s r n * , MASARYKUNIVERSITY
<5 — J5
JMI-^-4 NA. 9
Faculty of Science
Department of Chemistry
Study of Processes Associated with
Freezing of Aqueous Solutions
Ph. D. Dissertation
Ľubica Vetráková
Supervisor: Mgr. Dominik Heger, Ph.D. Brno 2017
Bibliographic Entry
Author: Mgr. Ľubica Vetráková (maiden name Krausková)
Faculty of Science, Masaryk University
Department of Chemistry
Title of Dissertation: Study of Processes Associated with Freezing of Aqueous
Solutions
Degree Programme:
Field of Study:
Supervisor:
Chemistry
Physical Chemistry
Mgr. Dominik Heger, Ph.D.
Academic Year: 2016/2017
Number of Pages:
Keywords:
85 + 71 pages of attachments
freezing; ice; acidity changes; pH shift; solid-state acidity;
crystallization of buffers; denaturation of enzymes; frost
flowers; acceleration of reactions; freeze-concentration;
selective ion incorporation; cryoprotection; indicator; acidity
probe; spectroscopy; diffuse reflectance; phosphate buffer;
DbjA; lyophilization
2
Bibliografický záznam
Autorka: Mgr. Ľubica Vetráková (rod. Krausková)
Přírodovědecká fakulta, Masarykova univerzita
Ústav chemie
Název práce:
Studijní program:
Studijní obor:
Vedoucí práce:
Akademický rok:
Studie procesů doprovázejících mrazení vodních roztoků
Chemie
Fyzikální chemie
Mgr. Dominik Heger, Ph.D.
2016/2017
Počet stran: 85 + 71 stran příloh
Klíčová slova: mrazení; led; změny kyselosti; skokpH; kyselost v pevné fázi;
krystalizace pufrů; denaturace enzymů; ledové květy;
zrychlení reakcí; zakoncentrování při mrazení; selektivní
zabudování iontů; kryoprotekce; indikátor; aciditní sonda;
spektroskopie; difusní reflektance; fosfátový pufr; DbjA;
lyofilizace
3
Abstract
Most of solutes present in an aqueous solution are very poorly soluble in ice. As a result,
interesting phenomena are observed when aqueous solutions are frozen. In my
dissertation, I describe and explain some of them - freeze-concentration, selective ion
incorporation, and freezing-induced acidity change. The thesis focuses mainly on the
study of the acidity changes during freezing of solutions of buffers and salts, and how
these acidity changes impact the stability and reactivity of other compounds present
in a solution. Solid state acidity was assayed spectroscopically with a use of acid-base
indicators, and numerically evaluated in terms of Hammett acidity function.
Four projects are described and discussed in this dissertation. The first project
deals with freezing- and sublimation-induced acidity changes during freezing and
lyophilization of phosphate buffers. The acidities of frozen and lyophilized samples
were monitored as a function of initial pH. Although the acidity of lyophiles was
somewhat similar to the initial pH, we found no resemblance between the initial pH
and the acidity in the frozen state, nor between the frozen-state acidity and that of
the resulting lyophile. This implies that monitoring only the acidity of a lyophile is not
sufficient to predict all the acidity changes throughout the whole lyophilization process.
In the second project, the frozen-state acidities of buffers were correlated to
recovery activity of the model enzyme, DbjA, that underwent several freeze-thaw
cycles in the corresponding buffer solution. Subsequently, the freezing-induced
acidity changes were prevented by addition of a properly chosen salt, and the activity
of the model enzyme after freezing and thawing was completely recovered. This
demonstrates the strong effect of the local frozen-state acidity on the stability of
frozen-thawed proteins.
The third project focuses on accelerated redox reaction between
4-chlorophenol and chromate in a frozen solution in comparison with the reaction in
an aqueous solution. The cause of the acceleration was sought. 100-fold increased
concentration of one reactant and a decrease of pH by 3 units were both needed for
the reaction to proceed in solution. Therefore, the acceleration of the reaction in ice
is reasoned by the freeze-concentration and the freezing-induced acidity change.
4
The last project deals with frost flowers - dendritic formations of ice crystals
found on freshly formed ice. The frost flowers are of interest for environmental
chemists due to their suspected role in a formation of sea-salt aerosol in polar areas.
We prepared the frost flowers in the laboratory and studied their integrity upon ice
sublimation with environmental scanning electron microscope. The results of the
observations imply that the frost flowers are probably not a direct source of a sea-salt
aerosol in polar areas.
5
Abstrakt
Většina látek rozpuštěných ve vodním roztoku je prakticky nerozpustná v ledu. Při
mrazení vodných roztoků můžeme proto pozorovat zajímavé jevy. Ve své disertaci
popisuji a vysvětluji některé z nich - zakoncentrování mrazením, selektivní zabudování
iontů a změnu kyselosti při mrazení. Tato práce se zaměřuje hlavně na změny kyselosti
při mrazení roztoků pufrů a solí a vliv těchto změn kyselosti na stabilitu a reaktivitu
ostatních látek přítomných v roztoku. Kyselost v pevném stavu byla stanovena
spektroskopicky s použitím acidobazických indikátorů, a numericky vyhodnocena jako
Hamettova aciditní funkce.
Disertační práce popisuje a diskutuje čtyři projekty. První projekt se zabývá
změnami kyselosti při mrazení a lyofilizaci fosfátových pufrů. Kyselosti mrazených a
lyofilizovaných pufrů byly monitorovány jako funkce původního pH roztoku. I když byly
kyselosti lyofilizátů do jisté míry podobné původnímu pH, nenašli jsme žádnou podobnost
mezi původním pH a kyselostí zmrazeného pufru, ani mezi kyselostí zmrazeného pufru a
výsledného lyofilizátů. Závěrem projektu je, že monitorování kyselosti výsledného
lyofilizátů není dostatečné na předpovídání všech změn kyselosti během celého procesu
lyofilizace.
Ve druhém projektu byly kyselosti zmrazených roztoků pufrů korelovány
s obnovenou aktivitou modelového enzymu DbjA, který byl podroben několika cyklům
zmrazení a rozmrazení v daném pufru. Změny kyselosti při mrazení byly následně
potlačeny přídavkem vhodné soli, což mělo za následek úplné obnovení aktivity
modelového enzymu po zmrazení a rozmrazení. Tyto výsledky demonstrují silný vliv
lokální kyselosti ve zmrazeném stavu na stabilitu zmrazených proteinů.
Třetí projekt se zaměřuje na hledání důvodu zrychlení redoxní reakce mezi
4-chlorofenolem a chromanem v ledu oproti reakci v nezmraženém roztoku. Tato reakce
začala probíhat v roztoku jen tehdy, když byla koncentrace jednoho z reaktantů zvýšená
100-násobně a pH roztoku pokleslo o 3 jednotky. Z toho vyvozujeme, že důvod pro
zrychlení této reakce v ledu je kombinace zakoncentrování roztoku a poklesu pH při
mrazení.
6
Poslední projekt se zabývá ledovými květy - dendritickými ledovými útvary, které
vznikají na čerstvém ledu. O ledové květy se zajímají environmentálni chemici kvůli jejich
předpokládané roli při tvorbě solných aerosolů v polárních oblastech. Proto jsme
pozorovali integritu laboratorně připravených ledových květů pomocí
environmentálního rastrovacího elektronového mikroskopu. Výsledky této studie
ukazují, že ledové květy pravděpodobně nejsou přímým zdrojem solných aerosolů
v polárních oblastech.
7
'I really like the idea that, once I die, I could meet all my loved ones again."
"Why not? Quantum chemistry does not exclude it..."
Ján Krausko & Tomáš Šolomek
© Ľubica Vetráková, Masaryk University, 2017
Acknowledgement
First of all, I would like to thank my supervisor Dominik Heger for the interesting topic
for my dissertation, his guidance during my studies, his enthusiasm for science, and neverending
discussions not only about science.
Next, I would like to thank all my colleagues and friends I have met during my
studies - Gabika, Vítek, Tomáš P., Lulu, Luboš, Lenka, Tomáš S., and the others. Thank you
for cooperation and your pleasant company not only in a lab.
My special thanks belong to all the members of the "chemical society" in my
hometown, Prievidza, for the wonderful time we have spent together. Probably I would
not have been doing chemistry at all, if I had not met Miroslav Kozák, a chemistry teacher.
He convinced me chemistry is what I want to do and that chemistry sessions are the best
way to spend Friday afternoons ©. I am especially grateful to Šolo, Jano and Peťo, great
"teachers" who introduced me to the mysteries of chemistry. Great thanks belong to Rasťo
and Paľo who try to gather the whole "crew" at least once a year. Chemistry is fun when
you have friends to share it with.
Most importantly, I wish to express my gratitude to my family. To my parents and
my husband Peťo for their endless support during my studies, patience, and their love.
And to my brother, rivalry with him always pushed me forward.
9
Table of contents
Table of Contents
1 Foreword 13
2 About the Dissertation 14
3 Review of the Literature 15
3.1 A Short Introduction to Ice 15
3.1.1 Importance of Freezing 15
3.1.2 How Many Ices Are There? 16
3.1.3 The surface of Ice 19
3.1.4 Frost Flowers 20
3.2 Processes Associated with Freezing of Aqueous Solutions 22
3.2.1 Freeze-Concentration 22
3.2.2 Freezing Potential 24
3.2.3 Freezing-Induced Acidity Change 27
3.2.4 Chemical Reactivity in Ice 29
3.3 Stabilization of Proteins by Freezing and Lyophilization 32
3.3.1 Lyophilization 32
3.3.2 Freezing 33
3.3.3 Stresses during Freezing and Lyophilization 34
3.3.4 Cryoprotection and Lyoprotection of Proteins 36
3.4 Techniques for the Measurement of Solid-State Acidity 39
3.5 Diffuse reflectance spectroscopy 41
4 Results and Discussion 43
4.1 Acidity of Frozen and Freeze-Dried Phosphate Buffers 43
4.1.1 Introduction 43
4.1.2 Characterization of Acidity Probes 43
10
Table of contents
4.1.3 Calculation of Hammett Acidity Functions Hx 45
4.1.4 Freezing-Induced Acidity Change 46
4.1.5 Effect of pHo 49
4.1.6 Effect of Temperature and Freezing Rate 50
4.1.7 Acidity of Lyophiles 51
4.1.8 Conclusion 52
4.1.9 Authors'Contributions 52
4.2 Suppression of Protein Inactivation during Freezing 53
4.2.1 Introduction 53
4.2.2 Model Enzyme 54
4.2.3 The effect of freezing parameters on DbjA activity 54
4.2.4 The relationship between acidity of a frozen buffer and DbjA activity 55
4.2.5 Neutralization of the buffer-induced acidification by addition of salts 56
4.2.6 Conclusion 58
4.2.7 Authors'Contributions 59
4.3 Accelerated redox reaction during freezing 60
4.3.1 Introduction 60
4.3.2 Reaction between 4-CP and Cr(VI) in solution and in the frozen state 60
4.3.3 Environmental applications 61
4.3.4 Conclusion 61
4.3.5 Authors'Contributions 62
4.4 Frost Flowers 63
4.4.1 Introduction 63
4.4.2 Preparation of the Frost Flowers 63
4.4.3 Observation of the Frost Flowers with ESEM 64
4.4.4 Conclusion 65
11
Table of contents
4.4.5 Authors'Contributions 66
4.5 Non-Ice-Related Projects 67
5 Literature 69
6 List of Abbreviations 81
7 Curriculum Vitae 82
8 List of Appendices 85
12
Foreword
1 Foreword
Ice as a research topic crossed my path during my undergraduate studies. At that time, I
was concerned with the properties of (partially) frozen solutions, freezing potential and
freezing-induced acidity changes, when solutions of various salts were frozen. During my
Ph.D. studies, I continued in the research of ice. I studied the freezing- and sublimationinduced
acidity changes in the buffer solutions widely used in biology and biochemistry,
and the effect of the acidity changes on denaturation of proteins during freezing. I
participated in the projects in the field of environmental ice chemistry, where the
acceleration of a reaction between environmental pollutants in ice was investigated and
a source of sea-salt aerosol in polar areas was sought. I hope that the results of these
projects will help to better understand the process of freezing and find their application
in the fields of biology, biochemistry, pharmaceutical industry, and environmental
chemistry.
Although the main tool of my work was UV-Vis absorption and diffuse reflectance
spectroscopy, I became skilled in a large variety of laboratory techniques in the field of
chemistry and biology - gas and liquid chromatography, dialysis, Bradford assay for
determination of protein concentration, enzymatic activity assay, etc.
It is not enough to measure some data, it is very important to process them and
understand them. Data analysis was essential part of my work. To analyse data more
efficiently, I learnt to write scripts in Matlab.
Experimental work is full of obstacles and often does not follow our plan. However,
every problem encountered while working on the projects was an opportunity to learn
new skills, bring up innovative ideas, and improve a design of the experiment.
Unanticipated results, that is what moves the science forward.
13
About the Dissertation
2 About the Dissertation
This dissertation is written as a compilation of freezing-related projects that have been
published of submitted for publication during my Ph.D. studies. The first part of the
dissertation, Review of the Literature, is not meant to serve as an exhaustive review,
rather it covers the background that enables the reader to understand the projects
discussed later. In the beginning, the structure and properties of ice are briefly
described. Then the interesting phenomena associated with freezing of aqueous
solutions are explained. Attention is paid to the stability of proteins during freezing
and lyophilization; the stresses causing protein denaturation are identified and
common approaches towards cryoprotection and lyoprotection of proteins are
summarized. In the end of the theoretical part, the techniques for the measurement
of the solid-state acidity are reviewed.
The selected projects are briefly commented on in the Results in Discussion
part. As these projects were already published or submitted for publication, most of
the experimental details are omitted and only the most important results are
presented to keep the story lucid. For more details, the interested reader is kindly
directed into the attached manuscripts. In the end, there is a section where some of
the non-ice-related projects I participated in are introduced shortly.
14
Review of the Literature
3 Review of the Literature
3.1 A Short Introduction to Ice
3.1.1 Importance of Freezing
Ice is all around us. It occurs naturally on Earth in the form of snow, lake and river ice, sea
ice, glaciers, permafrost, hoarfrost, or high etage clouds.1
On average, 7% of ocean's
surface is frozen; about 10% of the land is covered by ice permanently and up to half of
the Northern hemisphere in midwinter.2
This ice and snow coverage is of great
importance in the prevention of the climate change. Ice and snow have very high albedo,
the ability to reflect solar radiation, and prevent the surface warming. Nowadays,
naturally occurring ice is a subject of great interest of environmental scientific community
not only with respect to global warming but also as a fresh-water supply, as an
environmental archive, because of avalanches, for its ability to release pollutants to the
aqueous environment, and due to its potential to modify atmospheric composition.3
It is
suspected that the presence of ice in the atmosphere is essential for the production of rain
and the thunderstorms - it is the collision of ice particles in clouds that are responsible
for thunderstorm electricity.1
Great deal of scientific interest lies in ice prepared artificially, as artificial freezing
has widespread applications across the scientific, industrial and medical fields.
Cryopreservation is a commonly used practice in biology, medicine, and in the food
industry, where organelles, cells, tissues, organs, or any other biological material is frozen
to very low temperatures at which biochemical reactions are effectively stopped and longterm
stability of the material is achieved. Moreover, freezing is the first step of
lyophilization (freeze-drying), which is currently the most common formulation strategy
to stabilize biopharmaceuticals.4
In spite of the major economic importance, the
mechanisms underlying both the freezing and the subsequent lyophilization are not
sufficiently understood; thus, in practice, these processes still remain within the empirical
realm, making product optimization slow, inefficient, and frequently unsuccessful.4 - 6
With
a deeper understanding of freezing more efficient lyophilization cycles can be developed,
and the quality and stability of lyophilized biopharmaceuticals can be improved.4
15
Review of the Literature
3.1.2 How Many Ices Are There?
Ice is crystalline form of water. In fact, there are 16 different crystalline structures of
water (16 different ices) confirmed in the literature up to now.7
Some of them are
illustrated in the phase diagram in Figure 1. All the crystalline structures fulfil two "ice
rules": 1. each water molecule in the ice lattice is bound to four other water molecules via
hydrogen bonds, offering its hydrogens to two other water molecules and accepting
hydrogen bonds from another two; 2. there is only one hydrogen atom placed between
two neighbouring oxygen atoms. Violation of the "ice rules" leads to the formation of
protonic point defects in the ice structure that are responsible for electrical conductivity
and polarization of ice.1
Pressure (GPa)
Figure 1: The solid-liquid phase diagram of ice. Adapted from ref. 3.
Ice we are familiar with is called Ih. It is the only ice occurring naturally on the
surface of Earth as far as we know. It is obtained by freezing water at atmospheric
pressure or by direct condensation from water vapour above 173 K.1
It was the first
known phase of ice, hence roman numeral T was assigned to it, and 'h' was added to
distinguish this hexagonal phase from the metastable cubic ice Ic. The hexagonal wurtzitelike
structure was proposed by Pauling8
in 1935 and definitely confirmed by neutron
diffraction experiment9
on D2O more than 20 years later. The structure of ice Ih is depicted
16
Review of the Literature
in Figure 2. Each oxygen atom is surrounded by four nearest oxygens at the corners of a
regular tetrahedron. There is one hydrogen atom positioned asymmetrically between
each two neighbouring oxygens, bound covalently to the nearest oxygen and by hydrogen
bond to the other. 0-0-0 angle in the structure (109.5°) is very close to average H-O-H
angle in the water molecule (104.5°), so hydrogen atoms are only very slightly off the
direct line between neighbouring oxygens.1
'7
The structure of ice Ih is very open (Figure
3), which is very relevant to why ice Ih floats on water.
Figure 2: Crystal structure of ice Ih. White circles represent oxygen atoms, black circles represent hydrogen atoms. Both
"ice rules" are followed in the structure. Adapted from ref. 1.
The other ice phases are generally produced by the application of high pressures
and low temperatures. To relieve the strain, molecules rearrange themselves into denser
packings than in ice Ih. As a result, interesting structures are formed. For example, ice VIII
is formed of two identical water molecule networks that fully interpenetrate to fill the
available volume. If more pressure is applied to ice VIII, water molecules are squeezed
closer together and the hydrogen bonds between them shorten up to the point where
hydrogen atoms are located in the mid-points of the bond. Ice X, a crystal of hydrogen and
oxygen atoms rather than of distinct water molecules, is formed. It is speculated that, with
even greater pressures, ice must eventually become metallic.1
'7
17
Review of the Literature
Figure 3: Structure of ice Ih as viewedfrom above. Oxygen atoms in the structure form open channels. Hydrogen atoms are
not displayed here. Adapted from ref. 7.
There is 17t h
structure of ice described in the literature - cubic ice Ic, a metastable
variant of ice Ih. There are some doubts about its existence in the scientific community, as
no one yet has managed to prepare a pure sample of it, and what previously appeared to
be cubic ice has been found to be ice with irregular stacking of hexagonal layers.7
Despite
the irregular structure, cubic ice is formed in the beginning of nucleation phase and it is
transformed to the regular hexagonal ice Ih as a crystallization nucleus grows. For this
reason, cubic ice can be grown in confined spaces.7
Cubic ice is supposed to be present in
the higher atmosphere as snow crystal nuclei. Exceptionally, high-altitude clouds may
contain examples of cubic ice, which is rarely seen as Scheiner's halo1 0
at 28° around the
sun or moon (alternative explanation for the appearance of 28° halo, which does not
include cubic ice, is reported1 1
). There is also evidence for cubic ice being a constituent of
cometary ice.7
When water is frozen very rapidly, non-crystalline (amorphous) ices can be
formed. In 1932 warm water vapour was deposited on a very cold surface to form
amorphous ice for a first time. This material is called amorphous solid water (ASW). When
thin jet of water is projected into a very cold liquid, hyperquenched glassy water (HGW)
is formed. Other forms of amorphous ice - low-density amorphous ice (LDA), high-density
amorphous ice (HDA), and very high-density amorphous ice (VHDA) - were also
prepared.1 2
18
Review of the Literature
Even though the structures and properties of the other ice forms are interesting
and some of them are naturally present in the universe, let's stay on the Earth. This
dissertation deals only with ice Ih and when ice is mentioned later on, it specifically refers
to ice Ih.
3.1.3 The surface of Ice
The surface of a solid is a region with properties different from the bulk because of
bonding forces that atoms and molecules at the free surface experience from just one side.
Atoms are displaced from their normal sides, energies and force constants are changed,
and the layers below are affected.1
As a result, a phenomenon called surface premelting is
observed as a common properly of many materials.1 3
The presence of liquid or liquid-like
layer on the surface of the solid may exist if the energy of solid-vapour interface is greater
than the sum of the energies of solid-liquid and liquid-vapour interfaces; in such a case,
formation of liquid or liquid-like layer reduces the total surface energy.1
Such a premelted
liquid layer on the surface of ice is, for example, important for human leisure time
activities in winter like skiing, skating or snowballing.3
However, surface properties of ice
extent to lower temperatures and to greater depths that can be explained just by
premelting.1 4
Thin liquid-like layer in the surface of ice is often called quasi-liquid layer
and presents an intermediate state between the solid and bulk liquid, retaining some of
the underlying crystal order.3
The snapshots of quasi-liquid layer at the air-ice interface
at various temperatures obtained by molecular dynamic simulations1 5
are depicted in
Figure 4. The properties of the quasi-liquid layer were studied by various techniques like
optical ellipsometry,1 6 - 1 8
X-ray diffraction,1 9
NMR,2 0 - 2 5
proton channelling
experiments,2 6 - 2 7
surface conductivity measurements,2 8 - 2 9
and many others. All these
methods prove the presence of the quasi-liquid layer on the surface of ice, although its
approximate thickness and the temperatures at which it was observed differ per used
technique (Figure 5). Moreover, properties of the quasi-liquid layer are critically
dependent on the contamination of the surface of ice.1
More detailed description of the
methods used to study the surface of ice can be found in the report written by Petrenko.1 4
19
Review of the Literature
Figure 4: Snapshots of the quasi-liquid layer at the air-ice interface simulated for the undercooling of-59 K through -2 K
relative to the melting point of the water model. Adapted from ref. 15.
h M*" + r + Enharcr-tl conductivity
Eliipsonwtty
Uxygcti disorder (X-ray)
! Ifsurficc conductivity f
IJjJfjijjfifPrtikIn channelling:?;;
tJi4
!ji I iliiJi^'ijEn-.npi-Ti MfMirjfiAii«iiFf
j m r n
10
i — r TTTTT
]<][)
rf t l - r (ici
J,F(*ur double hyen
Ellipsomctry {0001}
t •• • *
m.r, flit quam
-J0v
C]
•{i iLOivftjcndiscnirr 10001J
f rrntr]—p r-rrrmi
O.i ] ID 100
AppHJSi. q.f.l. thitkreö (nm)
Figure 5: Presence of quasi-liquid layer indicated by various techniques, a, The temperature range below the melting point
Tm over which different techniques reveal special properties of the surface layer on ice. b, Approximate thickness of the
quasi-liquid layer deduced at -10 °C and -2 °C. Adapted from ref. 1.
3.1.4 Frost Flowers
Frost flowers are fern-like dendritic formations of ice crystals found on freshly formed ice
(Figure 6).3 0
"3 6
Unlike hoarfrost condensing from a supersaturated atmosphere, the frost
flowers are formed on sublimating ice surfaces.36
Specific conditions must be fulfilled for
20
Review of the Literature
the frost flowers to be grown - the surface of ice must be warm relative to the atmosphere
and the air above the ice must be locally supersaturated while the far-field atmosphere is
unsaturated.3 6
For that reason, the frost flowers are formed on very young sea ice, whose
surface is relatively warm due to the proximity of sea water,3 6
at wind speeds lower than
3-5 m s"1
.3 2
'3 6
Previously it was suggested that brine transport through the pores of sea ice
is important for the frost flower formation.3 1
'3 3
However, the frost flowers were observed
also on frozen rivers and fresh water lakes,3 0
and it was shown theoretically and
confirmed experimentally in a laboratory that the frost flowers can grow on pure ice.3 6
In
a laboratory, the frost flowers were usually grown at the air temperatures between -15°C
and -30°C.3 1
"3 2
'3 6
Sea salt frost flowers have very high salinity up to 115 g/kg3 3 - 3 4
compared with seawater salinity which is less than 35 g/kg.3 6
The frost flowers became
interesting for scientists recently because of their possible role in ozone-depletion events
in polar regions by supplying bromine compounds to the atmosphere,3 7
and as a possible
source of sea salt aerosol in the atmosphere.3 4
'3 8
However, recent studies have shown that
the frost flowers are unlikely to be the major direct source of sea salt aerosol and also the
main source of direct halogen production in the sea ice zone.3 5
Figure 6: Frost flowers on rivers in Alaska. Adapted from ref. 30.
21
Review of the Literature
3.2 Processes Associated with Freezing of Aqueous Solutions
3.2.1 Freeze-Concentration
Generally, most of solutes present in an aqueous solution are very poorly soluble in ice.
Only a few small molecules like HF, HC1, HNO3, formaldehyde, and NH3 may be dissolved
in ice lattice in appreciable amounts. Other solutes are not incorporated into the lattice of
growing ice. They are expelled to the liquid portion of the mixture. As the temperature is
lowered and more water crystallizes to ice, the liquid portion forms a layer on the surface
of ice, is trapped between growing ice grains, or forms inclusions in the ice structure.1
'3 9
The volume of the liquid portion is further reduced and the local concentration of the
solutes in this limited volume may increase by several orders of magnitude compared to
their initial concentration prior to freezing.4 0
This process is known as freezeconcentration
and the liquid portion is often termed as freeze-concentrated solution
(FCS).4 1
The freezing point of such FCS may be much lower than the freezing point of pure
water, so FCS can form a network of veins of concentrated liquid down to very low
temperatures.1
As the cooling proceeds, three types of phase behaviour can be
distinguished:4 2 - 4 3
1. the FCS forms a kinetically stable (but thermodynamically unstable) amorphous
phase called maximally freeze-concentrated solution;
2. the FCS forms a "doubly unstable" glass, in which partial ice/solute crystallization
may occur later during annealing or drying; this glass is unstable in both kinetic
and thermodynamic sense;
3. crystallization of water and a solute (eutectic crystallization) occurs during further
cooling, three phase system (ice, crystalline solute, and amorphous FCS) is formed.
Complete crystallization of ice and all the solutes is rare.4 2
Techniques of optical cryo-microscopy,4 1
scanning electron microscopy (Figure 7),4 4
environmental scanning electron microscopy (Figure 8),4 5
and confocal Raman
microscopy4 6
were applied for direct observation of FCS in the veins and triple junctions
between ice crystals. Indirectly, freeze-concentration was studied by spectroscopic
methods like UV-Vis absorption spectroscopy4 0
and fluorescence spectroscopy,4 7 - 5 0
where the extent of freeze-concentration was deduced from the presence of characteristic
peaks of excimers and aggregated molecules in the spectra. The extent of freezeconcentration
is dependent on the rate of freezing;4 0
'4 5
the local concentration of
Review of the Literature
methylene blue increased by 3 orders of magnitude compared to unfrozen solution during
fast freezing at 77 K and by at least 6 orders of magnitude during slow freezing at 243 K.4 0
Such concentration enhancement leads to contra-intuitive acceleration of chemical
reactions in the frozen state compared to reactivity in unfrozen solution at elevated
temperatures, and reaction pathways different from the ones observed in diluted
solutions. These phenomena are discussed in the chapter 2.2.4.
Figure 7: Crystalline NaCl-2H20 in the veins between ice crystals, as observed with scanning electron microscope. The ice
particles were prepared by spraying 0.043 M solution of NaCl into liquid nitrogen and sintered for 20 h at -25 °C The
darker areas are ice, the lighter areas along ice grain boundaries and as isolated patches on the ice surface are NaCh2H20
(left). Partial sublimation of ice in scanning electron microscope revealed the morphology of the area between the ice
crystals where FCS containing NaCl was located [right]. Adapted from ref. 44.
Figure 8: Observation of frozen uranyl salt solution with environmental scanning electron microscope. Dark area
represents ice crystals; white area corresponds to FCS containing uranyl salt. Detector of backscattered electrons sensitive
to heavy atoms was used. Adapted from ref. 45.
23
Review of the Literature
3.2.2 Freezing Potential
Freezing potential is a phenomenon first described by Workman and Reynolds in 1948
and studied further in detail two years later.5 1 - 5 2
It is an electrical potential generated
across an ice-water interface during freezing of a solution containing ionic solutes due to
unequal distribution coefficients for cations and anions between an ice crystal and an
unfrozen solution. One type of the ions (either the cation or the anion) is preferentially
incorporated into the ice lattice, whereas the excess of counter-ions accumulates in the
unfrozen solution next to the interface. The impurity ions trapped in ice are then
neutralized by highly mobile H.30+ and OH" groups supplied by thermal dissociation of
water molecules in ice. This produces ionization defects in the ice lattice. Uncompensated
ionization defects are then moved by the electric field to the growing interface, thus the
charge layer follows the growth front. The potential difference between the layer of
counter-ions ahead of the interface and the charge layer in ice is the freezing potential.5 3
Although there are several studies in the literature concerning the freezing
potential,5 1 - 6 9
this phenomenon is still not understood completely. Generally, the freezing
potentials measured by various investigators correspond as regards sign but differ as
regards a magnitude of the potential because the measurement of the freezing potentials
is experimentally difficult. The freezing potential was found to be a function of the species
and concentrations of the salts, presence of non-ionic solutes, freezing rate, time, and
crystal orientation. Magnitudes larger than 100 V were reported for solutions of 10~5
M
containing ammonium cation.5 2
-5 8
In case of a low freezing rate, only small freezing potential is measured6 5
due to the
neutralization of the charge imbalance via the flow of H30+/OH" ions through the
ice-water interface.5 3
As the freezing rate increases, the freezing potential rises, as the
migration of H30+/OH" and the neutralization of the potential is limited due to short time
and counterions accumulated at the interface.5 3
According to some studies, the freezing
potential only increases with increasing freezing rate.7 0 - 7 1
On the other hand, other
researchers reported the freezing potential to increase with increasing freezing rate up to
some point, and then to decrease again (Figure 9).6 6
-7 2
This behaviour is explained by the
theoretical model of Bronshteyn and Chernov in terms of the instability and break down
of the crystallization front at high freezing rates, and facilitated neutralization of the
charge imbalance through the interface.5 3
Review of the Literature
The usual dependence of the freezing potential on concentration of a salt (in the
logarithmic scale) resembles Gauss curve (Figure 10), with the maximum freezing
potential measured for concentrations between 10~5
to 10~3
M. For the concentrations
higher than 10~2
M, there is none or negligible freezing potential reported.5 9
'6 6
The reason
of such behaviour is still unclear, although there are some theories about it.5 3
'5 9
'7 2
According to one of the theories, the crystallization front becomes unstable due to the
high concentration of a salt, and the charge neutralization via the flow of H3O/OHoccurs,5
3
similar to when large freezing rate is applied.
20 30 40
Freezing rate / fims"'
50
Figure 9: Freezing potential ofNaCl (c = 5xl0~5
M] as afunction of freezing rate. Adapted from ref. 66.
30 -,
106
10 5
104
10J
102
101
10°
c (NaCl) / mol.dm3
Figure 10: Freezing potential as afunction of concentration ofNaCl. Adapted from ref. 66.
25
Review of the Literature
An anion is usually incorporated into the ice lattice more than a cation, and the
resulting freezing potential is positive*. Most likely it is the matter of the
electronegativity;5 2
'5 7
'6 3
'6 6
the electronegativity of the ionic atom or molecule needs to be
large so that it will be able to replace the largely electronegative oxygen atom in the ice
lattice.6 6
However, ionic radius7 2
and the orientation of a hydration shell5 9
can also play a
role. Negative freezing potentials are measured in one of the following cases:
1. An anion is rejected by ice. Such a behaviour is observed when a nitrate or an
acetate is frozen; the anion is relatively large and its structure is not compatible
with the water molecule.5 2
'5 8
2. Ammonium salt is used. Due to isomorphism of ammonium with hydronium
cation, NH4+
fits into the ice lattice and large negative freezing potentials are
measured.5 2
'5 8
'6 0
'6 6
The only known ammonium salt with positive freezing
potential is NH4F; fluoride anion is, due to its high electronegativity resembling the
one of the oxygen, even more soluble in ice than ammonium cation.5 2
3. In some cases, negative potentials were detected when the concentration of a salt
(with otherwise positive potential) was too low,5 2
'5 9
or when pure water was
frozen.5 2
'5 4
-5 9
t/s
Figure 11: Illustrative scheme of selective ion incorporation [left], and the evolution of the freezing potential in time
[right].69
The white hexagon symbolizes a growing ice, the blue colour symbolizes an unfrozen solution. Selective ion
incorporation into the ice leads to a charge imbalance between the solution and ice, which can be measured as the freezing
potential (of the solution with respect to ice]. Later, the charge imbalance is neutralized via a flow ofH*/OH~ through the
ice-water interface and the acidity of the remaining solution is altered. When NaCI solution is frozen, chloride anions are
incorporated to a greater extent into the ice, while freezing of NH4CI solution results in a greater incorporation of
ammonium cation into ice due to its resemblance to H3O+ cation.52
* There is some ambiguity in the literature about the signs of the potentials. Here, the freezing potential is expressed as
the potential of liquid with respect to ice, as it was originally defined by Workman and Reynolds. Some authors present
the freezing potential as the potential of ice with respect to liquid. Hence, the sign of the potential is reversed.
26
Review of the Literature
3.2.3 Freezing-Induced Acidity Change
A constant pH is essential for the stability of many (bio)molecules. In aqueous solutions,
buffers are typically used to maintain pH at a constant value. However, when the solution
is frozen, its pH can change by several pH units whether towards acidic or basic values,
even in the presence of a buffer. Three mechanisms were identified in the literature to be
responsible for freezing-induced acidity change: freeze-concentration effect,73
selective
ion incorporation (Workman-Reynolds effect),52-53,74 a n ( j sequential crystallization of
buffer components.7 5 - 7 9
Freeze-concentration effect as a phenomenon resulting in acidity change was
proposed by Heger et al.7 3
They applied UV-Vis spectroscopy to study the extent of
protonation of the acid-base indicator cresol red (CR) in frozen solutions as a function of
pHo (the subscript "0" indicates initial conditions prior to freezing). They found out that
apparent pKa1
of CR increased from ~1.1 in a solution to ~4 in the frozen state, while the
change of apparent pKa
2
during freezing was negligible. The extent of CR protonation (first
transition) increased by about 3 orders of magnitude during freezing. They attributed this
behaviour to the increase of the local concentrations of both CR and acids used for
adjusting pHo. Similar study was done by Govindarajan et al.,8 0
where the impact of
lyophilization on the protonation of sulfonephthalein indicators was examined in
trehalose/citrate systems with pHo 3.4-6. The protonation was consistently higher in the
lyophiles than in the corresponding solutions from which they were freeze-dried. The
protonation of the indicators in the lyophiles correlated with pHo - an increase in pHo
resulted in a decrease in the indicator protonation in the corresponding lyophile. Such
correlation between pHo and the acidity of resulting lyophiles is often termed "pH
memory"8 0
-8 1
The concept of selective ion incorporation (Workman-Reynolds effect) was
already introduced in the previous chapter. Preferential incorporation of one type of the
ion into the ice leads to the charge imbalance between the ice and the remaining solution,
which can be measured as the freezing potential. The neutralization of the charge
imbalance results in the change of solution's pH, as is schematically illustrated in Figure
11. The mechanism of the charge imbalance neutralization was theoretically explained
by Bronshteyn and Chernov5 3
as follows. Let's consider freezing of an aqueous solution of
NaCl with pHo = 7. It is known that CI" ions are incorporated in ice more than Na+
ions, so
there is an excess of negatively charged CI" ions in the ice and an excess of Na+
ions in the
Review of the Literature
solution. The mobility of the incorporated ions in the ice is extremely small and they form
a "freezing-in" charge in the ice. The excessive Na+
ions in the solution are concentrated
mainly in the Debye layer near the ice surface. In an ideal dielectric, the excessive Na+
ions
in the Debye layer would shield the liquid from the electric field, and the pH in the liquid
phase would not change. However, due to the highly mobile polarization and ionization
defects in the ice lattice, there is the charge transfer between the boundary layer of the
crystal surface and the inside of the crystal. The electric current results from the
redistribution of existing H30+
and OH" ions and of those generated by the dissociation of
water molecules in both phases. The excessive negative charge in the ice is neutralized by
the H30+
flow and the release of OH" from the ice to the solution. The rate of the
neutralization is limited by the rate of spontaneous thermal dissociation of water
molecules in the ice and at the boundary layer. As a result, the electric field is decreased
and Na+
and OH" leave the interface, so that the solution becomes alkaline.5 3
The relationship between the selective ion incorporation and the acidity change
was experimentally proven by Workman and Reynolds as soon as in 1950; NaCl solution
(c = 7xl0"5
M, pHo = 6.3) was partially frozen in an inert atmosphere, the unfrozen
solution and the ice were separated, allowed to equilibrate are room temperature, and
their pHs were measured. pH of the frozen fraction was 6.2, pH of the unfrozen fraction
was 7.O.52
Basification of NaCl solution during freezing was confirmed in other studies.6 8 -
70,73-74A s regards the effects of other salts, basification of LiCl,6 8
"6 9
KC1,6 8
"6 9
RbCl,6 9
MgCb,6 9
CaCb,6 9
BaCb,6 9
(CHs^NCl,6 9
NaF,6 9
NaBr,6 9
and Nal,6 9
and acidification of NH4 C1,6 9
-7 3
Na2S04,74
and (NH4)2S0474
were reported. The results of the acidity measurement are in
accordance with the results of the freezing potential measurements - selective
incorporation of an anion into the ice leads to rise of solution's pH and vice versa.5 2
Sequential crystallization of buffer components is the third abovementioned
mechanism responsible for the freezing-induced acidity change. The pH of a buffered
solution is determined by the concentration ratio of the deprotonated form of the buffer
(A") and the protonated form of the buffer (HA) according to Henderson-Hasselbach
equation
Vn = VKa + log[
^r ( 1 )
During freezing under equilibrium conditions, the composition of a maximally freezeconcentrated
buffer is defined by its eutectic composition, which is independent of the
Review of the Literature
pHo and the concentration of the buffer,7 8
and may substantially differ from the
composition of the unfrozen buffer solution. While the pH of potassium phosphate buffer
(K-P) at the eutectic point is close to 7.5, and only minor acidity changes were detected
when a neutral K-P solution was frozen,7 8
'8 2
the pH of sodium phosphate (Na-P) at the
eutectic point was found to be 3.6 due to the poor solubility of Na2HP04 • I2H2O at subzero
temperatures.7 8
As a result, a pH drop of up to 3 pH units was observed during freezing
an initially neutral solution of Na-P.7 5
"7 6
'7 8
The sequential crystallization of buffer
components may result in a "pH swing", as observed during the freezing of succinate
buffers, where the pH during the process first increased from 4 to 8 and subsequently
decreased to 2.2. The increase of the pH was attributed to the crystallization of succinic
acid, followed by the crystallization of monosodium succinate accompanied by the pH
drop. The "pH swing" in the opposite direction was observed when a succinate buffer with
pHo = 6 was frozen.8 3
Freezing usually occurs under non-equilibrium conditions, and both the pHo and
the buffer concentration play an important role in the extent of the buffer crystallization
and the resulting acidity in the frozen state; the higher the concentration of the buffer, the
more prone to pH jumps the buffer is. For that reason, lowering the buffer concentration
often results in milder pH jumps.75,83-84
The pH jumps were shown to be detrimental for the stability of proteins during
freezing and lyophilization.8 5 - 8 6
Thus, buffers that do not crystallize during freezing and
exhibit constant pKa regardless of the temperature are recommended for use in biology,
biochemistry, and the pharmaceutical industry. Citrate buffer is supposed to have these
properties, and therefore it was identified as the preferable buffer for lyophilized
formulations at acidic and near-neutral pHs. However, we are not aware of a buffer
candidate suggested for use at basic pH values.8 7
3.2.4 Chemical Reactivity in Ice
Ice and snow are environmentally important reaction media. Cryo-reactions (chemical
reactions in/on ice) became a subject of scientific interest in the past few decades due to
an unexpected chemistry occurring in ice. They can be classified into two groups:3 9
1. reactions where unexpected chemical transformation occurs as a result of
cooling/freezing;
29
Review of the Literature
2. reactions where the apparent rate of a known reaction is accelerated.
Due to the "grasshopper effect", semi-volatile organic compounds travel long distances
from the warmer regions of the Earth by multiple volatilization-transport-deposition
cycles and accumulate in polar areas.8 8 - 8 9
These compounds are often resistant to
environmental degradation; therefore, they are termed persistent organic pollutants
(POPs). Examples of such POPs are organochlorine pesticides,9 0 - 9 4
polychlorinated
biphenyls,9 5 - 9 6
polybrominated diphenyl ethers,9 7
polycyclic aromatic hydrocarbons,9 8
and dioxins and furans.9 9
They are deposited to ice and snow where photochemical
transformations different from those in aqueous solutions were observed,1 0 0 - 1 0 8
and
photoproducts with significantly higher toxicity are formed.1 0 9
Cryo-reactions could play
an important role in atmospheric chemistry where snowpack, polar stratospheric clouds,
cirrus clouds, frost flowers, freezing fogs, snowflakes, and hailstones are potential
surfaces for heterogenous chemistry to take place on.3 9
Probably the most famous
example of the role of ice particles the atmospheric chemistry is the phenomenon of
Antarctic ozone hole,1 1 0
which is crucially dependent on the role of polar stratospheric
clouds. The reaction of HC1 and CIONO2 molecules leads to formation of nitric acic/nitrate
adsorbed to the ice surface and release of ozone-depleting species (Cb, HCIO) into the
atmosphere.3 9
According to Arrhenius equation
k = A e - E
- l R T
, ( 2 )
rate constant of a chemical reaction decreases when temperature is lowered. For that
reason, chemical reactions are commonly decelerated at lower temperatures. It was
shown, however, that certain chemical reactions are accelerated in ice compared to those
in aqueous solution. This contra-intuitive behaviour is reported especially for bimolecular
reactions. Examples of such reactions are oxidations of nitrite by O 2 , 1 1 1 - 1 1 3
1,1diphenylethylene
by O 3 , 1 1 4
sulfide by H 2 O 2 , 1 1 5
elemental mercury by H 2 O 2 , 1 1 6
chromate by
H 2 O 2 , 1 1 7
chromate by arsenite,1 1 8 - 1 1 9
or hydrolysis of fluorescein diacetate.1 2 0
The effect of
freeze-concentration is thought to be the largest contributor to the acceleration of
reactions in frozen solutions.3 9
During freezing the reactants are confined in the solution
surrounded by walls of ice grains where the concentrations of the reactants become
extremely high.1 1 2
This process is schematically illustrated in Figure 12. A reaction must
30
Review of the Literature
satisfy the three following criteria in order to be accelerated by the freeze-concentration
effect:39
1. The reactions are required to be second order or higher, as first order reactions
are not accelerated in ice.
2. The total initial concentrations of reactants must be low.
3. A small activation energy is necessary for the reaction.
(a) (b) (c) (d)
Figure 12: Illustration of the freeze-concentration effect leading to acceleration of chemical reactions in the frozen state.
Concentrated phase (C) isformed around single ice crystals (I). As the ice crystals grow, concentrated solution (S) becomes
confined in a vein between the ice crystals and the concentration enhancement is sufficient for a reaction to be accelerated
(RJ. Adapted from ref. 112.
Apart from freeze-concentration, other factors are suspected to be responsible for
acceleration of chemical reactions in ice. As most of the observed accelerated reactions
are redox reactions, the freezing potential effect could play a role in the increased
reactivity in ice.3 9
Freezing-induced acidity change was identified as an important factor
in the acceleration of certain chemical reactions.5 3
'1 1 2
'1 2 1
The hydrolysis of frozen
potassium ferricyanide K3[FeCN6] in presence of NaCl was observed at pHo > 10, while it
proceeds only at pH > 12 in an aqueous solution.5 3
Similarly, the oxidative decomposition
of gallic acid occurs only in alkaline solutions without freezing. However, small amount
gallic acid decomposed even at pHo = 4.5 during freezing in presence of NaCl, and about
2/3 of gallic acid were decomposed after 50 freeze-thaw cycles. Both the presence of NaCl
and freezing were required for the decomposition.1 2 1
In the both abovementioned studies,
NaCl is assumed to increase pH of a solution during freezing due to Workman-Reynolds
effect. Chemical reactivity due to the freezing-induced acidity change is called freezing
hydrolysis.5 3
Catalytic effect of the ice surface was ruled out as a potential factor for
reaction acceleration, as the addition of crushed ice to the solution at 273 K had no effect
on the reactivity.1 1 2
31
Review of the Literature
3.3 Stabilization of Proteins by Freezing and Lyophilization
3.3.1 Lyophilization
Lyophilization is an important and well-established process to improve the stability and
long-term storage stability of labile drugs, especially therapeutic proteins.4
'1 2 2
Although
an aqueous liquid formulation is the easiest and most economical to handle during
manufacturing and it is the most convenient for the end user, many proteins are
susceptible to chemical (e.g., deamination or oxidation) and/or physical (e.g., aggregation
or precipitation) degradation during shipping and long-term storage in liquid
formulations.1 2 3
These stability problems can theoretically be avoided by properly
prepared lyophilized formulation, as degradative reactions are avoided or slowed down
sufficiently in the dried product, and the protein remains stable for months or years at
ambient temperatures.1 2 3
Nowadays, lyophilization is the most common formulation
strategy to prepare biopharmaceuticals; about 50% of the currently marketed
biopharmaceuticals are lyophilized.4
However, some proteins suffer irreversible damage
during lyophilization or afterwards during storage as a freeze-dried powder. The stability
problems are normally minimized by a combination of proper process control and
formulation optimalization.1 2 4
In general, lyophilization is a very time consuming and expensive process.4
'1 2 3
For
pharmaceutical purposes, lyophilization usually consists of three main steps: freezing
primary drying, and secondary drying.1 2 2
'1 2 5
Freezing itself is the major dehydration step
in lyophilization; about 80% water is converted to ice 4
The frozen product typically
contains remaining water and all the solutes in a single amorphous face.1 2 5
The freezing
process is presumable the most complex and the most important step of lyophilization.4
It is described in the next chapter in detail. An annealing step - holding the system for a
period of time at a temperature between the ice melt and the glass transition temperature
[Tg] of FCS, is often performed after the freezing step for bulking agents to crystallize out
and larger ice crystals to grow.1 2 2
'1 2 5
During the primary drying the temperature is held
2-5°C below the glass transition of FCS and a vacuum is applied to remove crystalline ice
by sublimation.1 2 5
After the primary drying step, the product can still contain
approximately 15-20% of unfrozen water.4
Most of the remaining water is removed by
desorption at elevated temperature and low pressure during the secondary drying step.
32
Review of the Literature
The objective of secondary drying is to reduce the residual moisture content to a level
optimal for stability, which is usually less than 1%.1 2 2
As large fraction of the remaining
water after primary drying may be associated with the protein, secondary drying may be
detrimental for the protein stability due to the reduction in stabilizing hydrogen bonds
between the protein and water molecules. For that reason, saccharides and polyols are
often used as lyoprotectants to substitute the lost hydrogen bonds with new hydrogen
bonds between the protein and -OH group of the lyoprotenctant.1 2 5
3.3.2 Freezing
Apart from the first step of lyophilization, freezing is often used as a standalone procedure
to store a biological material for extended periods of time.1 2 6
. In both cases, freezing can
cause substantial protein denaturation, resulting in a loss of activity. Therefore, the
understanding of the effects on freezing and thawing on protein stability is critical for
many applications in research and medicine.8 5
Freezing is defined as the process of ice crystallization from a supercooled solution.
It involves cooling of the solution, ice nucleation, and ice crystal growth.4
Generally, the
solution does not freeze spontaneously at the equilibrium freezing point.1 2 7
The
difference between the equilibrium and actual ice formation temperature is called degree
of supercooling.4
'1 2 2
The degree of supercooling depends on the solution properties and
cooling rate (it is important to distinguish between cooling rate and freezing rate; the
cooling rate is the rate at which the solution is cooled, while freezing rate refers to postnucleation
ice crystal growth1 2 6
). Generally, the highest supercooling is achieved when
small volumes of the solution are frozen in liquid nitrogen.1 2 2
Higher degree of
supercooling results in large number of small ice crystals, while lower degree of
supercooling results in lower number of large ice crystals. Nevertheless, it is necessary to
distinguish between global supercooling and local supercooling. When global
supercooling occurs, the entire volume achieves a similar level of supercooling and the
solidification progresses through the already nucleated volume. In this case, a slower
cooling rate leads to a faster freezing rate and vice-versa. In contrast, local supercooling
leads to a directional solidification; in this case, a faster cooling rate results in a faster
freezing rate. This is the case for high cooling rates, e.g. liquid nitrogen freezing.4
Degree
of supercooling can be controlled by the operator when seeding with ice is applied.1 2 6
33
Review of the Literature
3.3.3 Stresses during Freezing and Lyophilization
Although some proteins can tolerate freezing and/or drying stresses without any
harm,1 2 8 - 1 3 1
others suffer partial or complete loss of their activity during freezing and/or
drying in the absence of stabilizers.8 5
"8 6
'1 3 2
"1 4 7
The cause of the destabilization or
denaturation of an unprotected protein is a variety of stresses related to freezing and
lyophilization. The tolerance of various proteins toward freezing- and drying-induced
stresses significantly differs,1 4 8
and the freeze-thawing or freeze-drying protocol and
formulation design (protein concentration, buffer type and concentration, presence of
stabilizers and other solutes, solution's pH) also play an important role in the recovery of
protein activity after freeze-thawing and freeze-drying.1 2 3
Optimization of all the
parameters is difficult, time-consuming and often done by trial-and-error approach.1 2 6
Moreover, parameters optimal for one protein may not work for the other. Thus,
understanding of physico-chemical stresses imposed on proteins during freezing and
drying and their contribution to overall denaturation of proteins is essential for successful
stabilization of proteins in the frozen and freeze-dried state.1 2 6
The most important of
these stresses are listed below.
Low temperature. The free energy of protein unfolding is typically a parabolic
function of temperature, so the proteins tend to unfold not only at high but also at low
temperatures.4
The destabilization of proteins at low temperatures is referred to as cold
denaturation.4
'1 4 8
Cold denaturation is related only to decreased temperatures and occurs
in the absence of freezing.4
It is enthalpy-driven, in contrast to normal or thermal
denaturation, that are both entropy-driven.1 4 8
It is reported for variety of proteins.1 2 6
However, there is some evidence in the literature that the contribution of cold
denaturation to the overall degradation can generally be considered negligible.1 2 6
Formation of ice-water interface. Adsorption of a protein to the ice-water
interface results in surface-induced denaturation of surface-sensitive proteins.4
'1 4 9
There
are two hypotheses about the mechanism of the surface-induced denaturation. One of
them involves freezing potential (§3.2.2) at the ice-water interface; the other assumes an
ordering of the water molecules in the vicinity of the protein upon ice formation, thereby
creating an entropically unfavourable environment. Adsorption of the protein on ice leads
to entropy increase, which provides a thermodynamic driving force for protein
adsorption and unfolding.4
'1 2 6
'1 5 0
The effects of ice formation strongly depend on the
degree of supercooling and freezing rate, the factors that determine the specific surface
Review of the Literature
area of the formed ice.1 2 6
Surface-induced denaturation can be reduced or prevented by
slow cooling, which generates smaller ice-water interface,1 4 8
or by addition of non-ionic
surfactants, which compete with proteins for adsorption at the interface.4
Freeze-concentration. Freeze-concentration effect (§3.2.1) leads to rapid increase
of the concentration of all solutes. It results in the increase of ionic strength of the solution,
which potentially destabilizes proteins.1 2 6
The rate of bimolecular reactions might
increase due to freeze-concentration; this is counteracted to some extent by the decrease
in temperature and the increase in viscosity,1 2 6
although the acceleration of (3-glutamic
acid oligomerization at -20 °C was reported.1 5 1
The oxygen concentration in a partially
frozen solution at -3 °C is reported to be 1150 times higher in comparison with that in
solution at 0 °C;1 5 2
that can result in the oxidation of sulfhydryl groups in proteins.1 4 8
On
the other hand, increasing the protein concentration will increase stability of many
proteins during freezing.126
'128
'140
'142
'146
'153
-156
pH changes. The origin of freezing-induced pH change was already discussed in
§3.2.3. Such pH change may be detrimental with regard to protein stability, as many
proteins are stable only in a narrow pH range, and tend to unfold or denature at extreme
pH due to increased electrostatic repulsion between like charges.1 4 8
The detrimental
effect of freezing-induced pH change was demonstrated by significantly lower stability of
proteins frozen in Na-P in comparison with proteins frozen in K-P at otherwise identical
conditions 8 5 - 8 6
Crystallization of a buffer is a major factor for pH change when freezing of
proteins is concerned. It can be minimized by optimum choice of a buffer, lowering the
buffer concentration, keeping the buffer components in the amorphous state, or by
adjusting the freezing method.4
'1 2 2
'1 2 6 - 1 2 7
Phase separation. Liquid-liquid phase separation in the unfrozen phase and
formation of multiple amorphous phases during freezing may deprive the protein of the
cryoprotective effects of the stabilizers and thus negatively affect protein stability.4
'1 2 6
Dehydration. A fully hydrated protein has a hydration shell - a monolayer of water
covering the protein surface. The amount of water in the hydration shell is 0.3-0.35 g per
1 g of protein.1 5 7
As the water content of a lyophilized protein is generally less than 10%,
lyophilization removes part of a hydration shell, which may disrupt the native state of a
protein and cause denaturation. A protein tends to abolish as many charges as possible
when exposed to water poor environment, which may facilitate protein-protein
35
Review of the Literature
hydrophobic interactions and lead to protein aggregation. Moreover, removing water
molecules from an active site in protein may result in protein inactivation.1 4 8
3.3.4 Cryoprotection and Lyoprotection of Proteins
Freezing and subsequent thawing was reported to cause partial or complete loss of
activity of alcohol dehydrogenase,1 4 6
L-asparaginase,133
catalase,1 4 6 - 1 4 7
'1 5 8
(3galactosidase,8
5
'1 3 3
'1 4 6 - 1 4 7
haloalkandehalogenase,145
hexokinase,1 5 9
lactate
dehydrogenase,1 3 2
'1 3 6
'1 4 0
'1 4 6 - 1 4 7
'1 5 9
or phosphofructokinase1 6 0
in the absence of stabilizers.
Further losses of activity were reported during the drying step.8 6
'1 6 1
To prevent or
minimize protein denaturation during freezing and/or drying stabilizers called
cryoprotectants and lyoprotectants are added to protein formulations. Cryoprotectants
stabilize a protein during freezing, while lyoprotectants stabilize a protein during drying.
Since freezing and dehydration stresses imposed on protein are different, mechanisms of
protein stabilization are not the same during freezing and drying. Therefore, many
effective cryoprotectants do not stabilize proteins during drying, and vice-versa, effective
lyoprotectants may not stabilize proteins during freezing. Some stabilizers can serve as
both cryo- and lyoprotectants; otherwise, two (or more) stabilizers have to be used to
protect proteins during freeze-drying.1 4 8
There are numerous studies concerning croprotective and lyoprotective effect of
various chemicals, and it is not the aim of this dissertation to review them here in detail,
rather compile the most important information in brief. More detailed information about
cryoprotection and lyoprotection are reviewed elsewere.1 2 6
'1 4 8
-1 6 0
Several groups of chemicals are commonly used as cryoprotectants and/or
lyoprotectants, the most important of which are sugars,132
'141
-142
'154
'156
'158
'160
'162
-167
p0 /y0 /5 < 141-142,149,158,160,162-163,168-169 pO /yW j e r 5 ) 132,140,158,170-172 protein itself,128
'140
'142
'146
'153
'
155,160,162,173 174 non-aqueous solvents148
surfactants132137158175
amino acids, 8 2 1 4 1
I42,i60,i73,i76-178 s a / ^ 142,160,179 amines142 160
and amphiphilic excipients.180
Nowadays, two classes of cryoprotection mechanisms are suggested thermodynamic
and kinetic. A cryoprotectant operating via thermodynamic mechanism
shifts the equilibrium from the unstable, unfolded conformation toward stable, native
state of a protein. In contrast, a cryoprotectant operating via a kinetic mechanism slows
36
Review of the Literature
the rate of inactivation, but does not change the equilibrium between a native and a
denatured state.1 2 6
The thermodynamic stability of a protein is determined by the difference between
the free energy of a native and denatured state, the free energy of unfolding (AGu). The
greater AGu, the more stable the native protein is. There are two ways to increase AGu of
a protein. One is to use a specific ligand binding to the native state and decreasing its free
energy, thereby increasing the value of AGu. The effect of such ligand is depicted in Figure
13 (A). However, such ligands are protein-specific, so that they may not stabilize other
proteins, and may even destabilize them. The other way is to add an additive that interacts
repulsively with a protein, or has the ability to increase the surface tension between the
protein and the aqueous solution. That increases the free energy of the native state, but
the free energy of denatured (unfolded) state is even more increased due to larger surface
area of the unfolded protein, and the overall AGu increases.1 6 0
'1 8 1
This mechanism is called
preferential exclusion1 2 6
'1 5 3
'1 6 0
'1 8 1
and is schematically illustrated in Figure 13 (B) and in
Figure 14. The preferential exclusion mechanism was suggested for stabilization of
proteins in solution, later it was extended to freeze-thaw processes.1 4 8
A major difference
between the ligand binding and the preferential exclusion mechanism is that the
preferential exclusion is protein-independent, so the preferentially excluded solutes are
universally effective as protein stabilizers. A drawback of this stabilization mechanism is
enhanced protein association, as protein-protein interaction results in decreased surface
area per molecule and hence reduces the unfavourable interactions.1 8 1
Several other
stabilization mechanisms were postulated in the literature - modification of the size of ice
crystals, reduction of surface tension, restriction of diffusion of reacting molecules by
increasing the viscosity of a solution, or suppressing pH changes during freezing.1 4 8
In addition to the thermodynamic stabilization, cryoprotectants may operate via
kinetic mechanisms. Binding of ligands to a native protein may trap the protein kinetically
to the more stable state, or rise the activation energy of unfolding.1 8 1
The most widely
discussed kinetic mechanism is the vitrification hypothesis, where freeze-concentration
and low temperature result in increase in viscosity, reduced mobility and slowing of all
dynamic processes. In the glassy state, the protein becomes virtually immobilized, and the
rate constant of denaturation is reduced without affecting AGu.1 2 6
Analogous to the kinetic stabilization of proteins during freezing a major
mechanism of protein stabilization by lyoprotectants is the formation of an amorphous
Review of the Literature
glass.1 4 8
-1 8 2 - 1 8 3
Another suggested lyoprotection mechanism is the water replacement
hypothesis.1 5 4
'1 7 0
The stabilizers substitute water molecules in formation of hydrogen
bonds with a protein during drying to satisfy the hydrogen bonding requirement of polar
groups on the protein surface. Other mechanisms involve increasing Tg of protein
formulations,1 8 4
inhibiting crystallization of other solutes,1 8 0
preserving a protein's
internal mobility,1 8 5
or forming multiple electrostatic interactions with the protein.1 8 6
There is also a term pharmaceutical stability mentioned in the literature. It refers
to the ability of a protein to be processed, distributed and used without irreversible
change in protein's structure, conformation, or state of aggregation. The protein may
exhibit thermodynamic instability and unfold during freeze-drying but refold completely
upon reconstitution. Hence, it is pharmaceutically stable.1 2 4
B
Unfavorable interaction
Unfolded state ,
T
AG,
AG,
AG,,
Native state
No additive
Favorable interaction
Preferentially excluded additive
Structure-stabilizing additive
Figure 13: Free energy diagram of different states of proteins in the absence and presence of stabilizers. AGu is the free
energy of unfolding. Adapted from ref. 181.
Associated state
More stable
Native state
t 4More stable
Unfolded state
t
Figure 14: Schematic illustration of preferential exclusion mechanism. Repulsive interactions between a protein and a
solute are depicted with arrows. Unfolding [denaturation] of the protein leads to increase of the protein surface area and
so the amount of energetically unfavourable interactions. On the other hand, association of protein molecules decreases
the surface area per molecule and hence the association is energetically favourable. Adapted from ref. 181.
Review of the Literature
3.4 Techniques for the Measurement of Solid-State Acidity
There is no standard and universally accepted method for the measurement of solid-state
acidity. Several indirect techniques are described in the literature; however, they often
give different outcomes1 8 7 - 1 8 8
as the results are dependent on the basic principles and
assumptions of the method used.1 8 9
Commonly used methods for the measurement of
solid-state acidity are listed below.
Low temperature pH electrode. A low temperature pH electrode can be used to
monitor pH of a partially frozen solution,7 5
as it can operate at temperatures down to
-30 °C. When the solution is fully frozen, its contact with the electrode deteriorates, which
can affect the results. Moreover, it was shown previously that pH electrodes measure
proton concentration rather than proton activity;1 9 0
as FCS is far from similar to a dilute
solution, the acidity measured by the electrode can be biased from actual pH.
Slurry method. In this method, the surface acidity of a solid is estimated by
potentiometric measurement of pH of the concentrated suspension (slurry) of the solid in
water.1 8 7
This approach relies on the assumption that, under equilibrium conditions, the
surface pH of the solid corresponds to the pH of the slurry.1 9 1
However, the slurry contains
a significantly higher amount of water than the solid, and the slurry pH is dependent on
the concentration of the solid, the solubility of each component, and the presence of
particles ("the suspension effect").1 8 9
Therefore, the reliability of the abovementioned
assumption is debatable.
In situ indicator probes. In situ indicator probes are molecules sensitive to the
concentration of protons in their proximity. A degree of protonation of the probe is
reflected in some of its physical properties that can be measured easily, e. g. UV-Vis
absorption spectra,7 3
-8 0
Raman spectra,1 9 2
relative fluorescence intensity,6 8
-1 9 3
EPR
signal,1 9 4
or NMR chemical shift.7 4
To evaluate the solid-state acidity probed by indicators,
Hammet acidity function (Hx) is commonly used. It was proposed as a measure of acidity
beyond dilute aqueous solutions1 9 5
and defined in terms of the ionization of a neutral
acidic indicator Hind as
where c, aH+, y, x, and Ka\nA stand for the concentrations of the indicator species, proton
activity, indicator activity coefficients, charge of the deprotonated form of the indicator,
Review of the Literature
and acidity constant of the indicator in a dilute aqueous solution, respectively.8 0
In dilute
solutions, where the activity coefficients are close to unity, Hx is equal to pH. In very
concentrated solutions or solid systems the ratio of the activity coefficients is not
necessarily equal to 1, and these two quantities may differ. Besides Hx, equivalent terms
like "surface pH",1 8 7
"microenvironmental pH",1 9 6
"equivalent pH",1 8 9
and "apparent pH"7 9
can be found in the literature to express the solid-state acidity. These terms do not
represent the absolute proton activity, rather the effect of the environment on the
ionization state of the indicator probe. They are numerically equal to such pH of a dilute
aqueous solution that would result in the same ionization extent of the indicator as
present in the solid matrix.1 8 9
-1 9 6
Sulfonephthalein indicators are commonly used to probe solid-state acidity and for
Hx calculation.7 3
'8 0
'1 4 5
'1 8 7 - 1 8 9
'1 9 7
Although Hx can be indicator-specific due to its dependence
on the ratio of the activity coefficients of the indicator (Eq. 3),8 0
'1 8 7
only minor differences
were observed when the structurally similar sulfonephthalein indicators were used.8 0
-1 8 9
Therefore, using a series of sulfonephthalein indicators with various pKas appears to be a
useful method to probe solid-state acidity in a wider pH range.
To demonstrate the usefulness of the indicator probe method, the surface acidity
of common excipients was determined by the slurry method and via probe molecules, and
correlated with the chemical stability of an acid-sensitive active pharmaceutical
ingredient. While there was a clear relation between the stability and the surface acidity
when the indicator probe method was used, no clear trend was observed when the
stability was plotted as a function of the suspension pH. The results support the use of Hx
obtained by the indicator probe method as an empirical scale to represent relative acidity
of different excipients and to predict the stability of acid-sensitive pharmaceutical
ingredients.1 8 8
Review of the Literature
3.5 Diffuse reflectance spectroscopy
Diffuse reflectance is commonly used in the UV-Vis, near- and mid-infrared regions to
obtain molecular spectroscopic information about solid samples. A reflectance spectrum
is obtained by the collection of surface-reflected light. Two types of reflection can occur:
1. Specular (regular) reflection - reflection from smooth, polished surfaces (e. g. a
mirror). Light is reflected only at angle of reflection being equal to the angle of
incidence.
2. Diffuse reflection - reflection from matt surfaces (e. g. a white wall). Light is
reflected at angles independent of the angle of incidence.1 9 8
A property called reflectance (R; a term remittance can also be found in a
literature) is measured in diffuse reflectance spectroscopy. It is the ratio of intensities of
reflected to incident light, analogous to transmittance in transmission spectroscopy.1 9 8
However, if apparent absorbance log(l/i?) is calculated from reflectance (analogously to
absorbance in transmission spectroscopy), the intensities of the peaks are not linearly
proportional to concentration. As absorption and scattering of light occur simultaneously,
diffuse reflectance spectra need to be treated with a two-constant theory. The most often
used two-constant theory to describe and analyse diffuse reflectance spectra is KubelkaMunk
theory,1 9 8
originally introduced in 1931.1 9 9
In this theory, Kubelka-Munk function
F(R) was derived:
F(R) = ( 1
~R)2
=« = 2
- 3 0 3 £ C
/ (4)
2 R S S
where R stands for the reflectance of the sample, K and S are absorption and scattering
coefficients, respectively, e is molar absorption coefficient, and c is sample concentration.
This function is linearly dependent on analyte concentration, provided that S remains
constant and all assumptions of the model are fulfilled.1 9 8
Although Kubelka-Munk theory
assumes no specular component in a diffuse reflected light, this is usually untrue, as light
can be specularly reflected from the surfaces of individual particles. The presence of the
specular component in a diffuse reflectance spectrum is problematic. Specularly reflected
beam interacts only once with the sample surface, while diffuse reflected beam undergoes
multiple reflections (Figure 15). Thus, specular reflection contains less spectroscopic
information that the diffuse reflected component, and spectra can be distorted.1 9 8
41
Review of the Literature
Therefore, samples for diffuse reflectance measurement need to be prepared with care in
order to minimize the specular component.2 0 0
If the reader is interested in more details about diffuse reflectance spectroscopy,
they can be found in the referred books.198,200-201
Figure 15: Scheme of diffuse reflectance [blue] and specular reflectance from individual particles [red]. Black arrows
represent incident beams, black circles represent individual particles of a sample.
42
Acidity of Frozen and Freeze-Dried Phosphate Buffers
4 Results and Discussion
4.1 Acidity of Frozen and Freeze-Dried Phosphate Buffers
The results of this project were accepted for publication to International Journal of
Pharmaceutics. The manuscript "Vetráková, Ľ.; Vykoukal, V.; Heger, D, Comparison of
Acidities of Aqueous, Frozen, and Freeze-Dried Phosphate Buffers. Is there a "pH Memory"
Effect?" is attached in Appendix A, Supplementary Information is included.
4.1.1 Introduction
Using of buffers during freezing and lyophilization may lead to large pH shifts that are
harmful for the stability of proteins and other biomolecules. As lyophilization comprises
two distinct steps, freezing and drying, the pH shift in both steps may affect the stability
of proteins in the resulting lyophile. There are several studies in the literature where the
stabilities of lyophilized compounds are correlated with the acidity of their freeze-dried
state,1 8 8
-1 8 9
-1 9 6
-2 0 2
but the effect of freezing-induced acidity change is usually omitted. The
acidity of lyophiles is often similar to pH of a pre-lyophilization solution; this similarity is
called "pH memory".8 0
"8 1
In spite of that, it is not clear if the acidity is not altered in the
frozen state as there is no study in the literature comparing acidities of all three states liquid,
frozen and lyophilized, to the best of our knowledge. Therefore, we studied the
acidities of frozen and lyophilized phosphate buffers, 50 mM Na-P and 50 mM K-P, as a
function of pHo. Indicator probe method was used to estimate the solid-state acidities. The
buffers were frozen and lyophilized in presence of sulfonephthalein indicators, and the
Hammet acidity function Hx was calculated from the measured UV-Vis spectra. The
detailed description of the spectroscopic measurement can be found in the attached
manuscript.
4.1.2 Characterization of Acidity Probes
Two indicators were used (individually) for the measurement - cresol red (CR; p/G1
= 1.1,
pKa
2
= 8.15; Figure 16), and bromocresol green (BCG; pKa = 4.7; Figure 17). The set of
these two indicators covers a pH range of approximately 0 to 10, as each indicator is able
43
Acidity of Frozen and Freeze-Dried Phosphate Buffers
to probe a pH range of approximately pKa ± 2 where two indicator forms are present in
the spectrum and Hx is calculated from their ratio (Eq. 3). CR has two pKa values far away
from each other; therefore, three distinct forms are distinguishable in the spectra diprotonated
H2(CR), monoprotonated H(CR)~, and fully deprotonated (CR)2 .
In this case,
H(CR)is
considered as deprotonated regarding first transition, but as protonated
regarding second transition. For BCG, diprotonated form is not present in our measured
spectra as pKa1
of BCG is -0.85.2 0 3
Thus, this transition will not be considered at all.
Absorption maxima of the indicator forms in a solution are listed below the corresponding
structures (Figure 16, Figure 17). Solid-state absorption maxima are red-shifted to some
extent compared to the positions in aqueous solutions.
518 nm 434 nm 573 nm
Figure 16: Structures of the deprotonated, monoprotonated, and deprotonated form ofcresol red (CR).
+ H+
PKa = 4.7
H(BCG)
443 nm
(BCG)
616 nm
Figure 17: Structures of the protonated and deprotonated form of bromocresol green (BCG).
44
Acidity of Frozen and Freeze-Dried Phosphate Buffers
4.1.3 Calculation of Hammett Acidity Functions Hx
Hx of a sample is calculated from a ratio of relative abundances of the deprotonated and
protonated forms of the indicator (Eq. 3). To calculate the relative abundances of the
indicator forms in the spectrum and the resulting Hx, following function in Matlab is used:
function [Hx] =find_Hx (ind_forml, ind_form2, sample_spectrum, pKaJnd)
c = Isqnonneg ([ind_forml, ind_form2], sample_spectrum);
Hx= pKa_ind+loglO(c(2)/c(l));
where ind_forml and ind_form2 are the vectors of absorption coefficients of the
protonated and deprotonated forms of the indicator, respectively, sample_spectrum is the
vector of absorbances of the sample Hx of which is being calculated, and pKaJnd is the
corresponding pKa value of the indicator. The result of the calculation, variable c, is a
vector with 2 components, c(l) and c(2), abundances of the protonated and deprotonated
form of the indicator in the sample spectrum. For the frozen and lyophilized samples it is
complicated to calculate the absolute abundances due to unknown optical path length;
nevertheless, the calculation of relative abundances is sufficient as Hx is calculated from
their ratio. From the absorption coefficients and calculated abundances of the indicator
forms, a theoretical spectrum of the indicator with acidity Hx is calculated and visually
compared to the experimentally measured spectrum (Figure 18).
1.2
1
0.8
0.6
0.4
0.2
0
-0.2
300 350 400 450 500 550 600 650
Figure 18: Abundances of the protonated and deprotonated form ofCR in the sample spectrum. The blue line represents a
measured absorption spectrum ofCR in frozen 50 mM Na-P (pHo = 7.0). The green lines represent calculated absorbances
belonging to the individual forms of CR in the spectrum (c(l)*ind_forml and c(2)*ind_form2], and the red line represents
their sum (c(l)*ind_forml + c(2)*ind_form2J. Resemblance of the measured [blue] and calculated [red] spectrum indicates
goodness of the fit.
45
Acidity of Frozen and Freeze-Dried Phosphate Buffers
4.1.4 Freezing-Induced Acidity Change
The UV-Vis spectra of CR in liquid, frozen, and freeze-dried 50 mM Na-P having pHo from
5.0 to 9.0 are displayed in Figure 19 and the corresponding Hx functions are presented in
Figure 20.The disappearance ofthe peak of (CR)2 and
appearance of H.2(CR) peak instead
indicates large acidification of the Na-P buffers during freezing in accordance with the
results published previously.7 5
'7 8
This large freezing-induced acidification of Na-P is
caused by the crystallization of the basic buffer salt Na2HP04 -12 H2O, which is poorly
soluble at sub-zero temperatures,2 0 4
while NaFhPCU, the indicator probe, and a fraction of
water is supposed to form FCS which eventually solidifies into an amorphous matrix. On
the contrary, Hx functions of frozen K-P buffer are more similar to pHo.
When the Na-P buffer was frozen under equilibrium conditions in the previous
study, eutectic composition was reached during freezing and pH of the maximally freezeconcentrated
solution was 3.6 independently of pHo and concentration of the buffer.7 8
Under non-equilibrium conditions the solution may solidify before the eutectic
composition is reached. In that case, pH of a maximally freeze-concentrated buffer prior
to solidification is expected to lie in between pHo and the eutectic pH. pH 4.2 was
measured with low temperature pH electrode for frozen 50 and 100 mM Na-P buffer with
pHo = 7.4 when non-equilibrium freezing was applied.7 5
However, the Hx functions of
frozen Na-P solutions presented in this study usually ranged from 1.5 to 2.5, that is lower
than the eutectic pH,7 8
and also lower than the pH values measured with a low
temperature pH electrode.7 5
We assume that this discrepancy is caused by measuring two
related, but still different, properties of the samples. The concentration of protons in the
medium surrounding ice is measured by the low temperature pH electrode,7 5
'1 9 0
while the
ability of a frozen solution to protonate the indicator probe is measured spectroscopically
as the Hx function. These properties are interconnected but do not need to be numerically
equal in environments other than dilute aqueous solutions, as the activity coefficients and
ice interface properties also play a role. We assume the ability of a frozen solution to
protonate the probe may be more important properly than actual proton concentration
when discussing the stability of proteins, for which the change of degree of protonation
often results in unfolding and denaturation;1 4 8
the degree of protonation experienced by
the indicator probe is assumed to mimic the protein's degree of protonation.1 9 2
46
Acidity of Frozen and Freeze-Dried Phosphate Buffers
50 mM Na-P
0.6 -I
PH„ = 5.00 H ( C R ) - rcR)* a
0.5 pH( = 6.00 i
pH0 = 7.00
*
0.4- P H 0 = 8.01
pH0 = 9.00
0.3-
0.2-
D.1
0.0- 1
1 1
1 1
1 1
1 1 i '
slow freezing
>
(233 K, ethanol bath)
300 350 400 450 500 550 600 650
XI nm
I fast freezing
Y (77 K, liq. N2)
lyophilization
>
300 350 400 450 500 550 600 650
XI nm
400 450 500 550 600 650
XI nm
ilization
300 350 400 450 500 550 600 650
XI nm
Figure 19: The UV-Vis spectra ofCR in the 50 mM Na-P according to pHo. a, The absorption spectra of the liquid solutions
at 295 K. h, The absorption spectra of the solutions frozen at 233 K. c, The absorption spectra of the solutions frozen at 77
K. d, The Kubelka-Munk functions of the lyophiles. The pHo values are color-coded in the legend in the panel a.
50 mM Na-P: • frozen at 77 K • frozen at 233 K • lyophile
Figure 20: The Hx values of the 50 mM Na-P buffer frozen at 77 K (black square), at 233 K (red circle), and freeze-dried
from the solution frozen at 77 K (blue diamond). CR was used as the indicator for all thefrozen samples and the lyophile
with pHo = 9. For the other lyophiles, BCG was used as the probe. The empty symbol with the arrow is used when the exact
Hx cannot be calculated, because only the H(CR)~form is present in the spectrum.
47
Acidity of Frozen and Freeze-Dried Phosphate Buffers
50 mM K-P
slow freezing
(233 K, ethanol hath)
fast freezing
(77 K, liq. N2)
PHC = 4.99 H ( C R ) . (CR)2
" C
pHB = 5.93 ,
" pH„ = 6.97 / \
1— pH„ = 8.01 / )
— pH0 = 8.99/
pH0 = 10.00
360 400 450 500 550 600 050
ÄI nm
lyophilization
1.2
-.0
I °
I °
c
1 0.4
02PH0
= « 9 (CR)2
" b
pH0=5.93 r
(CR)2
" b
" pH, = 6.97 / ^ \
— pH0 = 8.01 / \
pH0 = 8 9S# \
— PH„=10# ft
300 350 400 450 500 550 600 650
11 nm
lyophilization
•=• 0.6
P H
» = 4
" H ( C R ) ( C R ) a
dp H B = 5 . 9 3 ,
p H a = 6.97 / ^ ~ \
p H 0 = 8.01 // NXs
300 350 400 450 500 550 600 650
XI nm
Figure 21: The UV-Vis spectra ofCR in the 50 mM K-P according to pHo. a, The absorption spectra of the liquid solutions at
295 K. b, The absorption spectra of the solutions frozen at 233 K. c, The absorption spectra of the solutions frozen at 77 K.
d, The Kubelka-Munk functions of the lyophiles. Diffuse reflectance spectrum of the lyophile with pHo 9.0 could not be
measured due to its hygroscopicity.
50 mM K-P:
7 8
PH0
frozen at 77 K • frozen at 233 K • lyophile
Figure 22: The Hx values of the 50 mM K-P buffer frozen at 77 K (black square], at 233 K (red circle], and freeze-dried at
77 K (blue diamond). CR was used as the indicator. The empty symbols with the arrows are used when the exact Hx cannot
be calculated, because only the H(CR]~form is present in the spectrum.
48
Acidity of Frozen and Freeze-Dried Phosphate Buffers
4.1.5 Effect of pHo
If the buffer solutions were frozen under equilibrium conditions, their acidity in frozen
state should be independent of pHo.7 8
In this study, non-equilibrium freezing was applied
and an anomalous dependence of the Hx of the frozen buffer on pHo was observed. The
peak of H2(CR) in the spectra of the frozen samples frequently increased as the pHo of the
solution increased (Figure 19), that is, the freezing of more basic solutions often produced
more acidic frozen samples. For example, Na-P buffer of pHo 8.0 frozen at 233 K was more
acidic than the same buffer at pHo 6.0 (Figure 20). To verify this phenomenon and explore
it in more detail, a new set of Na-P buffer solutions was prepared with pHo ranging from
6.5 to 8.5 and scaled by 0.25 (Figure 23). For the samples frozen at 233 K, a close-toparabolic
dependence of Hx on pHo with a minimum near pHo = 8 was observed. Such
anomalous behaviour was indicated previously; the frozen 100 mM Na-P buffer of pHo =
5.7 was less acidic than the frozen 100 mM Na-P buffers of pHo = 6.7 and 7.4.7 5
The
explanation of this phenomenon could be as follows. According to Henderson-Hasselbach
equation (Eq. 1, p. 28), higher pHo results in higher concentration of Na2HP04. Thus,
higher supersaturation with respect to Na2HP04 is achieved during non-equilibrium
freezing and the crystallization of Na2HP04 -12 H2O is facilitated.7 5
Another explanation
of the anomalous dependence of Hx on pHo may lie in an inhibition of
Na2HP04 crystallization in presence of higher concentrations of Nal-tePCU.205
50 mM Na-P frozen at: • 77 K • 233 K A 233 K — 77 K
Figure 23: The Hx values of the 50 mM Na-P bufferfrozen at 77 K (black square], 233 K (red circle), and at 233 K and
subsequently cooled down to 77 K (blue tringel). The empty symbols with the arrows are used when the exact Hx cannot be
calculated, because only the H(CR)~ form is present in the spectrum.
49
Acidity of Frozen and Freeze-Dried Phosphate Buffers
There was no clear trend observed for the dependence of the Hx functions of the
Na-P buffer frozen at 77 K on pHo, although the Hx functions of the samples with pHo < 7.25
were higher than those of the samples with pHo > 7.50 (Figure 23). We assume the values
were more scattered due to a predominance of H(CR)~ form in the spectra, that resulted
in larger uncertainty in Hx.
In contrast with Na-P, no anomalous dependence on pHo was observed when K-P
buffer was frozen. The Hx functions increased with pHo. The Hx functions of frozen K-P
buffer are usually lower than the corresponding pHos, although one would expect them to
rise for pHo < 7 as the eutectic pH is 7.5.
4.1.6 Effect of Temperature and Freezing Rate
The extent of the indicator protonation in the frozen Na-P buffer was strongly dependent
on the temperature during freezing; the freezing of the Na-P buffer at 233 K led to greater
protonation of CR in comparison with the freezing 77 K (Figure 19). This might be due to
more extensive crystallization of Na2HP04 during slower freezing (the effect of the
freezing rate) or temperature dependent pKa of the indicator (the effect of the sample
temperature). Therefore, Na-P buffer was either fast frozen directly at 77 K or slowly
frozen at 233 K and then cooled down to 77 K. As the Hxs of the buffers fast frozen at 77 K
are markedly higher in comparison with those slowly frozen and then cooled down to 77
K (Figure 23), it implies that a lower freezing rate is the cause of the enhanced protonation
of the indicator at 233 K. A lower freezing rate leads to more thorough, closer-tothermodynamic,
crystallization of the buffer salts, while faster freezing quenches the
crystallization process.
The Hx functions of the slowly frozen samples slightly decreased after the
additional cooling from 233 K to 77 K, so there is also a weak temperature effect. Its
explanation remains unclear; three possible scenarios are proposed:
1. The pKa1
of CR may be slightly temperature dependent, although we found only the
temperature dependence of pKa
2
in the literature.2 0 6
2. At 233 K, there might still be a small amount of non-frozen FCS present, even
though the temperature is lower than the eutectic temperature.2 0 7 - 2 0 8
As the
temperature drops and more ice forms, the volume of such a solution decreases,
and the concentration of protons and other solutes rises.
Acidity of Frozen and Freeze-Dried Phosphate Buffers
3. Further crystallization of Na2HPC>4-12 H2O is very improbable, but cannot be
completely excluded.
For the frozen K-P buffer, no clear dependence of Hx on freezing rate was observed.
We suppose, as both K-P buffer salts have a similar tendency to crystallize during
freezing2 0 4
a quenching of the crystallization does not alter Hx much.
4.1.7 Acidity of Lyophiles
The acidity changes observed for the frozen Na-P buffer were significantly
mildened after the drying stage (Figure 20). To explain this behaviour, we applied X-ray
powder diffraction to identify the crystalline components in the lyophiles with pHos 5.0,
7.0, and 9.0. In the Na-P lyophile with pHo 5.0, the presence of crystalline NaH2P04
(anhydrous) was confirmed. For the lyophile with pHo 7.0, there were clear peaks in the
diffractogram indicating some crystalline phase, but we were unable to assign them to
any of the buffer salts. The diffraction patterns similar to those of Nal-hPCUand Na2HP04
were observed, but their positions were shifted. We assume a mixed co-crystal was
formed. At pHo 9.0 only the presence of Na2HP04 (anhydrous) was detected. When we
compare the results of the acidity measurements of the Na-P lyophiles with those of the
X-ray diffraction experiments, it seems that the indicator is not confined to the residues
of solidified FCS anymore in a lyophile, rather it probes the surface of the identified
crystals.
It is rather surprising there is no signal for Na2HP04 crystals observed in the
lyophile with pHo 5.0 as the freezing-induced acidity changes indicate the presence of
crystalline Na2HP04 • 12 H2O in the frozen sample. Either its signal in the resulting lyophile
is too low compared to the monosodium salt, or it dehydrated to an amorphous anhydrate
during the drying, as was published previously.2 0 9
Hx functions of the K-P lyophiles were found in a range from 6.75 to 7.75 (Figure
20Figure 22), which is close to eutectic pH (7.5). They were just about 1 unit higher
(except for the one with pHo 10.0) in comparison with those of corresponding frozen
solutions, so the sublimation-induced acidity changes were much smaller in comparison
with Na-P. We are not sure if the observed change in Hx during drying results from
crystallization of a buffer salt, or the apparent pKa of the indicator slightly changes during
drying. When the overall acidity change is considered, Hxs of the lyophiles were higher
Acidity of Frozen and Freeze-Dried Phosphate Buffers
than pHos for pHo < 7 and lower for pHo > 7. An explanation for this behaviour may lie in
excessive crystallization of the more abundant buffer salt (KH2PO4 for pHo < 7, K2HPO4 for
pHo > 7), as both salts have similar tendency to crystallize (pKa
2
of phosphoric acid is close
to 7, eutectic pH of K-P buffer is 7.5), and the indicator being bound in the amorphous
matrix with not yet crystallized salts. Indeed, only crystalline KH2PO4 was detected by Xray
diffraction in the lyophiles with pHo 5.2 and 6.8 (unfortunately, we could not analyse
the diffraction patterns of the lyophile with pHo 8.7 due to its hygroscopicity).
However, there is a discrepancy between the behaviour of the Na-P and K-P
lyophiles. The comparison of lyophiles' acidity and X-ray measurements suggested the
indicator shall be present in the vicinity of the crystallized salts in the Na-P buffer, while
in the K-P buffer it shall be present in the amorphous matrix. Further investigation is
needed to resolve this problem.
Anyway, the presented experiments demonstrate that, although the acidity of a
lyophile is somewhat similar to pHo of pre-lyophilization solution, it could be significantly
altered in the frozen state. Thus, the concept of pH memory is not valid for the whole
freeze-drying process. We found no resemblance between pHo and the acidity of frozen
buffer, nor between the acidity of frozen state and that of the corresponding lyophile.
Therefore, the acidity after the both steps, freezing and drying, should be monitored if pHinduced
denaturation is to be avoided.
4.1.8 Conclusion
Although the acidity of the Na-P buffer changed significantly towards acidic values during
freezing these acidity changes were not reflected in the acidity of corresponding
lyophiles. These acidity changes, potentially harmful to the stability of proteins and other
biomolecules, were masked after the sample has been dried. Therefore, the measurement
of the final lyophile's acidity without the knowledge of the freezing-induced acidity
changes may be misleading when stability of acid-sensitive compounds is considered.
4.1.9 Authors' Contributions
t.V. measured the UV-Vis absorbance and diffuse reflectance spectra, calculated the Hx
functions and analysed the results. V.V. provided the powder X-ray diffraction
measurements and analysed the diffractograms. E.V. and D.H. wrote the manuscript.
Suppresion of Protein Inactivation during Freezing
4.2 Suppression of Protein Inactivation during Freezing
The results of this project were published in: Krausková, L.; Procházková, J.; Klašková, M.;
Filipová, L.; Chaloupková, R.; Malý, S.; Damborský, J.; Heger, D., Suppression of protein
inactivation during freezing by minimizing pH changes using ionic cryoprotectants. Int. J.
Pharm., 2016.1 4 5
The manuscript is attached in Appendix B.+ Supplementary data are
available at http://dx.doi.Org/10.1016/j.ijpharm.2016.05.031. A Czech national patent
N°30618 (patent application PV 2015-55) was granted for the described method of
cryoprotection.
4.2.1 Introduction
Freezing either as a standalone process or as the first step of lyophilization, is commonly
used to enhance a long-term stability of biomolecules. However, the freezing-induced
stresses (§3.3.3) often result in structural changes and a loss of a biological function of
these molecules. The exact contribution of individual stresses to degradation of the
biomolecules is not fully understood.1 2 6
As I have focused on freezing-induced acidity
changes during my Ph. D. studies, the effect of the frozen-state acidity on the stability of a
model enzyme was examined in this study. Two types of phosphate buffers, Na-P and K-P,
were used in the experiments. They are similar in composition in the liquid state, but
differ substantially when frozen. Na-P acidifies extensively during freezing due to
precipitation of Na2HP04, while there is only a minor change in acidity during freezing of
K-P.2 0 4
Thus this pair of buffers makes it possible to evaluate only the effect of the acidity
change, with other factors being very similar. The frozen-state acidities were evaluated as
the Hammett acidity functions Hx described previously. The stability of the enzyme after
freezing and thawing was expressed in terms of recovery activity - the ratio of the activity
of the frozen-thawed sample to that of the non-frozen sample at otherwise identical
conditions. Lower value of the recovery activity represented greater
inactivation/denaturation of the enzyme. Subsequently, acidity changes arisen from the
crystallization of the buffer were counteracted and the effect on the stability of
frozen-thawed enzyme was monitored.
t In the manuscript, Hx is denoted as Ho, and the CR forms are labelled A, B, and C instead of H2(CR), H(CR)~, and (CR)2 -
,
respectively.
Suppresion of Protein Inactivation during Freezing
4.2.2 Model Enzyme
Haloalkanedehalogenase DbjA (EC 3.8.1.5) from Bradyrhizobium japonicum was used as
the model enzyme. It has a broad pH optimum with maximum activity at pH 9.7, and
retains more than 90% of the maximum activity in the pH range from 7.7 to 10.4. DbjA
loses its tertiary structure when pH is below 6.2 or above 10.7. When pH is lower than 5.3,
DbjA aggregates.210
DbjA's dehalogenation activity was assayed as a rate of enzymecatalysed
dehalogenation of 1,2-dibromoethane to 1-bromoethanol. The reaction
progress was monitored either by Iwasaki method2 1 1
or by GC analysis. The detailed
description of the activity assay can be found in the attached manuscript.
4.2.3 The effect of freezing parameters on DbjA activity
Samples of DbjA were exposed to freeze-thaw stress under various conditions to study
the impact of freezing-induced acidity changes on denaturation of this model enzyme.
Two freezing methods were mostly used: fast freezing in liquid nitrogen (77 K) and slow
freezing in an ethanol cooling bath (233 K). To achieve very slow cooling rates, freezing
in a cryostat with a cooling rate 0.5 K/min was used. The freeze-thaw cycles were
repeated three times to pronounce the effect of freezing on the enzymatic activity. Under
Figure 24: Effect offreezing rate and buffer type on the structural changes and recovery activity of DbjA. Recovery activities
were measured in 6, 6, 9, and 6 replicates, respectively, error bars represent the standard deviations. Preserved a-helical
content is the ratio of a-helical content in the frozen-thawed sample to that in the non-frozen sample, as obtained from CD
spectroscopy.
Suppresion of Protein Inactivation during Freezing
all conditions examined, the recovery activity of DbjA fast frozen at 77 K was significantly
lower than that from the slowly frozen samples (Figure 24). This is in agreement with
results of several previous studies,1 4 6 - 1 4 7
'1 4 9
'1 5 6
'1 7 5
'1 7 9
but in contradiction with some
others.8 5
'2 1 2
We believe this repugnance is a consequence of different sensitivity of
proteins to various freezing-induced stresses. During slow freezing, the surface-induced
denaturation is expected to be lower due to smaller ice surface area;8 4
on the other hand,
an enhanced acidity change is observed due to more extensive precipitation of buffer salts
(§4.1). The loss of DbjA activity during freezing was in good agreement with the structural
changes of the enzyme as observed by CD spectroscopy (Figure 24).
Additional cooling of the samples frozen at 233 K to 77 K and subsequent holding
in the frozen state had no effect on the recovery of DbjA activity. It indicates that the
enzyme degradation predominantly occurs during the freezing process. DbjA
denaturation was irreversible - the enzyme did not regain its activity within 24 hours after
thawing when incubated at 277 K.
4.2.4 The relationship between acidity of a frozen buffer and DbjA activity
Acidity functions Hx of frozen buffer solutions were calculated from the corresponding
UV-Vis absorption spectra as described in §4.1.3. A clear relationship between the Hx of
the frozen sample and DbjA recovery activity was observed (Figure 25). The greatest loss
80
60
O
CO 40
£>dl>oü
£ 20
H 1.4 6.1 6.6 6.8
ví*
Figure 25: Recovery activities of DbjA in various buffer solutions with pHo = 7.5 after 3 freeze-thaw cycles at 77 K (16 ± 9,
36 ± 5, 66 ± 6, and 61 ±8 per cent, respectively], and the corresponding Hx valuesfor the frozen buffer solutions at 77 K.
Error bars represent standard deviations of the recovery activities from 9,12, 6, and 6 replicates, respectively.
Suppresion of Protein Inactivation during Freezing
of DbjA activity was observed when the enzyme was frozen in 50 mM Na-P, which is the
buffer that exhibited the largest acidification during freezing. On the contrary, DbjA frozen
in 50 mM K-P buffer recovered most of its original activity as this buffer exhibited only
minor acidity changes during freezing. Lowering the Na-P concentration to 1 mM, even
using no buffer at all (a dialysis against pure water with pHo of 7.5 adjusted using NaOH)
resulted in both smaller acidification during freezing and much higher recovery activity
after thawing. Thus, a clear relationship between the freezing-induced acidity changes
and the stability of the model enzyme is demonstrated.
4.2.5 Neutralization of the buffer-induced acidification by addition of salts
To counteract the acidity changes arisen from the crystallization of the Na-P buffer, a salt
shifting the frozen-state acidity towards more basic values was sought. As was mentioned
earlier, a selective incorporation of an anion into the ice leads to excessive negative charge
in ice, which is later neutralized by the H30+
flow and the release of OH" from ice to the
solution (§3.2.3). This approach does not alter pH of non-frozen solution, only the frozenstate
acidity. The frozen state acidity was tested for a series of common salts;6 9
among
them, TMAC1 exhibited very high level of basification when frozen. The explanation of this
behaviour lies in a bulkiness of the tetramethylammonium cation; its tendency to
incorporate into the ice lattice is expected to be very low in comparison with the chloride
anion (Figure 26). The extent of this basification was dependent on the concentration of
TMAC1, with a higher concentration of TMAC1 resulting in a more basic frozen solution (
Table 1).
Concentration of TMAC1 Hx of frozen solution
0.001 M 7.5
0.01 M 7.9
0.1 M 8.1
Table 1: The effect of TMAC1 concentration on Hxofa frozen solution. pHoofall the solutions was 5.0.
Suppresion of Protein Inactivation during Freezing
f \ TMA+ C \
TMAOH
TMACI >
^N
' ) v >—' J
Figure 26: The scheme of selective incorporation of ions of TMACI into the ice and a consequent charge neutralization
leading to acidity change. The white hexagon symbolizes a growing ice, the blue colour symbolizes an unfrozen solution.
TMACI at various concentrations was added to the 50 mM Na-P solution and the
acidity of the frozen mixed solution was monitored. Addition of TMACI to the Na-P buffer
resulted in less acidic frozen solution in comparison with the frozen buffer alone. Almost
complete inhibition of the freezing-induced acidification of the buffer was observed when
TMACI concentration was 0.1 M.
To demonstrate the protective effect of TMACI, fast freezing at 77 K was used as it
resulted in the greatest loss of DbjA activity when the enzyme was frozen in buffer alone.
The effect of adding TMACI to 50 mM Na-P buffer on the recovery of DbjA activity after
freezing and thawing was positive over the whole range of tested concentrations (from
0.01 M to 1 M). The enzyme's activity was completely recovered at TMACI concentrations
> 0.1 M, so there was an agreement between the inhibition of the acidification and the
stability of the enzyme during freezing. TMACI concentration of 0.1 M had no negative
effect on the activity of DbjA enzyme. In the presence of TMACI at higher concentrations,
however, the specific activity of the non-frozen enzyme was decreased, probably due to
an inhibition of the dehalogenation reaction by chloride anions added to the solution. For
these reasons, TMACI with 0.1 M concentration was the most appropriate for suppression
of DbjA enzyme inactivation. The effect of adding 0.1 M TMACI to 50 mM phosphate
buffers on the recovery activity of DbjA after three freeze-thaw cycles is illustrated in
Figure 27. The recovery activity of DbjA in 50 mM Na-P buffer alone was (16 ± 9)%, in the
presence of 0.1 M TMACI it increased to (105 ± 24)%. Similarly, the recovery activity of
DbjA in 50 mM K-P increased from (66 ± 6)% to (106 ± 14)% when 0.1 M TMACI was
added to the solution prior to freezing.
The effect of TMACI was also validated for very slow cooling rates (0.5 K/min). The
recovery activity of DbjA slowly frozen in 50 mM Na-P alone was (43 ± 16)%; after adding
0.1 M TMACI, the enzymatic activity was recovered to (112 ± 27)%.
Suppresion of Protein Inactivation during Freezing
120 -
50 mM Na-P 50 mM K-P
Figure 27: Effect of addition of 0.1 M TMAC1 on the recovery activity ofDbjA in the phosphate buffers after 3 freeze-thaw
cycles at 77 K. Error bars represent standard deviations of the recovery activities from 9,12, 6, and 6 replicates, respectively.
4.2.6 Conclusion
In this project, we have demonstrated the important role of the frozen-state acidity for
the stability of frozen-thawed proteins. The loss of activity of the frozen-thawed enzyme
correlated with the acidity changes during freezing. When the acidity changes arisen from
the crystallization of the buffer were prevented by the addition of tetramethylammonium
chloride (TMAC1), which counteracts pH changes during freezing due to selective
incorporation of its ions into the ice, the enzymatic activity was completely recovered.
This approach was tested for very fast (immersing into liquid N2) and very slow
(0.5 K/min) cooling rates, and multiple freeze-thaw cycles were used to pronounce the
overall effect.
Our model enzyme, DbjA, experienced full recovery of its activity when the acidity
changes during freezing were prevented. The other freezing-induced stresses (low
temperature, formation of ice-water interface, phase separation, etc.) were apparently
much less significant in comparison with the acidity change for the denaturation of DbjA
enzyme. It demonstrates the strong effect of the acidity changes during freezing on the
proteins' stability. As the sensitivity of various proteins towards these stresses
considerably differs, our approach of minimizing the freezing-induced acidity changes
may not result in a full recovery of all proteins. However, it serves as a proof of principle
Suppresion of Protein Inactivation during Freezing
that minimization of the freezing-induced acidity changes may lead to more successful
protein stabilization both in research laboratories and in industry.
4.2.7 Authors' Contributions
L\K. (L\V.) measured the frozen-state acidity, calculated the Hx functions and coordinated
the experimental work. E.K. (E.V.), J.P., and M.K. performed the activity assays and
analysed the data. R.C. and M.K. measured CD spectra. S.M. provided a statistical analysis.
R.C. and J.D. provided the methodology for the activity assay. D.H. directed the research.
L\V. and D.H. wrote the manuscript.
Accelerated Redox Reactions during Freezing
4.3 Accelerated redox reaction during freezing
The results of this project were published in: Ju, J.; Kim, J.; Vetráková, Ľ.; Seo, J.; Heger, D.;
Lee, C; Yoon, H.-L; Kim, K; Kim, J., Accelerated redox reaction between chromate and
phenolic pollutants during freezing./, haz. mat, 2017.2 1 3
The manuscript is attached in
Appendix C. Supplementary data are available at:
http://dx.doi.Org/10.1016/i.ihazmat.2017.01.031.
4.3.1 Introduction
Chromium and phenolic compounds are industrial pollutants commonly found in the
aquatic environments,2 1 4 - 2 1 5
that are transported and eventually accumulated in the polar
regions,1 0 2
'2 1 6
where their reactivity in ice might be significantly different from their
reactivity in an aqueous solution (§3.2.4).1 0 1 - 1 0 2
Therefore, the rate of the reaction
between 4-chlorophenol (4-CP) and chromate (Cr(VI)) in an aqueous solution at 25 °C
and in the frozen state at -20 °C was investigated and a cause for an acceleration of the
reaction in ice was sought.
In this project, I determined the frozen state acidities using the indicator probe
method; I did not participate in the reactivity assays. For that reason, only short
description of the project is provided. More details can be found in the attached
manuscript.
4.3.2 Reaction between 4-CP and Cr(VI) in solution and in the frozen state
The redox reaction between 4-CP and Cr(VI) in ice was compared to that in an aqueous
solution under the environmentally relevant conditions - initial concentrations of both
compounds were 20 uM and pHo was 3.5 to mimic the acidic conditions in wastewaters.
While the rate of reaction between the two compounds was negligible in an aqueous
solution, the reaction proceeded rapidly in the frozen state (Figure 28). The rate of the
reaction in ice increased when the concentrations of the reactants were increased or pHo
was decreased. The reaction in ice still proceeded when the concentration of one of the
reactants was decreased to 5 uM or pHo increased to 5.
Both the freeze-concentration and the acidity change were suspected to be a cause
of the reaction acceleration in ice. To resolve the role of the acidity change, the frozen-
Accelerated Redox Reactions during Freezing
state acidity was calculated; the Hx function for the frozen solution was about 0.5. When
the pH 0.5 was applied for the aqueous solution, there was no significant change in the
reaction rate. Thus, just acidification during freezing is not enough for the redox reaction
to proceed. When the concentration of one of the reactants was increased 100-fold and
pH was decreased to 0.5, the reaction in water proceeded. It implies that the combination
of the freeze-concentration of the reactants and the more acidic environment is
responsible for acceleration of this type of reactions. However, the experiment in which
only the concentrations of the reactants would be increased at pH 3.5 was not done, so
the effect of the freeze-concentration alone cannot be evaluated.
Reaction time (h)
Figure 28: Time profiles of Cr(VI] concentrations in the solution and ice. Experimental conditions were as follows:
[4-CP] = 20 \iM, [CrfVI]]= 20 \iM, pHo = 3.5, and reaction temperature = 25 °C in water and -20 °C in ice. Adapted from
reference 213.
4.3.3 Environmental applications
The accelerated redox reaction of 4-CP and Cr(VI) in ice was observed also for real
polluted water, its rate was not reduced when other metals and organic compounds were
present. That makes this reaction important in real environments, e.g. for a
transformation of Cr(VI) to much less hazardous Cr(III) in cold environments.
4.3.4 Conclusion
Although the reaction between phenolic pollutants and Cr(VI) barely proceeded in a
solution, it was markedly accelerated when the solution was frozen. The reaction progress
61
Accelerated Redox Reactions during Freezing
was observed in a solution only if the concentration of one of the reactants was increased
100-fold and pH was decreased by 3 units. Therefore, the combination of the freezeconcentration
effect and the enhanced acidity in the frozen state was identified to play a
key role in the reaction acceleration. The environmental relevance of this reaction was
indicated.
4.3.5 Authors' Contributions
J.J., K.K., and Ju.K. designed and organized the experiments and wrote the manuscript. Ja.K.
performed the kinetic experiments. J.S. and C.L. analysed the products. E.V. and K.K. did
the spectroscopic measurements of the frozen-state acidity. E.V. calculated the H*
functions. H.-I.Y. contributed to the discussion of results. D.H. organized the collaboration
and contributed to the interpretation and discussion of results.
62
Frost Flowers
4.4 Frost Flowers
The results of this project were published in: Yang, X.; Neděla, V.; Runštuk, J.;
Ondrušková, G.; Krausko, J.; Vetráková, Ľ.; Heger, D., Evaporating brine from frost flowers
with electron microscopy and implications for atmospheric chemistry and sea-salt
aerosol formation. Atmos. Chem. Phys., 2017.2 1 7
The manuscript is attached in Appendix
D. Supplementary data are available at: http://dx.doi.org/10.5194/acp-17-6291-2017.
4.4.1 Introduction
Frost flowers, dendritic formations of ice crystals found on freshly formed ice, are
suspected to play a role in ozone-depletion events in polar regions by supplying bromine
compounds to the atmosphere,3 7
and as a possible source of sea salt aerosol in the
atmosphere.3 4
'3 8
In this study, the frost flowers were prepared in a laboratory in a way to
mimic naturally grown frost flowers. Subsequently, they were transferred to an
environmental scanning electron microscope (ESEM) and their integrity upon
sublimation was observed in order to find out if the frost flowers could be the direct
source of the sea salt aerosol.
4.4.2 Preparation of the Frost Flowers
Specific conditions must be fulfilled for the frost flowers to be grown - the surface of ice
must be warm relative to the atmosphere, the air above the ice must be locally
supersaturated while the far-field atmosphere is unsaturated.3 6
In this experiment, the
frost flowers were grown from 3.5% NaCl solution, the salinity of which is similar to that
of seawater, in a walk-in chamber cooled down to -30 °C. The samples were poured into
Styrofoam boxes and placed into the chamber; the Styrofoam served as an insulation so
that the samples were predominantly cooled from above by a cold air. The formation of
the frost flowers was sensitive to air flow in the chamber, as it decreased the local
supersaturation of the air above the ice. Therefore, only few of the frozen samples yielded
the frost flowers on the surface of ice.
63
Frost Flowers
4.4.3 Observation of the Frost Flowers with ESEM
The frost flowers were observed in ESEM at temperatures -5.2 °C and -17.0 °C and
pressures in order of hundreds of Pascals. The relative humidity was set to be slightly
below the sublimation curve, so that the water vapour did not desublimate on the frost
flowers, rather a slow sublimation of ice from the frost flowers was observed. The
conditions exactly matching the sublimation curve are difficult to be established inside
the microscope chamber.
The shape of the frost flowers was irregular; the surface was covered with
dendritic finger-like spikes. The shape of the spikes was relatively stable even when the
bulk frost flowers slowly sublimated. The spikes were presumably covered with more
concentrated brine which reduced the vapour pressure and their tendency to
evaporate/sublimate. The presence of the spikes significantly increases the available
surface area at which heterogenous reactions (e.g. bromine liberation) may occur.
The spikes were cohesive and hard to break. They were easily bended by the air
flow in the chamber (Figure 29). When two spikes were close to each other, they
combined and formed one spike. Apparently, the frost flowers do not tent to split into
multiple sub-particles upon sublimation under these conditions. The result of complete
frost flower sublimation inside the microscope chamber was a large chunk of salt.
Figure 29: The dynamical in situ images of the spikes during slow sublimation of the frost flower at -5.2 °C. Scale bar is
100 p.m. Adapted from ref. 217.
64
Frost Flowers
No NaCl crystals were observed at the temperature -5.2 °C; this is in accordance
with expectations based on NaCl/water phase diagram, as only ice and brine are supposed
to be present above eutectic temperature (-21.21 °C). If the system was in equilibrium,
the brine concentration at this temperature should be 8.3%. At the temperature -17 °C
the surface of the frost flowers was partially covered with crystalline NaCl (Figure 30).
This is interesting, as there is no region above the eutectic temperature in the phase
diagram where ice and crystalline NaCl occur at the same time. Presumably, evaporation
of water from the brine caused brine oversaturation (the equilibrium concentration of
NaCl in the brine at -17 °C is 20%, the saturation point is 24%) which resulted in the
formation of NaCl crystals. Moreover, the close-to-rectangular shape of the crystals
indicates the presence of anhydrous NaCl, even though the phase diagram predicts the
presence of NaCl • 2H2O at sub-zero temperatures and concentrations lower than 61.9%.
Apparently, the frost flowers at -17 °C were under far-from-equilibrium conditions.
Figure 30: The crystalline NaClformed on the frost at -15 "C. Scale bar is 200 p.m. Adapted from ref. 217.
4AA Conclusion
We prepared salty frost flowers in the laboratory and observed them during sublimation
with an electron microscope. The presented observations indicate that, without external
forces, the frost flowers are not a direct source of sea salt aerosol, as the spikes present in
the frost flowers are cohesive and do not tend to separate from the bulk. The result is in
accordance with a previous study, where no sea salt aerosol from the frost flowers was
detected in wind tunnel experiments.3 5
65
Frost Flowers
4.4.5 Authors' Contributions
J.K., G.O. and E.V. prepared the frost flowers. J.R. did the electron microscopy. D.H., J.K. and
E.V. analysed the microscopic observations. X.Y. analysed the environmental applications.
D.H. and X.Y. wrote the manuscript.
66
Non-Ice-Related Projects
4.5 Non-Ice-Related Projects
UV-Vis absorption and diffuse reflectance spectroscopy were the main tools for me to
study the properties of frozen aqueous solutions. For that reason, several spectroscopic
projects not related to freezing crossed my path during my Ph. D. studies. They are briefly
mentioned here.
One of the projects focused on azobenzene, a IUPAC recommended actinometer. It
was intended to be used as an actinometer for ice photochemistry in our group instead of
commonly used 2-nitrobenzaldehyde;2 1 8
however, the published molar absorption
coefficients of cis- and trans-azobenzene (ais and arans) turned out to be incorrect. As the
knowledge of the molar absorption coefficients is essential for the calculation of the
quantum yields, ais and arans needed to be re-determined. The detailed descriptions of the
experimental procedures and calculations are provided in the attached manuscript
(Appendix E). Novelized ais were substantially different from the previously published
values (Figure 31), especially in the range from 300 to 360 nm. We assume that
c/s-azobenzene contaminated with a small amount of trans-isomer was used for the
calculation previously. The accuracy of the newly obtained values was validated by
analysis of a spectral series where the concentration of c/s-azobenzene ranged from 15 to
98%. The total concentration of azobenzene calculated from the spectra with the use of
£ds and arans differed by less than 1.5% throughout the whole series; it indicates the redetermined
values of ais and arans are correct.
250 300 350 400 450 500 550 250 300 350 400 450 500 550
Figure 31: The re-determined molar absorption coefficients (black line] of trans- (left] and cis-azobenzene (right] in
methanol. Values previously reported by other authors are shown for comparison (orange,219
blue,220
and pink221
].
67
Non-Ice-Related Projects
The photoisomerization quantum yields of azobenzene were novelized
accordingly. The manuscript concerning the novelized quantum yields is under
preparation.
In another project, diffuse reflectance spectroscopy was one of the techniques used
to study photoinduced electron transfer in a mixed crystal of methyl viologen
hexafluorophosphate and dodecamethylbambus[6]uril.2 2 2
The published paper is
available at http://dx.doi.org/10.1021/jacs.6b08589.
Literature
5 Literature
1. Petrenko, V. F.; Whitworth, R. W., Physics of ice. Oxford University Press: Oxford,
1999.
2. Bartels-Rausch, T., Ten things we need to know about ice and snow. Nature 2013,
494(7435).
3. Bartels-Rausch, T.; Bergeron, V.; Cartwright, J. H. E.; Escribano, R.; Finney, J. L.;
Grothe, H.; Gutierrez, P. J.; Haapala, J.; Kuhs, W. F.; Pettersson, J. B. C; Price, S. D.;
Sainz-Diaz, C. I.; Stokes, D. J.; Strazzulla, G.; Thomson, E. S.; Trinks, H.; Uras-Aytemiz,
N., Ice structures, patterns, and processes: A view across the icefields. Rev. Mod. Phys.
2012,84(2).
4. Kasper, J. C; Friess, W., The freezing step in lyophilization: Physico-chemical
fundamentals, freezing methods and consequences on process performance and
quality attributes of biopharmaceuticals. Eur. J. Pharm. Biopharm. 2011, 78 (2).
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80
List of Abbreviations
6 List of Abbreviations
4-CP 4-chlorophenol
ASW amorphous solid water
BCG bromocresol green
CR cresolred
CrfVI) chromate
FCS freeze-concentrated solution
GC gas chromatography
HDA high-density amorphous ice
HGW hyper-quenched glassy water
K-P potassium phosphate buffer
LDA low-density amorphous ice
Na-P sodium phosphate buffer
pHo initial pH of a solution prior to freezing
POPs persistent organic polutants
TMAC1 tetramethylammonium chloride
VHDA very high-density amorphous ice
81
Curriculum Vitae
7 Curriculum Vitae
Personal Information
Name: Ľubica Vetráková
Maiden name: Ľubica Krausková
Academic title: Mgr.
Born: 24t h
February 1988 in Bojnice, Slovakia
Education
Since 2012 Faculty of Science, Masaryk University, Brno - Physical Chemistry
Ph. D. study
2010 - 2012 Faculty of Science, Masaryk University, Brno - Physical Chemistry,
Upper Secondary School Teacher Training in Chemistry
master degree, Mgr., summa cum laude
2007-2010 Faculty of Science, Masaryk University, Brno - Chemistry
bachelor degree, Be.
2003 - 2007 Grammar School of V. B. Nedožerský, Prievidza, Slovakia
specialization - Information technolology
Employment
2014-2016 research specialist, Research Centre for Toxic Compounds in the
Environment (RECETOX), Masaryk University, Brno
2017 - Institute of Scientific Instruments of the Czech Academy of Sciences,
Brno
Teaching Experience
Introductory Chemistry Laboratory, Physical Chemistry - laboratory course,
Photophysical Spectroscopic Methods, Statistical Thinking and Data Treatment
82
Curriculum Vitae
List of Publications
1. Krausková. L.: Procházková, J.; Klašková, M.; Filipová, L.; Chaloupková, R.; Malý, S.;
Damborský, J.; Heger, D., Suppression of protein inactivation during freezing by
minimizing pH changes using ionic cryoprotectants. Int. J. Pharm., 2016, 509, 41-
49, DOI: 10.1016/j.ijpharm.2016.05.031
2. Ju, J.; Kim, J.; Větráková, L.: Seo, J.; Heger, D.; Lee, C; Yoon, H.-L; Kim, K; Kim, J.,
Accelerated redox reaction between chromáte and phenolic pollutants during
freezing./, haz. mat, 2017,329, 330-338, DOI: 10.1016/j.jhazmat.2017.01.031
3. Fiala, T.; Ludvíkova, L.; Heger, D.; Švec, J.; Slanina, T.; Větráková, L. u.; Babiak, M.;
Nečas, M.; Kulhánek, P.; Klán, P.; Šindelář, V, Bambusuril as a one-electron donor
for photoinduced electron transfer to methyl viologen in mixed crystals. /. Am.
Chem. Soc. 2017,139, 2597-2603, DOI: 10.1021/jacs.6b08589
4. Yang, X.; Neděla, V.; Runštuk, J.; Ondrušková, G.; Krausko, J.; Větráková. L.: Heger, D.,
Evaporating brine from frost flowers with electron microscopy and
implications for atmospheric chemistry and sea-salt aerosol formation. Atmos.
Chem. Phys. 2017,17, 6291-6303, DOI:10.5194/acp-2017-35
5. Větráková, L.; Vykoukal, V.; Heger, D, Comparison of Acidities of Aqueous, Frozen,
and Freeze-Dried Phosphate Buffers. Is there a "pH Memory" Effect? Accepted
for publication to International Journal of Pharmaceutics
Conferences
1. Krausková L.: Heger D., Freezing Potential Measurements. At XIIt h
Workshop of
Physical Chemists and Electrochemists, Brno, Czech Republic, 2012 (oral
presentation)
2. Krausková L.: Heger D., Processes Accompanying Freezing of Aqueous Solutions. At
International Meeting on Atomic and Molecular Physics and Chemistry, Lille, France,
2013 (poster presentation)
3. Krausková L.: Procházková J.; Klašková M.; Heger D., Acidity of Frozen Solutions and
Its Connection to Degradation of Enzymes during Freezing. At XIVt h
Workshop of
Physical Chemists and Electrochemists, Brno, Czech Republic, 2014 (oral
presentation)
Curriculum Vitae
4. Krauskova E.: Heger D., Acidity of Frozen Solutions and Its Connection to Degradation
of Enzymes during Freezing. At International Meeting on Atomic and Molecular
Physics and Chemistry, Birmingham, UK, 2015 (poster presentation)
5. Krauskova E.: Heger D., Spectroscopic studies of acidity of frozen and freeze-dried
samples. At Central European Conference on Photochemistry, Bad Hofgastein,
Austria, 2016 (poster presentation)
Awards
2012 Department of Chemistry's Award, Faculty of Science, Masaryk University
2007 International Chemistry Olympiad, Moscow, Russia, bronze medal
84
List of Appendices
8 List of Appendices
Appendix A: Vetráková, Ľ.; Vykoukal, V.; Heger, D, Comparison of Acidities of Aqueous,
Frozen, and Freeze-Dried Phosphate Buffers. Is there a "pH Memory" Effect? Submitted
manuscript
Appendix B: Krausková, Ľ.; Procházková, J.; Klašková, M.; Filipová, L.; Chaloupková, R.;
Malý, S.; Damborský, J.; Heger, D., Suppression of protein inactivation during freezing by
minimizing pH changes using ionic cryoprotectants. Int. J. Pharm., 2016, 509, 41-49
Appendix C: Ju, J.; Kim, J.; Vetráková, Ľ.; Seo, J.; Heger, D.; Lee, C; Yoon, H.-L; Kim, K;
Kim, J., Accelerated redox reaction between chromáte and phenolic pollutants during
freezing./, haz. mat, 2017,329, 330-338
Appendix D: Yang X.; Neděla, V.; Runštuk, J.; Ondrušková, G.; Krausko, J.; Vetráková, Ľ.;
Heger, D., Evaporating brine from frost flowers with electron microscopy and implications
for atmospheric chemistry and sea-salt aerosol formation. Atmos. Chem. Phys. 2017, 17,
6291-6303
Appendix E: Vetráková, Ľ.; Ladányi, V ; Al Anshori, J.; Dvořák, P.; Wirz, J.; Heger, D, The
Absorption Spectrum of the Cis-form of Azobenzene. Manuscript
85