporation and therefore stronger cooling of the droplet. The critical point
that can clearly be seen in the measured curves appears later for increasing ethanol content
also because of the slower evaporation of ethanol in comparison to dichloromethane.
The critical points in the radius squared curves correspond with the temperature
increases as well as with the aspect ratio increases. The graphs for the HPMC solution
droplets are not analyzable due to the strong droplet oscillations, but the curves show that
the “critical point” must be early in the drying process in comparison to the copolyvidone
solutions. Note that the end of the depicted graphs occurs after a third of the measurement,
the first drying stage and the critical point of the different formulations are shown in detail.
Also for the HPMC solutions the droplet surface temperature increases up to about 50 °C
by the end of the measurement.
Figure 5.76: Comparison of the drying behaviour of the copolyvidone formulations
having different solvent ratios, drying gas conditions: 50 °C and 0.5 % relative
humidity

Results and Discussion 140
Figure 5.77: Comparison of the drying behaviour of the HPMC formulations having
different solvent ratios, drying gas conditions: 50 °C and 0.5 % relative humidity
The SEM-pictures in Figure 5.78 show the different particle shapes for both substances in
the start formulation. The surface of the copolyvidone as well as for the HPMC particle is
smooth as expected for amorphous substances. The particle shape is more crinkled for the
HPMC particles than for the copolyvidone particles. The droplet movement for the HPMC
solutions due to the crust collapsing leading to crinkled particles could be the reason for
the strong droplet oscillations at the beginning of the measurements. The oscillations stop
when the final particle size is reached.
(a) copolyvidone start (100x)
(b) HPMC start (100x)
Figure 5.78: SEM-pictures of the start formulation particles containing copolyvidone
or HPMC as polymer, drying gas conditions: 50 °C and 0.5 % relative humidity

Results and Discussion 141
The particle shape is not dependent on the solvent ratio as the pictures for the start
formulations and the 30:70 or 40:60 formulations for the polymers in Figure 5.79 show.
SEM-pictures for the other solvent ratios (not shown) confirm this, they all look similar to
the related start formulations.
(a) copolyvidone 40:60 (100x)
(b) copolyvidone 40:60 (1000x)
(c) HPMC 30:70 (75x)
(d) HPMC 30:70 (1000x)
Figure 5.79: SEM-pictures of particles of different polymer formulations, drying gas
conditions: 50 °C and 0.5 % relative humidity
5.4 Itraconazole formulation experiments
Itraconazole formulations containing as a polymer excipient either copolyvidone or HPMC
were analyzed at different ratios of the solvents dichloromethane and ethanol. The aim of
this single droplet drying experiment is to find a dependence of the kind of polymer
excipient in the formulation and the solvent ratio on the final particle shape. Previous spray
drying experiments of different itraconazole formulations had shown different powder

Results and Discussion 142
appearances. The information from the levitation experiments can be integrated in the
development of an optimum formulation for spray drying of itraconazole.
The formulations contained 3.2 % itraconazole, 4.8 % copolyvidone or HPMC and
92 % solvent mixture of different solvent ratio of dichloromethane and ethanol. The
solvent ratios were 58.8:41.2 (start formulation), 70:30, 60:40, 50:50, 40:60 and 30:70, the
same as for the pure excipient solutions. The drying gas was nitrogen and the experiments
were performed at a drying air temperature of 50 °C and 0.5 % relative humidity without a
direct drying air stream. The initial droplet volume was 2 μl, and the initial SPL, as set to
the lowest setting for stable levitation, was 159.5 dB. The comparison of the drying curves
for both start formulations is given in Figure 5.80 and Figure 5.81. The
itraconazole / copolyvidone formulation shows similar behaviour to the pure copolyvidone
formulation. The critical point can clearly be seen in the graphs for the radius squared, the
droplet surface temperature and the aspect ratio. In contrast to the excipient formulation,
the critical point appears earlier in the curves due to the higher solid content achieved by
itraconazole addition. The particle stays more spherical, so that the aspect ratio curve is
almost constant around 1.1, in contrast to the pure copolyvidone solutions.
Figure 5.80: Drying behaviour of itraconazole / copolyvidone start formulation
droplets, drying gas conditions: 50 °C and 0.5 % relative humidity

Results and Discussion 143
Figure 5.81: Drying behaviour of itraconazole / HPMC start formulation droplets,
drying gas conditions: 50 °C and 0.5 % relative humidity
The itraconazole / HPMC formulation is also similar to its basic pure excipient
formulation. Again the droplet surface temperature increases directly after the particle was
inserted into the acoustic field. The radius squared and aspect ratio curves show droplet
oscillations, but much less than for the pure excipient solution. The critical point is not
clearly detectable in the curves. The aspect ratio increases from 1.1 to about 1.3, in contrast
to the formulation containing copolyvidone. Note that the particles were dried for a longer
time than depicted.
Figure 5.82 and Figure 5.83 show the comparison of the formulations having
different solvent ratio. The itraconazole / copolyvidone formulations show that the critical
point in the curves appears later for increasing amount of ethanol in the solvent mixture.
This is due to its slower evaporation as already shown for the pure excipient solutions. The
30:70 formulation needs nearly twice the drying time to the critical point than the 70:30
solution. The solubility of itraconazole could have an opposite effect, because itraconazole
is less soluble in ethanol (0.2 g/l) than in dichloromethane (250 g/l). But this influence
cannot be seen in the drying time to the critical point. The critical point can be clearly seen
in all measured curves for different solvent ratios. The curve progression is similar to the
pure copolyvidone solutions, showing that the evaporation behaviour of the solution is
dependent on the polymer and not on the itraconazole.

Results and Discussion 144
Figure 5.82: Comparison of the drying behaviour of itraconazole / copolyvidone
formulations, drying gas conditions: 50 °C and 0.5 % relative humidity at different
solvent ratios
Figure 5.83: Comparison of the drying behaviour of itraconazole / HPMC
formulations, drying gas conditions: 50 °C and 0.5 % relative humidity at different
solvent ratios

Results and Discussion 145
Figure 5.84 gives the comparison of the evaporation coefficients depending on the ethanol
content for the pure copolyvidone and the itraconazole / copolyvidone formulations. The
absolute values for both experimental series are similar, but the pure copolyvidone values
show more fluctuations. For both series the evaporation coefficient decreases clearly with
higher ethanol content. The itraconazole / HPMC solutions show less oscillation in
comparison to the pure HPMC solutions. Here the curves can be better compared, but still
the calculation of the evaporation coefficients is imprecise. For none of the measured
curves the critical point is detectable. The radius squared curves, the droplet surface
temperature and the aspect ratio have similar curve progressions for all solvent ratios. The
absence of a clear critical point can again be explained by an immediate surface film
forming on insertion of the solution droplet in the acoustic field.
Figure 5.84: Evaporation coefficients of copolyvidone and itraconazole / copolyvidone
formulations depending on the ethanol content, drying gas conditions: 50 °C and
0.5 % relative humidity
The SEM-pictures given in Figure 5.85 show the particle shapes of the start formulation
particles either containing copolyvidone or HPMC. The particle shape departs completely
from that of the pure excipient solutions. The itraconazole / copolyvidone particle is almost
spherical but much more crinkled on the surface. The itraconazole / HPMC particle is also
more spherical than the pure excipient formulation, but has a less crinkled surface. For the
HPMC formulation the addition of itraconazole leads to smoothing of the surface, whereas

Results and Discussion 146
for the copolyvidone formulation the particle becomes more crinkled. For a higher ethanol
ratio the particles are more crinkled than in the start formulations for both of the
itraconazole / polymer formulations as shown Figure 5.86.
(a) itraconazole / copolyvid. start (100x)
(b) itraconazole / copolyvid. start (1000x)
(c) itraconazole / HPMC start (100x)
(d) itraconazole / HPMC start (1000x)
Figure 5.85: SEM-pictures of start formulation particles of itraconazole with
copolyvidone and HPMC, drying gas conditions: 50 °C and 0.5 % relative humidity
The itraconazole / copolyvidone particle at a solvent ratio of 30:70 is more spherical than
the particle produced from the HPMC formulation. The difference in the particle shape for
the formulation containing HPMC is noticeable, where for high ethanol content the surface
structure changes from smooth to crinkled for the 40:60 and the 30:70 formulation. This
can be due to the sample that changes for high ethanol content in both formulations to a
suspension because the itraconazole is not sufficiently soluble in the solvent mixture due to
the high ethanol ratio. In the case of the formulation containing HPMC and itraconazole

Results and Discussion 147
the dispersed particles in the suspension may to lead to the crinkled surface in comparison
to the solution formulations with higher dichloromethane content.
(a) itraconazole / copolyvid. 30:70 (100x)
(b) itraconazole / copolyvid. 30:70 (1000x)
(c) itraconazole / HPMC 30:70 (100x)
(d) itraconazole / HPMC 30:70 (1000x)
(e) itraconazole / HPMC 50:50 (100x)
(f) itraconazole / HPMC 50:50 (1000x)
Figure 5.86: SEM-pictures of itraconazole / HPMC and itraconazole / copolyvidone
formulation particles, drying gas conditions: 50 °C and 0.5 % relative humidity

Results and Discussion 148
As a result the itraconazole formulations containing copolyvidone or HPMC show a
completely different drying behaviour. For the copolyvidone formulation the dependence
of the drying time to the critical point on the solvent mixture is clearly visible. The drying
rate is dependent on the polymer and not on the itraconazole, shown by the similar curve
progression for both the pure copolyvidone and the itraconazole / copolyvidone graphs. In
contrast the final particle shape differs between the pure excipient and the
itraconazole / excipient formulations and also between the solutions and suspensions for
itraconazole / HPMC. Here the itraconazole has a strong influence on the particle surface
appearance.
5.5 Drying of protein solutions
5.5.1 Bovine carbonic anhydrase (bCA) droplets
The aim is to analyze the drying behaviour, the particle formation and the final particle
shape of different bCA solution formulations. Any protein stabilization in the drying
process due to trehalose addition is analyzed via measurement of the residual activity of
bCA. The results of formulations with different trehalose content are compared using
levitated particles as well as spray dried powders. In further experiments the kinetics of
protein inactivation in the drying process are examined by removing the droplet or particle
from the acoustic field at different times. The characterisation of bCA is given in 4.1.1.1.
Trehalose is able to stabilize proteins in solution, during the drying process and in
the dry product. The three main hypotheses for the protection mechanisms of trehalose are
water-replacement, water-layer (or preferential exclusion), and mechanical-entrapment (or
glass immobilisation) [Lins et al. 2004]. The water-replacement hypothesis attributes the
stabilisation of the protein to direct interaction via hydrogen bonds between the protein and
the hydroxyl-groups of trehalose [Allison et al. 1999; Arakawa et al. 2001]. The waterlayer
hypothesis assumes that a layer of water molecules is trapped close to the protein
surface due to the preferential exclusion of trehalose from the vicinity of the protein
[Belton and Gil 1994]. Trehalose is an amorphous substance that builds up a glass state
during the drying process that reduces protein unfolding due to mechanical entrapment
[Hagen et al. 1995; Lins et al. 2004].
The bCA and trehalose were dissolved in 50 mM trizma-buffer pH 7.5 and stored on ice
until the measurement begins. The experiments were performed first with solutions of bCA

Results and Discussion 149
and trehalose total content of 10 % (w/v). Further experiments used a bCA content of
10 % (w/v) plus trehalose to provide either a constant total solids content or a constant
protein content. The experiments were performed at drying air conditions of 60 °C and
10 % relative humidity without a direct drying air stream. The initial droplet volume was
1.5 μl, the initial SPL was about 167.0 dB, and the experiments were performed six times
each. Experiments with bCA and also other tested proteins are much more difficult than
the excipient experiments using sugars or polymers. The droplets start to oscillate strongly
and at / after the critical point the particles often break and fall out of the acoustic field so
that the experiment has to be repeated. The measurement of the distance to the next upper
pressure node for the calculation of the evaporation rate is therefore not possible due to
strong oscillations.
Figure 5.87 shows the drying behaviour of a 10 % (w/v) bCA solution. Both the
/0²-curve and the droplet surface temperature curve show the critical point in the
drying process as a curve, as already seen with amorphous substances. The aspect ratio is
about 1.4, decreases before the critical point, and rises again to 1.4 for the final particle.
The aspect ratio is here not useful for clear detection of the critical point.
Figure 5.87: Drying behaviour of a droplet containing bCA 10 % (w/v) in 50 mM
trizma buffer pH 7.5, drying air conditions: 60 °C and 10 % relative humidity

Results and Discussion 150
The SEM-picture of a 10 % (w/v) bCA solution particle is given in Figure 5.88, as well as
a SEM-picture of spray dried powder particles using a 10 % (w/v) bCA solution. In both
cases the bCA particles have a smooth surface and a hollow interior. The pure levitated
protein particles are very fragile and often break after the critical point or during storage in
Eppendorf-tubes before SEM-pictures can be taken. Even a small amount of trehalose in
the formulation (20 % of total solids) leads to much better levitation behaviour and storage
stability of the particles. Schiffter and Lee [2007b] found also hollow particles for pure
catalase as levitated particles and as a spray dried formulation. This particle shape seems to
be common for pure or buffered aqueous protein solutions and will also be shown for pure
LDH particles in 5.5.2.
(a) 60 °C, 10 % rel. humidity (100x)
(b) spray dried T in = 130 °C (3000x)
Figure 5.88: SEM-pictures of (a) a levitated particle and (b) the spray dried powder
of bCA 10 % (w/v) in 50 mM trizma buffer pH 7.5
A trehalose solution 10 % (w/v) in 50 mM trizma buffer pH 7.5 was also dried in the
levitator at 60°C and 10% relative humidity and in the spray dryer at T in = 130 °C. The
SEM-pictures of a levitated particle and of the spray dried powder are given in Figure 5.89.
Both the levitated and the spray dried particles show similar particle shape compared to the
experiments using trehalose in pure water in 5.3.1. The levitated particle is (as for the
trehalose in pure water) not completely spherical and has a smooth surface, but it is not as
much flattened as the trehalose in water particle. The spray dried particles are almost
spherical.

Results and Discussion 151
(a) 60 °C, 10 % rel. humidity (100x)
(b) spray dried T in = 130 °C (3000x)
Figure 5.89: SEM-pictures of (a) a levitated particle and (b) the spray dried powder
of trehalose 10 % (w/v) in 50 mM trizma buffer pH 7.5
5.5.1.1 bCA-trehalose 10 % (w/v) total solids formulations
In the first set of experiments trehalose was added to the bCA solutions achieving a total
solids’ content of 10 % (w/v) (except buffer salts). The bCA-trehalose ratios used are 8:2,
6:4, 4:6 and 2:8. Figure 5.90 shows the comparison of the droplet surface temperature and
the /0²-curves for the different formulations. The droplet surface temperature is
little influenced by the amount of trehalose in the mixtures. For higher trehalose content
the temperature increase is slightly shifted to later drying times. The /0²-curves
show a similar slope at the beginning. The critical point tends to appear also later for
higher trehalose content. The aspect ratio of the droplet is about 1.3 - 1.4 at the beginning
of the measurement and increases after the critical point as shown in Figure 5.91. For high
bCA content the increase cannot clearly be seen, but with increasing trehalose content the
aspect ratio increases up to about 2.4 for the 2:8 formulation. Yet pure trehalose
10 % (w/v) solution droplets do not show such an increase in the aspect ratio after the
critical point.
The dried particles were removed from the acoustic field to determine both enzyme
activity assay and to take SEM-pictures. The total drying time was set to 563 s.
Additionally, the protein formulations were spray dried at 130 °C inlet temperature to
produce powders for comparison of the residual activity of bCA and of the particle shape.
Figure 5.92 shows the comparison of the residual activity of the bCA formulations after the
drying process in the levitator or spray dryer.

Results and Discussion 152
Figure 5.90: Comparison of the drying behaviour of bCA-trehalose 10 % (w/v) total
solids formulation droplets, drying air conditions: 60 °C and 10 % relative humidity
(r(t)²/r(0)² and droplet surface temperature)
Figure 5.91: Comparison of the drying behaviour of bCA-trehalose 10 % (w/v) total
solids formulation droplets, drying air conditions: 60 °C and 10 % relative humidity
(aspect ratio)

Results and Discussion 153
For a trehalose portion of ≥ 40 % of the total solids the residual activity for both the
levitated and the spray dried samples is about 100 %. With ≤ 20 % trehalose a lower
residual activity for the levitated samples and the pure bCA 10 % (w/v) solution is clearly
shown. In spite of the scatter in the measured activity values this figure shows the
stabilizing effect of even small amounts of trehalose on bCA.
The similarity between the levitation and spray drying experiments is remarkable.
During spray drying the droplets are exposed to a much higher drying air temperature.
Additionally the droplet surface area, where proteins can adsorb and unfold [Adler and Lee
1999], is much higher in spray drying (related to the same sample volume) due to the
smaller droplet size. This was assumed to lead to a higher protein deactivation during the
spray drying process. However, the results compared to the levitation experiments are
similar, possibly because of the longer drying time in the levitator. An influence of the
ultrasonic field was excluded by Weis and Nardozzi [2005], who found similar enzyme
kinetics in a bulk protein solution and in levitated protein droplets. Residual activity
experiments using catalase and trehalose were performed by Schiffter [2006]. Schiffter
achieved sufficient protein stability at a trehalose content of 20 % of total solids.
Figure 5.92: Residual activity of bCA depending on the trehalose content of the
formulations in comparison of levitated and spray dried samples (10 % (w/v) total
solids)

Results and Discussion 154
The SEM-pictures of the levitated particles and the spray dried powders are given in Figure
5.93 and Figure 5.94. All of the levitated particles show a smooth and less crinkled surface.
The 8:2 sample has, as already shown for the pure bCA particles, a hole at the top and
shows its hollow interior. For higher trehalose content the holes in the particles disappear
and the particles are more flattened. This relation was already shown in the aspect ratio
curves in Figure 5.91. However, the pure trehalose particle (Figure 5.89 (a)) is more
spherical than the particles having high trehalose content. The spray dried powders show
crinkled particles for all bCA formulations. Particles with a high amount of bCA show
fewer but deeper dimples than the particles with high trehalose content. In comparison, the
pure trehalose powder particles are nearly spherical (Figure 5.89 (b)). The dimples seen in
the spray dried powder particles cannot directly be found in the levitated particles.
(a) bCA-trehalose 10 % 8:2 (100x)
(b) bCA-trehalose 10 % 8:2 (3000x)
(c) bCA-trehalose 10 % 6:4 (100x)
(d) bCA-trehalose 10 % 6:4 (3000x)
Figure 5.93: SEM-pictures of the (a, c) levitated and (b, d) spray dried bCA-trehalose
10 % (w/v) total solids formulations, drying air conditions: 60 °C and 10 % relative
humidity

Results and Discussion 155
(a) bCA-trehalose 10 % 4:6 (100x)
(b) bCA-trehalose 10 % 4:6 (3000x)
(c) bCA-trehalose 10 % 2:8 (100x)
(d) bCA-trehalose 10 % 2:8 (3000x)
Figure 5.94: SEM-pictures of the (a, c) levitated and (b, d) spray dried bCA-trehalose
10 % (w/v) total solids formulations, drying air conditions: 60 °C and 10 % relative
humidity
A similarity between the levitated particle samples and the spray dried powder is the
inclination of the particle top side that can be seen in the 6:4 and 2:8 formulation particles.
Spray drying experiments with catalase [Schiffter 2006] led to similar results for the
change of the particle shape. With increasing trehalose content the particle surface is more
crinkled.
The period in which protein inactivation takes place was analyzed by experiments
with different drying times between 0 - 563 s. Before the critical point the droplets were
collected directly into the PCR tubes, whereas after the critical point the particles were
removed using a spoon net. The experiments were performed for all bCA-trehalose
formulations at 6 different points of time and the results are given in Figure 5.95. The 6:4,
4:6 and 2:8 formulations do not show any substantial decrease in the residual protein

Results and Discussion 156
activity during the drying process. The 8:2 formulation and pure bCA 10 % (w/v) show a
decrease in residual activity towards the end of the measurement. The critical points for
these two solutions appear at about 265 s drying time. The residual activity results show
that inactivation takes place predominantly after the critical point.
Protein inactivation during the drying process may take place mostly via surface
adsorption of the proteins to the droplet surface and also the influence of temperature [Maa
and Hsu 1997; Mumenthaler et al. 1994]. Surface adsorption of the protein will take place
before the critical point, but the holes and the hollow interior of the particles having high
bCA content show an increase in the surface area at crust formation. Therefore a larger
area for surface adsorption of the protein is available after the critical point. The
temperature of the droplet or particle increases after the critical point leading to higher
thermal stress on the protein especially when it is still able to unfold in the liquid parts of
the droplet / particle. The results show therefore that single droplet drying experiments are
a helpful method to examine the drying process and its influence on residual protein
activity.
Figure 5.95: Residual activity of bCA depending on the drying time for the bCAtrehalose
10 % (w/v) total solids formulations, drying air conditions: 60 °C and 10 %
relative humidity

Results and Discussion 157
5.5.1.2 bCA 10 % (w/v) plus trehalose formulations
The protein stabilizing experiments were repeated using a constant bCA 10 % (w/v)
content plus trehalose in the ratios of 10:10, 10:5 and 10:3.3 with a 50 mM trizma buffer
pH 7.5 as solvent. The advantage of these experiments is a constant bCA content in the
protein activity assays. The drying behaviour of this formulations is compared in Figure
5.96 and Figure 5.97. The /0²-curves for all mixtures have critical points that lie
close together, though the solution droplets have different solids content. In the droplet
temperature curves the critical point can clearly be seen. These curves show an earlier
temperature increase for a higher trehalose content and therefore for a higher solids
content, as expected. For formulations with high trehalose content the temperature increase
is prolonged. However, pure trehalose droplets have a faster temperature increase in
comparison to the 10:5 and 10:10 mixtures. The formulations having higher trehalose
content have a higher aspect ratio than the pure protein or trehalose droplets, but no clear
dependency can be seen.
Figure 5.96: Comparison of the drying behaviour of bCA 10 % (w/v) plus trehalose
formulation droplets, drying air conditions: 60 °C and 10 % relative humidity
(r(t)²/r(0)² and droplet surface temperature)
The residual protein activity of the formulation particles does not substantially change with
increasing trehalose content as shown in Figure 5.98. The residual activity of the pure bCA
particles is much lower than that of the stabilized formulations. The results for the

Results and Discussion 158
levitation and the spray drying experiments are again similar to the bCA-trehalose 10 %
total solids formulations.
Figure 5.97: Comparison of the drying behaviour of bCA 10 % (w/v) plus trehalose
formulation droplets dried at 60 °C and 10 % relative humidity (aspect ratio)
Figure 5.98: Residual activity of bCA depending on the trehalose content in
comparison of levitated and spray dried samples (bCA 10 % (w/v) plus trehalose)

Results and Discussion 159
The results for the residual protein activity measurements of bCA at different drying times
are given in Figure 5.99. Here none of the bCA-trehalose formulations shows a decrease in
the residual protein activity, because of the relative high trehalose content. Even the 10:3.3
formulation shows sufficient stabilization of the protein. This confirms the results for the
bCA-trehalose 10 % (w/v) total solids formulations, where the minimum trehalose ratio
necessary for stabilization is between 8:2 and 6:4.
Figure 5.99: Residual activity of bCA depending on the drying time for the bCA
10 % (w/v) plus trehalose formulations, drying air conditions: 60 °C, 10 % relative
humidity
Figure 5.100 gives the comparison of the SEM-pictures of levitated particles and spray
dried powders for the bCA 10 % (w/v) plus trehalose formulations. The levitated particles
of all three formulations show a smooth surface and have an inclination of the top side that
leads to a hole with increasing bCA content. The partially fractured particle of the 10:3.3
formulation shows clearly the hole and the hollow interior of the particle. The spray dried
powders show deeper dimples for low trehalose content in the 10:3.3 formulation than for
high trehalose content in the 10:10 formulation. With increasing trehalose content the
powder particles are more crinkled.

Results and Discussion 160
(a) bCA 10 % plus trehalose 10:3.3 (100x)
(b) bCA 10 % plus trehalose 10:3.3(3000x)
(c) bCA 10 % plus trehalose 10:5 (100x)
(d) bCA 10 % plus trehalose 10:5 (3000x)
(e) bCA 10 % plus trehalose 10:10 (100x)
(f) bCA 10 % plus trehalose 10:10 (3000x)
Figure 5.100: SEM-pictures of the (a, c, e) levitated and (b, d, f) spray dried bCA
10 % (w/v) plus trehalose formulations, drying air conditions: 60 °C and 10 %
relative humidity

Results and Discussion 161
5.5.2 L-Lactic dehydrogenase (LDH) droplets
L-Lactic dehydrogenase from rabbit muscle is chosen as a second model protein to achieve
a higher activity loss in the stabilization experiments. LDH is assumed to be more sensitive
to thermal stress [Adler and Lee 1999]. Porcine LDH was used in spray drying
experiments by Adler and Lee [1999] using trehalose as a carrier to analyze powder
particle shape, the residual protein activity after the drying process and the storage
stability. They found an opposing effect of the inlet temperature that led for a low
temperature setting to high residual protein activity after the drying process, but the higher
water content of the spray dried powder produced less storage stability.
In the present experiments LDH was (after dialysis and concentration of the
original LDH-suspension) diluted with a 100 mM potassium phosphate buffer pH 7.0.
Trehalose was added for the two test series of LDH-trehalose 10 % (w/v) total solids
(except buffer salts) and 10 % (w/v) LDH plus trehalose. The experiments were performed
at a drying air temperature of 60 °C and a relative humidity of 10 % without a direct drying
air stream. The initial droplet volume was 1.5 μl and the initial SPL was about 167.0 dB.
The drying behaviour measured via the ²/0²-curve, droplet surface temperature and
aspect ratio, and the residual activity of the protein for the levitated and spray dried
formulations was analyzed. Strong oscillations occurred and especially the pure LDH
10 % (w/v) solution particles break after the critical point or fell out of the pressure node.
For this reason the LDH measurements were difficult to perform.
The drying behaviour of LDH 10 % (w/v) in 100 mM potassium phosphate buffer
pH 7.0 is presented in Figure 5.101. The ²/0²-curves and the droplet surface
temperature curves show a bend at the critical point that indicates an amorphous state of
the protein. The aspect ratio increases in comparison to the bCA 10 % (w/v in 50 mM
trizma buffer pH 7.5) droplets at the critical point from 1.3 up to 1.8. Particle oscillations
can be seen in the graph, reflecting the levitation problems for the pure LDH solution
droplets. As mentioned before, after the critical point the particles started to oscillate and
often fell out of the pressure node. Fragile particles break, making it difficult to achieve a
sufficient drying time for the LDH 10 % (w/v) particles. As already shown for the bCA
particles, the addition of small amounts of trehalose led to better drying behaviour in the
levitator.

Results and Discussion 162
Figure 5.101: Drying behaviour of a LDH 10 % (w/v) in 100 mM phosphate buffer
pH 7.0 droplet, drying air conditions: 60 °C and 10 % relative humidity
The SEM-picture of the levitated LDH 10 % (w/v) particle in Figure 5.102 shows the
fragility of the sample. Even if the particle could be removed intact after drying was
completed, it breaks during the preparation for the SEM analysis. But the broken particle
still shows that pure LDH 10 % (w/v) solution droplets lead to hollow particles with a hole
at the top side. The spray dried powder of the same formulation (inlet temperature 130 °C)
shows particles with dimples and holes that correspond with the shape of the levitated
particle.
(a) 60 °C, 10 % rel. humidity (100x)
(b) spray dried T in = 130 °C (3000x)
Figure 5.102: SEM-pictures of (a) a levitated particle and (b) the spray dried powder
of LDH 10 % (w/v) in 100 mM phosphate buffer pH 7.0

Results and Discussion 163
Trehalose 10 % (w/v) dissolved in 100 mM potassium phosphate buffer pH 7.0 shows a
very strange particle shape of the levitator dried droplet shown in Figure 5.103. The droplet
formed a surface film immediately after it was brought into the pressure node, and starts to
flatten. The resulting particles look like a disc. This appearance is due to the potassium
phosphate buffer. Trehalose dissolved in 50 mM trizma buffer pH 7.5 or in pure water
leads in the levitation experiments to much less flattened particles. The strange particle
shape of the levitated samples is not seen in the spray dried powder. The spray dried
particles are nearly spherical.
(a) 60 °C, 10 % rel. humidity (50x)
(b) spray dried T in = 130 °C (3000x)
Figure 5.103: SEM-pictures of (a) a levitated particle and (b) the spray dried powder
of trehalose 10 % (w/v) in 100 mM phosphate buffer pH 7.0
5.5.2.1 LDH-trehalose 10 % (w/v) total solids formulations
In the first set of experiments trehalose was added to the LDH solution using 100 mM
potassium phosphate buffer pH 7.0 as solvent to a solids content of 10 % (w/v) (except
buffer salts). The LDH-trehalose ratios examined are 8:2, 6:4, 4:6 and 2:8 as for the bCA
solution experiments. Figure 5.104, Figure 5.105 and Figure 5.106 give the drying
behaviour of the droplets in comparison of the different formulations. The strange curve
progression of pure 10 % (w/v) trehalose droplets is remarkable. The drying behaviour of
this solution is explained by sudden film formation after the droplet was inserted in the
pressure node that leads to a reduction of solvent evaporation and therefore to an increase
in the initial droplet surface temperature. The increasing aspect ratio is due to the extreme
flattening of the droplet during the drying process that was already shown in the SEM-

Results and Discussion 164
picture of the pure trehalose particle (Figure 5.103 (a)). The /0²-curves do not
show a clear dependency on the trehalose content of the formulations. Only the 2:8
formulation has an clearly earlier critical point in comparison to formulations containing a
lower amount of trehalose. The droplet surface temperature curves show that with
increasing amount of trehalose in the formulation the temperature increase at the critical
point is shifted to earlier drying times. The temperature increase is slower in comparison to
formulations with higher protein content. Here the formation of a surface film similar to
the pure trehalose solution droplets can be seen as the reason for the earlier surface
temperature increase. The aspect ratio for the 8:2 formulation surprisingly does not change
at the critical point, whereas the 6:4 formulations shows an increase in the aspect ratio to
about 2.0. At high trehalose content the aspect ratio curves for the 4:6 and 2:8 formulations
show clearly the strong flattening of the droplet / particle with increasing trehalose content
leading to disc-shaped particles. The irregular flat particle shape can lead to oscillations
and the particles can fall out off the pressure node, so that sufficiently long drying times
can hardly be reached.
Figure 5.104: Drying behaviour of LDH-trehalose 10 % (w/v) total solids formulation
droplets, drying air conditions: 60 °C and 10 % relative humidity (r(t)²/r(0)²)

Results and Discussion 165
Figure 5.105: Drying behaviour of LDH-trehalose 10 % (w/v) total solids formulation
droplets, drying air conditions: 60 °C and 10 % relative humidity (droplet surface
temperature)
Figure 5.106: Drying behaviour of LDH-trehalose 10 % (w/v) total solids formulation
droplets dried at 60 °C and 10 % relative humidity (aspect ratio)

Results and Discussion 166
In the SEM-pictures of the levitated LDH formulation particles (Figure 5.107 and Figure
5.108) the flattening at increasing trehalose content can clearly be seen. The LDHtrehalose
8:2 particle (Figure 5.107) has a similar particle shape to the pure LDH particle
(Figure 5.102 (a)) with the hole at the top side and the hollow interior. As soon as trehalose
is added to the formulation, the particles do not break anymore. Even a small amount of
trehalose has a strong influence on the particle stability in the 8:2 formulation. The 6:4
formulation particle is obviously more flattened than the 8:2 particle and shows an
inclination of the particles top side. The spray dried powders for both formulations show
strongly dimpled particles, most for the 6:4 formulation. Some particles, especially in the
8:2 powder formulation, seem to have a hollow interior.
(a) LDH-trehalose 8:2 (100x)
(b) LDH-trehalose 8:2 (3000x)
(c) LDH-trehalose 6:4 (100x)
(d) LDH-trehalose 6:4 (3000x)
Figure 5.107: SEM-pictures of the (a, c) levitated particles and (b, d) spray dried
powders of LDH-trehalose 10 % (w/v) total solids formulations, drying air conditions:
60 °C and 10 %relative humidity

Results and Discussion 167
The levitated particles with a higher trehalose content of 4:6 and 2:8 (Figure 5.108) are
clearly more flattened and have a mostly smooth surface. The flatness may lead to the
oscillations that have already been seen in the aspect ratio curves. The spray dried powders
show shallower dimpled but more wrinkled particles for higher trehalose content. This
relation could also be seen for the bCA-trehalose formulations before.
SEM-pictures of LDH / trehalose formulations using porcine LDH were previously
given by Adler and Lee [1999] for spray drying solutions containing 10 % (w/w) total
solids. The trehalose ratio was very high at 99.7 % and 95 % of the total solids. The spray
dried powder particles are for higher trehalose content more wrinkled than the nearly pure
trehalose powder particles containing only 0.3 % LDH. This confirms the results from the
present experiments.
(a) LDH-trehalose 4:6 (100x)
(b) LDH-trehalose 4:6 (3000x)
(c) LDH-trehalose 2:8 (100x)
(d) LDH-trehalose 2:8 (3000x)
Figure 5.108: SEM-pictures of the (a, c) levitated particles and (b, d) spray dried
powders of LDH-trehalose 10 % (w/v) total solids formulations, drying air conditions:
60 °C and 10 % relative humidity

Results and Discussion 168
The surface structure of the levitated particles dependent on the trehalose ratio in the
formulation is given in the detailed SEM-pictures in Figure 5.109. The 8:2 formulation
particle has a smooth, but cracked surface. The same mainly smooth but less cracked
surface is shown for the 6:4 formulation particle, whereas the 4:6 particle has no cracks
and is more wrinkled. The 2:8 formulation particle shows the smoothest surface of all
mixture ratios. The surface structure of the particles reflects the increasing amount of
trehalose in the formulations. Pure trehalose particles have a highly smooth surface and are
stable in the levitation process, whereas the pure LDH particles show a high fragility. The
fragility of LDH can be seen in the cracks of the particle surface that decrease with
increasing trehalose content in the formulation. The particle-stabilizing influence of
trehalose is responsible for less particle breakage after the critical point in comparison to
pure LDH particles.
(a) LDH-trehalose 8:2 (1000x)
(b) LDH-trehalose 6:4 (1000x)
(c) LDH-trehalose 4:6 (1000x)
(d) LDH-trehalose 2:8 (1000x)
Figure 5.109: Surface structure of levitated LDH-trehalose 10 % (w/v) total solids
particles, drying air conditions: 60 °C and 10 % relative humidity

Results and Discussion 169
The residual activity of LDH in the different levitated and spray dried LDH-trehalose 10 %
total solids formulations was also analyzed. The results for the spray dried powder
formulations are given in Figure 5.110. A stabilizing effect of trehalose addition on the
residual LDH activity can be seen for most formulations, but a dependency on the amount
of trehalose in the mixture is ambiguous. The levitated LDH formulation particles were
also analyzed, but the results show large discrepancy of the measured activity values. The
measured residual activity of the fresh sample solution was reproducible, but the measured
activity values of the levitated particles deviated greatly.
Adler and Lee [1999] performed LDH activity studies using a spray dried powder
produced from a solution containing 0.3 % porcine LDH stabilized by 99.7 % trehalose
(10 % total solids). Using spray drying conditions of T in = 90 °C, T out = 60 °C led to an
activity loss of 11 % for the protein. At a higher inlet temperature of 150 °C the activity
loss increases to about 25 %. This is a much higher activity loss than found for the
stabilized formulations containing a higher ratio of LDH in the present experiments, but it
has to be considered that in the present experiments LDH from rabbit muscle was used.
Figure 5.110: Residual activity of the spray dried LDH-trehalose 10 % (w/v) total
solids formulations depending on the trehalose content

Results and Discussion 170
5.5.2.2 LDH 10 % (w/v) plus trehalose formulations
The drying behaviour described in the ²/0²-curves, the droplet surface temperature
curves and the aspect ratio curves for the LDH 10 % (w/v) plus trehalose solutions using
100 mM potassium phosphate buffer pH 7.0 with the LDH-trehalose ratios of 10:10, 10:20
and 10:30 is given in Figure 5.111, Figure 5.112 and Figure 5.113. As expected, with
increasing total solids of the formulations the critical point appears earlier in the ²/
0²-curves and the final particle is larger. In the droplet surface temperature curves the
critical point appears consequently also at earlier drying times for higher solids content and
the temperature increases substantially slower. The aspect ratio increase of the droplets is
not as high as for the LDH-trehalose 10 % total solids formulations, the highest value is
only about 2.5. For the 4:6 and 2:8 droplets (10 % total solids formulations) the droplet is
more flattened in comparison with the LDH-trehalose ratio 10:30. For a higher solids
content the flattening of the particle is therefore reduced. The levitated particles produced
from the high concentrated solutions are often not completely dry at the end of the
measurement and stick at the tube wall before the SEM-pictures could be taken. This leads
subsequently to particle deformation, so that the visible particle shape is not necessarily
representative of the particle shape directly after the drying process.
Figure 5.111: Drying behaviour of LDH 10 % (w/v) plus trehalose formulation
droplets, drying air conditions: 60 °C and 10 % relative humidity (r(t)²/r(0)²)

Results and Discussion 171
Figure 5.112: Drying behaviour of LDH 10 % (w/v) plus trehalose formulation
droplets, drying air conditions: 60 °C and 10 % relative humidity (droplet surface
temperature)
Figure 5.113: Drying behaviour of LDH 10 % (w/v) plus trehalose formulation
droplets, drying air conditions: 60 °C and 10 % relative humidity (aspect ratio)

Results and Discussion 172
The SEM-pictures of the spray dried powders for each formulation are given in Figure
5.114. The 10:10 formulation leads to dimpled powder particles. With increasing trehalose
content the dimples are reduced in the 10:20 formulation, and the 10:30 formulation shows
spherical but fused particles. This could be due to the higher water content in the spray
dried powder produced from the high concentrated solution. A possible explanation is also
the sticky nature of trehalose [Maa et al. 1997].
(a) LDH plus trehalose 10:10 (3000x)
(b) LDH plus trehalose 10:20 (3000x)
(c) LDH plus trehalose 10:30 (3000x)
Figure 5.114: Spray dried powders of LDH 10 % (w/v) plus trehalose formulations,
T in = 130 °C
For the levitated particles the activity values again show discrepancies so that the results
are not useful for interpretation of the stabilizing effect of trehalose. The residual activity
of the spray dried samples is around 80 % and there is no increase of activity due to
trehalose addition visible (Figure 5.115). No further increase in the amount of trehalose in
the formulation was analyzed because of the influence of the trehalose content on the

Results and Discussion 173
drying process leading to stickiness of the particles (shown for the 10:30 formulation). In
the 10 % (w/v) formulations an increase in the trehalose content would lead to smaller
amounts of LDH and therefore lower activity of the sample particle and still higher
measurement inaccuracies. For this reason no further experiments using LDH formulations
were performed.
Figure 5.115: Residual activity of spray dried LDH 10 % (w/v) plus trehalose
formulations depending on the trehalose content
The protein activity measurement at different drying times was performed for the pure
10 % (w/v) LDH in 100 mM potassium phosphate buffer solution. The results are given as
an example in Figure 5.116 to illustrate the behaviour that occurs. An unambiguous
decrease in the LDH activity is not seen. The activity of the droplets removed well before
the critical point (about 250 s) are reproducible and show no reduction of the LDH activity.
However, for particles removed close to the critical point the standard deviations of the
measured LDH activity values become very large. Additionally, as mentioned before after
the critical point the particles break or fall out of the pressure node, so that it was
impossible to analyze the whole drying process. Trehalose addition (8:2 formulation, 10 %
total solids) leads to longer drying times, but not to an improvement of the reproducibility

Results and Discussion 174
of the LDH activity values at and after the critical point. LDH is therefore not suitable for
the analysis of protein inactivation during the whole drying process with this technique.
Figure 5.116: Residual activity of LDH 10 % (w/v) solution droplets dependent on the
drying time, drying air conditions: 60 °C and 10 % relative humidity
5.5.3 Trypsinogen droplets
Trypsinogen was investigated for its suitability in protein stabilization experiments.
Tzannis and Prestrelski [1999] used a 2 % (w/v) trypsinogen solution in 1 M HCl for
protein stabilization studies with sucrose as a stabilizing excipient in spray drying
formulations (T in = 120 °C, T out = 85 °C). They found a reduction of the residual protein
activity in the spray dried pure trypsinogen powder to about 85 % related to the untreated
solution. Even small amounts of sucrose stabilized the protein. The highest residual
activity of 99.9 % was found for the 1:1 mixture. This activity reduction is promising for
the projected levitation experiments.
The drying behaviour of the 10 % (w/v) trypsinogen solution droplet using water
p.a. as solvent is given in Figure 5.117. The drying air conditions were 60 °C and 20 %
relative humidity without a direct drying air stream. Lower humidity settings do not lead to
sufficiently long drying times due to droplet oscillations. The ²/0²-curve and the
droplet surface temperature curve both show the curve progression for an amorphous

Results and Discussion 175
substance. The ²/0²-curve shows a bend at the critical point and a slow droplet
surface temperature increase can be seen. The aspect ratio decreases during the
measurement from 1.3 to about 1.1. A slight sudden displacement of the curve to lower
values appears at the critical point.
Figure 5.117: Drying behaviour of a trypsinogen 10 % (w/v) solution droplet, drying
air conditions: 60 °C and 20 % relative humidity
The SEM-pictures of the pure trypsinogen particles are given in Figure 5.118. The
levitated particles and the spray dried powder particles show a smooth surface. The
partially fractured levitated particle has a hollow interior. A hole as seen for the pure bCA
and LDH solution particles is not visible in the pictures. The spray dried powder consists
of hollow particles with a hole, as already seen for the 10 % (w/v) carbonic anhydrase and
LDH powders.
The measured residual activity of a 10 % (w/v) trypsinogen solution in water p.a.
both for the levitated particles dried at 60 °C and 20 % relative humidity without a direct
drying air stream and the spray dried powder (T in = 130 °C, T out = 80 °C) was, however,
100 %. For this reason trypsinogen is also not useful for protein stabilization experiments
in the levitator. No explanation for this behaviour or that of LDH was sought further in this
work.

Results and Discussion 176
(a) 60 °C, 20 % rel. humidity (100x)
(b) 60 °C, 20 % rel. humidity (100x)
(c) spray dried T in = 130 °C (3000x)
Figure 5.118: SEM-pictures of the (a, b) levitated and (c) spray dried trypsinogen
10 % (w/v) solution particles

Summary and Conclusions 177
6 Summary and Conclusions
This thesis deals with the analysis of the evaporation / drying behaviour and the particle
formation of pure solvents, excipient solutions and solutions containing an active
pharmaceutical ingredient or model protein. The analytical method used for this purpose is
single droplet drying using an ultrasonic levitator. During the whole drying process the
levitated droplets are monitored by a CCD-camera for droplet size and position
measurement, and an IR-camera for observation of the droplet surface temperature. The
dried particles are removed for either SEM-pictures or for residual activity measurements
of protein. The first part of this thesis discusses the evaporation behaviour of pure solvent
droplets. In the second part experiments using excipient solutions are presented. Solutions
containing the active pharmaceutical ingredient itraconazole or the model proteins bovine
carbonic anhydrase (bCA), L-lactic dehydrogenase from rabbit muscle (LDH) or bovine
trypsinogen are discussed in the third part of this thesis.
In the first part of this thesis water, the most common solvent for protein
formulations, was examined. To investigate the influence of different droplet sizes on
evaporation, droplets with an initial diameter of 500 μm, 800 μm and 1200 μm were
analyzed at different drying air conditions (temperature and relative humidity) without a
direct airstream to the droplet. The droplet surface temperature is constant for the most part
of the measurement. The initial diameter of the droplets shows no influence on their
droplet surface temperature. The experimental droplet surface temperatures (taken from the
constant temperature part) were compared to the wet-bulb temperature taken from a
psychrometric chart for the respective ambient conditions. Additionally the values are
compared to the theoretical droplet surface temperature taking into account the ultrasonic
field according to Yarin et al. [1999]. The experimental values are higher than the wet-bulb
temperature and the theoretical surface temperature calculated according to Yarin et al.
[1999]. The deviation of the surface temperatures can be explained by the energy supply
from the acoustic wave to the droplet. This leads to a heating-up of the droplet and
therefore to higher surface temperature.
For all initial droplet sizes an almost linear decrease of the ²/0²-graph can
be seen. The experimental evaporation coefficients (negative slope of the ²/0²graph)
are higher for larger initial diameters, because evaporation is accelerated by the

Summary and Conclusions 178
higher SPL setting that is necessary for stable levitation of larger droplets. The
experimental values of the evaporation coefficient are higher than the calculated values
using the wet-bulb temperature or the surface temperature according to Yarin et al. [1999].
Using the experimental surface temperature Sherwood numbers lie in the range of 2,
indicating correspondence of the evaporation process with the d²-law. Therefore, the
accelerated evaporation cannot be sufficiently explained only by the influence of the inner
acoustic streaming. It is also dependent on the energy supply by the acoustic wave.
By using a drying airstream directed to the droplet the evaporation can be
accelerated and therefore the evaporation coefficient increases. In experiments at an
ambient temperature of 25 °C the measured droplet surface temperature is lower using a
drying airstream than without one. Due to the reduction in the amount of water vapour in
the outer acoustic stream there is an increased cooling of the droplet. In contrast, at an
ambient temperature of 60 °C the values using a direct drying airstream are higher. The
higher supply of hot drying air to the droplet via direct airstream compared to
measurements without airstream appears to predominate.
The organic solvents acetone, 2-butanone, dichloromethane, ethanol, ethyl acetate,
2-propanol and tetrahydrofuran were also analyzed and show different evaporation
coefficients and droplet surface temperatures dependent on their liquid properties. In
comparison to the calculated values there is also a deviation, with higher values for the
experimental evaporation coefficients and droplet surface temperatures. Some solvent
mixtures show a break in the ²/0²-graphs indicating the fast evaporation of the
more volatile solvent in the mixture at the beginning of the measurement. As soon as the
more volatile fraction has almost completely evaporated, a surface temperature increase
takes place. Other solvent mixtures show an almost linear decrease in the ²/0²graph.
In this case the evaporation is faster with increasing amount of the more volatile
solvent in the mixture and the droplet surface temperature is lower.
In the second part of this thesis excipient solutions were examined using trehalose
and sucrose as amorphous substances and mannitol as a crystalline substance. The curve
progression of the ²/0²-graphs, of the droplet surface temperatures and the aspect
ratios were compared. In the first drying stage solution droplets show a linear decrease in
the ²/0²-graph. After the critical point the droplet size stays almost constant. The
droplet surface temperature increases at the critical point, because the evaporation of the
solvent is slowed down due to crust formation. Depending on the sugar properties,

Summary and Conclusions 179
amorphous or crystalline, the curve progression at the critical point differs. An amorphous
substance like trehalose shows a bend in the ²/0²-graph and the droplet surface
temperature increase is slow. The aspect ratio changes before and after the critical point,
because the substance does not form a stable particle but it is still deformable. In contrast,
the crystalline substance mannitol shows a sharp break in the ²/0²-graph and an
immediate droplet surface temperature increase. The aspect ratio is almost constant in the
second drying stage. This drying behaviour of mannitol is due to the rapid formation of a
solid crystalline crust at the critical point. The present experiments show that droplet
surface temperature measurement is, in addition to the droplet size measurement, a helpful
analytical method to detect the critical point in the drying process of solution droplets.
The polymer excipients copolyvidone (Kollidon® VA 64) and HPMC (Pharmacoat
615) dissolved in various dichloromethane-ethanol-mixtures were analyzed. The
experimental results show a completely different drying behaviour of the two substances.
The ²/0²-graph of the copolyvidone solution shows a break at the critical point and
the droplet surface temperature increases as expected. In contrast, the HPMC solution
droplets form a surface film immediately after the droplets were inserted into the acoustic
field. This leads to a continual droplet surface temperature increase and to a large bend in
the ²/0²-graph, so that a critical point for the HPMC solutions is not clearly
detectable. With increasing amount of the less volatile solvent ethanol in the
dichloromethane-ethanol-mixture there is a shift of the critical point to later drying times
for the copolyvidone solutions. The HPMC solutions do not show substantial changes in
the curve progression.
In the third part of this thesis solutions of itraconazole as an active pharmaceutical
ingredient and solutions of the model proteins bCA, LDH and trypsinogen together with
excipients were examined. Itraconazole was analyzed using either copolyvidone or HPMC
as a polymer excipient. The droplet surface temperature curve, the ²/0²-graph and
the change of the aspect ratio during the measurement are not influenced by the
itraconazole addition, but depend on the added polymer. The final particle shape is,
however, predominated by itraconazole. Particles containing itraconazole show a rougher
surface in comparison to the pure polymer particles. The analysis of the two
itraconazole / polymer formulation series in the levitator illustrates a potential application
of single droplet drying for the optimization of formulations.

Summary and Conclusions 180
The drying behaviour of the pure 10 % (w/v) bCA solution as well as of different bCAtrehalose
formulations shows the characteristic curve progression for amorphous
substances. SEM-pictures show that with increasing trehalose content the particles have a
more smoothed surface, but are more flattened. Levitated pure carbonic anhydrase particles
have a hole at the top side and are hollow, as also observed with spray dried powder
particles. The residual protein activity increases for both the levitated particles and the
spray dried powder samples with trehalose addition. A trehalose addition of more than
20 % of the total solids does not lead to higher protein protection. In further experiments
the bCA-trehalose formulations were tested for protein stability during the drying process.
At different time points in the drying process droplets or particles were removed out of the
levitator and the residual activity of the protein was measured. For the pure 10 % (w/v)
bCA solution a decrease in the residual activity after the critical point can be seen. The
stabilized formulations show almost constant activity during the drying process as
expected.
LDH was chosen as second model protein, because it should show higher losses of
activity during drying. However, the activity measurements of the levitated particles show
wide deviations of the measured values and cannot be interpreted. The spray dried samples
of the formulation show a stabilization effect even for a low trehalose ratio of 20 % of the
total solids. SEM-pictures of the levitated particles show an extreme flattening with
increasing trehalose content. The particles produced from the pure 10 % (w/v) trehalose
solution droplets are also very flattened and disc-shaped. This behaviour is a result of using
the 100 mM potassium phosphate buffer as solvent, because trehalose dissolved in pure
water or in 50 mM trizma buffer lead to predominantly spherical particles in the levitator.
The flat particles started to oscillate making measurements difficult. Pure LDH particles
have a hole at the top side and are hollow, as also seen with the pure bCA particles.
Trypsinogen was analyzed as the third protein, but no activity decrease due to the
drying process could be observed. The drying behaviour of the pure 10 % (w/v)
trypsinogen solution droplets is similar to the other two proteins.
Single droplet drying experiments including droplet surface temperature
measurements are a valuable tool for the analysis of the evaporation behaviour of solvents
and of the drying process of pharmaceutical solution formulations for spray drying. The
results for particle shape and residual protein activity from spray dried powders and
levitated particles correspond well. Residual protein activity measurements are possible,

Summary and Conclusions 181
because the droplet or particle can be removed out of the acoustic field at any time. For this
reason formulations containing expensive pharmaceutical proteins can be optimized by
using very small amounts of substance. The kinetics of protein inactivation during the
drying process needs, however, further analysis using a more temperature-sensitive model
protein. Furthermore, surfactants should be analyzed as excipients in the formulations, also
in combination with sugars like trehalose, for the protein stabilizing effect due to their
accumulation at the droplet surface. Via integration of a cooling system for the piezocrystal
in the transducer, experiments could be performed at higher drying air
temperatures.

Zusammenfassung 182
7 Zusammenfassung
Diese Arbeit beschäftigt sich mit der Untersuchung des Verdampfungs- sowie
Trocknungsverhaltens und der Partikelbildung von reinen Lösungsmitteln, von
Hilfsstofflösungen und von Lösungen, die einen Wirkstoff oder ein Modellprotein
enthalten. Die für diesen Zweck verwendete analytische Methode ist die Trocknung von
Einzeltropfen in einem Ultraschall-Levitator. Während des gesamten Trocknungsprozesses
werden die schwebenden Tropfen durch eine CCD-Kamera für die Messung der
Tropfengröße und Position, und durch eine IR-Kamera für die Beobachtung der
Temperatur auf der Tropfenoberfläche überwacht. Die getrockneten Partikel werden für
SEM-Aufnahmen oder für Messungen der Restaktivität bei Proteinen entnommen. Im
ersten Teil dieser Arbeit wird das Verdampfungsverhalten von reinen Lösungsmitteltropfen
behandelt. Im zweiten Teil werden Versuche mit Hilfsstofflösungen beschrieben.
Lösungen, die den Wirkstoff Itraconazol oder die Modellproteine Carboanhydrase vom
Rind (bCA), L-Lactat-Dehydrogenase aus Kaninchenmuskel (LDH) oder Trypsinogen
vom Rind enthalten, werden im dritten Teil dieser Arbeit behandelt.
Wasser, das am weitesten verbreitete Lösungsmittel für Proteinformulierungen,
wurde zuerst untersucht. Um den Einfluss verschiedener Tropfengrößen auf die
Verdampfung zu ermitteln, wurden Tropfen mit einem initialen Durchmesser von 500 μm,
800 μm und 1200 μm bei verschiedenen Trocknungsluftbedingungen (Temperatur und
relative Luftfeuchte) ohne direkten Luftstrom auf den Tropfen untersucht. Die
Oberflächentemperatur des Tropfens ist über den größten Teil der Messung konstant. Ein
Einfluss des initialen Durchmessers der Tropfen auf ihre Oberflächentemperatur konnte
nicht festgestellt werden. Die experimentell ermittelten Oberflächentemperaturen der
Tropfen (aus dem konstanten Temperaturbereich) wurden mit der Kühlgrenztemperatur
verglichen, die mit Hilfe eines psychrometrischen Diagramms für die jeweiligen
Umgebungsbedingungen ermittelt wurde. Zusätzlich erfolgte ein Vergleich mit der
theoretischen Oberflächentemperatur der Tropfen unter Berücksichtigung des Einflusses
durch das Ultraschallfeld, berechnet nach Yarin et al. [1999]. Die experimentell ermittelten
Temperaturwerte sind größer als die Kühlgrenztemperatur und als die theoretische
Oberflächentemperatur berechnet nach Yarin et al. [1999]. Die Differenz der Oberflächentemperaturen
lässt sich durch den Energieeintrag aus der akustischen Welle in den Tropfen

Zusammenfassung 183
erklären. Dieser führt zu einem Aufheizen des Tropfens und folglich zu einer höheren
Oberflächentemperatur.
Bei allen initialen Durchmessern der Tropfen ist eine nahezu lineare Abnahme des
²/0²-Graphen zu erkennen. Die experimentellen Verdampfungskoeffizienten
(negative Steigung des ²/0²-Graphen) sind bei größeren initialen Durchmessern
höher, da die Verdampfung durch eine stärkere SPL-Einstellung, die für ein stabiles
Schweben größerer Tropfen erforderlich ist, beschleunigt wird. Im Vergleich sind die
experimentell ermittelten Werte des Verdampfungskoeffizienten höher als die berechneten
Werte unter Verwendung der Kühlgrenztemperatur oder der Oberflächentemperatur
berechnet nach Yarin et al. [1999]. Bei Verwendung der experimentell ermittelten
Oberflächentemperatur liegen die Sherwood-Zahlen im Bereich von 2, was auf eine
Übereinstimmung des Verdampfungsprozesses mit dem d²-Gesetz hinweist. Die
beschleunigte Verdampfung kann daher mit dem Einfluss der inneren akustischen
Strömung allein nicht zufriedenstellend erklärt werden. Sie ist auch abhängig von dem
Energieeintrag durch die akustische Welle.
Unter Verwendung eines auf den Tropfen gerichteten Trocknungsluftstrahls kann
die Verdampfung beschleunigt werden und folglich erhöht sich der Verdampfungskoeffizient.
In Versuchen bei 25 °C Umgebungstemperatur ist die gemessene
Oberflächentemperatur der Tropfen bei Verwendung eines Trocknungsluftstrahls geringer
als ohne. Aufgrund der Reduzierung des Wasserdampfanteils in der äußeren akustischen
Strömung kommt es zu einer verstärkten Kühlung des Tropfens. Bei 60 °C
Umgebungstemperatur sind die Werte mit direktem Trocknungsluftstrom dagegen höher.
Die größere Zufuhr an heißer Trocknungsluft an den Tropfen durch den direkten Luftstrom
im Vergleich zu Messungen ohne Luftstrom scheint hier dominierend zu sein.
Die organischen Lösungsmittel Aceton, 2-Butanon, Dichlormethan, Ethanol,
Ethylacetat, 2-Propanol und THF wurden ebenfalls untersucht und zeigen verschiedene
Verdampfungskoeffizienten und Oberflächentemperaturen der Tropfen abhängig von ihren
Flüssigkeitseigenschaften. Im Vergleich zu den berechneten Werten gibt es auch hier eine
Abweichung, die Werte für die experimentellen Verdampfungskoeffizienten und die
Oberflächentemperaturen der Tropfen sind höher. Einige Lösungsmittelmischungen zeigen
Kurven in den ²/0²-Graphen, was auf eine schnelle Verdampfung des leichter
flüchtigen Lösungsmittels in der Mischung zu Anfang der Messung hinweist. Sobald der
leichter flüchtige Anteil fast vollständig verdampft ist, findet ein Anstieg der

Zusammenfassung 184
Oberflächentemperatur statt. Andere Lösungsmittelmischungen zeigen eine fast lineare
Abnahme des ²/0²-Graphen. In diesem Fall ist die Verdampfung mit steigendem
Anteil des leichter flüchtigen Lösungsmittels in der Mischung schneller und die
Oberflächentemperatur der Tropfen ist geringer.
Im zweiten Teil dieser Arbeit wurden Hilfsstofflösungen unter Verwendung von
Trehalose und Saccharose als amorphe Substanzen und Mannitol als kristalline Substanz
untersucht. Die Kurvenverläufe der ²/0²-Graphen, der Oberflächentemperaturen
und der Achsenverhältnisse der Tropfen wurden verglichen. In der ersten Trocknungsphase
zeigen Lösungstropfen eine lineare Abnahme des ²/0²-Graphen. Nach
Überschreiten des kritischen Punktes bleibt die Tropfengröße nahezu konstant. Die
Oberflächentemperatur der Tropfen steigt am kritischen Punkt an, da die Verdampfung des
Lösungsmittels durch die Krustenbildung verlangsamt wird. Abhängig von den
Eigenschaften des Zuckers, amorph oder kristallin, unterscheidet sich der Kurvenverlauf
am kritischen Punkt. Eine amorphe Substanz wie Trehalose zeigt eine Kurve im ²/
0²-Graphen und der Anstieg der Oberflächentemperatur der Tropfen ist langsam. Das
Achsenverhältnis ändert sich vor und nach dem kritischen Punkt, da die Substanz nicht
sofort einen festen Partikel bildet, sondern dieser immer noch formbar bleibt. Im
Gegensatz dazu zeigt die kristalline Substanz Mannitol einen scharfen Knick im ²/
0²-Graphen und einen sofortigen Temperaturanstieg der Tropfenoberfläche. Das
Achsenverhältnis ist nahezu konstant in der zweiten Trocknungsphase. Dieses
Trocknungsverhalten von Mannitol ist bedingt durch den sofortigen Aufbau einer festen
kristallinen Kruste am kritischen Punkt. Die vorliegenden Versuche zeigen, dass die
Messung der Tropfenoberflächentemperatur, zusätzlich zu der Tropfengrößenbestimmung,
ein hilfreiches analytisches Verfahren ist, um den kritischen Punkt im Trocknungsprozess
von Lösungstropfen zu bestimmen.
Die polymeren Hilfsstoffe Copolyvidon (Kollidon® VA 64) und HPMC
(Pharmacoat 615) wurden, gelöst in unterschiedlichen Dichlormethan-Ethanol-
Mischungen, analysiert. Die Versuchsergebnisse zeigen ein ganz unterschiedliches
Trocknungsverhalten der zwei Substanzen. Der ²/0²-Graph der
Copolyvidonlösung weist einen Knick am kritischen Punkt auf und die
Oberflächentemperatur der Tropfen steigt wie erwartet an. Im Gegensatz dazu bilden die
HPMC–Lösungstropfen unmittelbar nach Einbringen des Tropfens in das Ultraschallfeld

Zusammenfassung 185
einen Oberflächenfilm aus. Dieser führt zu einem kontinuierlichen Anstieg der
Oberflächentemperatur des Tropfens und zu einer weiten Kurve im ²/0²-Graphen,
so dass der kritische Punkt für die HPMC-Lösungen nicht klar bestimmbar ist. Bei den
Copolyvidonlösungen erfolgt mit ansteigendem Anteil des geringer flüchtigen
Lösungsmittels Ethanol in der Dichlormethan-Ethanol-Mischung eine Verlagerung des
kritischen Punktes zu späteren Trocknungszeitpunkten. Die HPMC-Lösungen zeigen keine
wesentlichen Änderungen im Kurvenverlauf.
Im dritten Teil dieser Arbeit wurden Lösungen mit Itraconazol als Wirkstoff und
Lösungen mit den Modellproteinen bCA, LDH und Trypsinogen zusammen mit
Hilfsstoffen untersucht. Itraconazol wurde unter Verwendung von Copolyvidon oder
HPMC als polymerem Hilfsstoff untersucht. Der Oberflächentemperaturverlauf der
Tropfen, der ²/0²-Graph und die Entwicklung des Achsenverhältnisses während
der Messung werden nicht durch den Itraconazolzusatz beeinflusst, sondern sind abhängig
vom eingesetzten Polymer. Das Aussehen des fertigen Partikels wird allerdings von
Itraconazol bestimmt. Partikel, die Itraconazol enthalten, zeigen eine rauere Oberfläche im
Vergleich zu reinen Polymerpartikeln. Die Untersuchungen der zwei Itraconazol/Polymer-
Formulierungsserien im Levitator veranschaulichen eine mögliche Anwendung der
Einzeltropfentrocknung bei der Optimierung von Formulierungen.
Das Trocknungsverhalten der reinen 10 %igen (m/V) bCA-Lösung und das
verschiedener bCA-Trehalose-Formulierungen zeigt den für amorphe Substanzen
typischen Kurvenverlauf. Die SEM-Aufnahmen zeigen, dass die Partikel mit ansteigendem
Trehalosegehalt eine glattere Oberfläche haben, insgesamt jedoch flacher sind. Levitierte
reine Carboanhydrasepartikel haben ein Loch an der Oberseite und sind hohl, ebenso wie
bei den sprühgetrockneten Pulverpartikeln beobachtet. Die Restaktivität des Proteins steigt
sowohl für die levitierten Partikel als auch für die sprühgetrockneten Pulverproben mit
Trehalosezusatz an. Ein Zusatz an Trehalose von mehr als 20 % des Gesamtfeststoffanteils
führt jedoch zu keiner verbesserten Proteinstabilisierung. Die bCA-Trehalose-
Formulierungen wurden in weiteren Versuchen auf die Proteinstabilität während des
Trocknungsvorgangs hin untersucht. Zu verschiedenen Zeitpunkten des
Trocknungsprozesses wurden Tropfen oder Partikel aus dem Levitator entnommen und die
Restaktivität des Proteins bestimmt. Bei der reinen 10 %igen (m/V) bCA-Lösung ist eine
Abnahme der Restaktivität nach dem kritischen Punkt zu sehen. Die stabilisierten

Zusammenfassung 186
Formulierungen zeigen wie erwartet eine nahezu konstante Aktivität während des
Trocknungsprozesses.
LDH wurde als zweites Modellprotein ausgewählt, da es höhere Aktivitätsverluste
während der Trocknung zeigen sollte. Die Aktivitätsmessungen der levitierten Partikel
zeigen jedoch große Abweichungen der Messwerte und können nicht interpretiert werden.
Die sprühgetrockneten Proben der Formulierungen zeigen einen Stabilisierungseffekt
schon bei einem geringen Trehaloseanteil von 20 % des Gesamtfeststoffanteils. Die SEM-
Aufnahmen der levitierten Partikel zeigen eine extreme Abflachung mit ansteigendem
Trehalosegehalt. Die aus Tropfen der 10 %igen (m/V) Trehaloselösung hergestellten
Partikel sind ebenfalls sehr flach und „disc“-förmig. Dieses Verhalten ist durch die
Verwendung des 100 mM Kaliumphosphat-Puffers als Lösungspuffer hervorgerufen, denn
Trehalose gelöst in reinem Wasser oder in 50 mM Trizma-Puffer führt zu vorwiegend
abgerundeten Partikeln im Levitator. Die flachen Partikel fingen an zu oszillieren, was die
Durchführung der Messungen erschwerte. Reine LDH-Partikel haben ein Loch an der
Oberseite und sind ebenso wie die reinen bCA-Partikel hohl.
Trypsinogen wurde als drittes Protein untersucht, allerdings konnte keine
Aktivitätsabnahme aufgrund des Trocknungsprozesses festgestellt werden. Das
Trocknungsverhalten von Tropfen der reinen 10 %igen (m/V) Trypsinogenlösung ist
vergleichbar mit den zwei anderen Proteinen.
Trocknungsexperimente an Einzeltropfen, die Messungen der
Oberflächentemperatur einschließen, sind ein nützliches Instrument bei der Analyse des
Verdampfungsverhaltens von Lösungsmitteln und des Trocknungsprozesses von
pharmazeutischen Lösungsformulierungen für die Sprühtrocknung. Die Ergebnisse
bezüglich des Aussehens der Partikel und der Restaktivität der Proteine von den
sprühgetrockneten Pulvern und den levitierten Partikeln stimmen gut überein. Messungen
der Restaktivität eines Proteins sind möglich, da die Tropfen oder Partikel jederzeit aus
dem akustischen Feld entnommen werden können. Daher können Formulierungen, die
teure pharmazeutische Proteine enthalten, unter Verwendung sehr kleiner Substanzmengen
optimiert werden. Die Kinetik der Inaktivierung des Proteins während des
Trocknungsprozesses erfordert hingegen weitere Untersuchungen mit einem noch
temperatursensibleren Modellprotein. Des Weiteren sollten Tenside als Hilfsstoffe in den
Formulierungen, auch in Kombination mit Zuckern wie Trehalose, auf die
proteinstabilisierende Wirkung aufgrund ihrer Anreicherung an der Tropfenoberfläche

Zusammenfassung 187
untersucht werden. Durch den Einbau einer Kühlung für den Piezokristall im Transducer
könnten die Versuche bei höheren Temperaturen der Trocknungsluft durchgeführt werden.

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Lebenslauf
Persönliche Daten
Name:
Eva Cornelia Wulsten, geb. Schmidt
Geburtsdatum/-ort: 11.04.1979 in Delmenhorst
Anschrift: Saßnitzer Straße 19
90425 Nürnberg
Familienstand:
verheiratet
Schulbildung
1985 - 1989 Grundschule Ganderkesee-Bookholzberg
1989 - 1991 Orientierungsstufe Ganderkesee-Bookholzberg
1991 - 1998 Gymnasium Ganderkesee
Abschluss mit Abitur am 22.06.1998
Studium
10/1998 - 11/2002 Studium der Pharmazie an der Technischen Universität
Carolo-Wilhelmina zu Braunschweig
1. Abschnitt der Pharm. Prüfung am 25.08.2000
2. Abschnitt der Pharm. Prüfung am 11.11.2002
3. Abschnitt der Pharm. Prüfung am 08.01.2004
Approbation als Apothekerin am 21.01.2004
Praktika
03/99 Famulatur in der Stern-Apotheke Bookholzberg
08/99 Famulatur in der Zentralapotheke des Zentralkrankenhauses
St.-Jürgen-Straße in Bremen
12/2002 - 05/2003 Pharmaziepraktikum bei der Lilly Forschung GmbH in
Hamburg, Abteilung Pharmazeutische Entwicklung
(Analytik)
06/2003 - 11/2003 Pharmaziepraktikum in der Apotheke und Sanitätshaus
Gamsen, Gifhorn-Gamsen

Berufliche Tätigkeiten
01/2004 - 06/2005 Apothekerin in der Apotheke und Sanitätshaus Gamsen in
Gifhorn-Gamsen
07/2005 - 09/2005 Apothekerin in der Apotheke im Hansa-Carré in Bremen
10/2005 - 02/2009 Wissenschaftliche Mitarbeiterin und Doktorandin am
Lehrstuhl für Pharmazeutische Technologie der Friedrich-
Alexander-Universität Erlangen-Nürnberg
Anfertigung der Doktorarbeit zum Thema „Determination
of droplet surface temperature and drying kinetics of
protein solutions using an ultrasonic levitator“
05/2006 - 08/2008 zeitweise Nebentätigkeit in Apotheken in Nürnberg und
Übernahme von Apothekennotdiensten
Weiterbildung
seit 02/2006
Weiterbildung zum Fachapotheker für Pharmazeutische
Technologie
Nürnberg, 20.02.2009