Calcium Carbonate

BIOCHEMISTRY RESEARCH TRENDS
CALCIUM CARBONATE
OCCURRENCE, CHARACTERIZATION
AND APPLICATIONS
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CALCIUM CARBONATE
OCCURRENCE, CHARACTERIZATION
AND APPLICATIONS
ALBERTA COHEN
EDITOR
New York
Copyright © 2016 by Nova Science Publishers, Inc.
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CONTENTS
Preface vii
Chapter 1 Characterization of Surface Properties
of Calcium Carbonate 1
Izabela Polowczyk, Anna Bastrzyk
and Tomasz Koźlecki
Chapter 2 Effect of Macromolecules on the Structures
of Calcium Carbonate 29
Zygmunt Sadowski, Anna Bastrzyk
and Izabela Polowczyk
Chapter 3 Structural Design of Siloxane-Containing Vaterite
for Application in Bone Reconstruction Remedies 49
Jin Nakamura, Shinya Yamada, Yoshio Ota,
Yoshio Sakka and Toshihiro Kasuga
Chapter 4 Porous Calcium Carbonate Cores As Templates
for Preparation of Peroral Proteins Delivery Systems:
The Influence of Composition of Simulated
Gastrointestinal Fluids on the Structure and
Morphology of Carbonate Cores 73
N. N. Sudareva, N. N. Saprykina, E. V. Popova
and A. D. Vilesov
Index 97
PREFACE
Calcium carbonate is one of the most abundant materials present in nature.
In this book, the characterization of surface properties of calcium carbonate
are reviewed, particularly, the Washburn method is described in detail. The
effect of natural and synthetic macromolecules on the structure of calcium
carbonate is described as well. The third chapter highlights the general criteria
for the application of vaterite (an artificially prepared compound, which has
the least thermodynamic stability among the three crystalline polymorphs of
calcium carbonate) for biomedical applications and the science of its structural
modification towards achieving tunable solubility. The final chapter examines
the porous calcium carbonate cores as templates for preparation of peroral
proteins delivery systems and the influence of ionic composition of intestinal
medium on the structure and morphology of carbonate cores and release
profiles of model and therapeutic proteins.
Chapter 1 – In this chapter the Washburn method was described in detail.
This method was compared with other techniques, and eventually used to
measure the contact angle of precipitated calcium carbonate treated with a
cationic surfactant. The values of contact angle for various wetting liquids
were calculated using a modified Washburn equation. These data were used to
estimate the surface free energy components using the van Oss equation. To
confirm the obtained data, the flotation experiments of modified and untreated
calcium carbonate particles were performed in a single bubble Hallimond tube.
The flotation recovery was calculated as a ratio of a mass of floating particles
to a mass of feed. In addition, the adsorption isotherm of dodecylammonium
hydrochloride onto precipitated calcium carbonate was determined. The zeta
potential of non-modified and surfactant-modified particles was measured to
confirm the mechanism of dodecylammonium hydrochloride adsorption on the
calcium carbonate surface. The obtained data revealed that capillary rise
viii Alberta Cohen
method (Washburn method) can be used to determine the contact angle and
surface energy of non-modified calcium carbonate as well as after its surface
modification.
Chapter 2 – Calcium carbonate is one of the most abundant materials
present in nature. In this review chapter, the effect of natural and synthetic
macromolecules on the structure of calcium carbonate will be described. Also,
an influence of concentration of additives on the morphology of precipitate
will be considered. The several mechanisms of molecule interaction with ions
and mineral surface, leading to creation of unique structure, will be also
discussed.
Chapter 3 – Vaterite, an artificially prepared compound, has the least
thermodynamic stability among the three crystalline polymorphs of calcium
carbonate (CaCO3). Its structure comprises of a hexagonal unit, consisting of
alternatively stacked Ca2+/CO32- uni-ionic planes along its c-axis. This crystal
exhibits a wide range of structural modifications by incorporating different
ionic compounds within the uni-ionic planes. Here, the authors describe the
preparation of vaterite micro-particles doped with aminopropyl-siloxane
(referred to as SiV) using a CO2 gas bubbling method, with the purpose of
using this as a biomaterial in bone reconstruction remedies. On contact with
body fluids, vaterite immediately releases Ca2+ ions, which is an essential raw
material for bone formation by osteoblast cells, while a trace amount of
soluble silicate ion enhances cellular activities to accelerate bone formation.
These effects are known to be dose-dependent. Therefore, the solubility tuning
of SiV is particularly important, achieved via design of coordination structures
between vaterite and siloxane. In the bubbling method, a precursor gel of
amorphous CaCO3 with silane monomer was produced, which spontaneously
crystallized into spherical SiV particles with average diameter of 1.5 μm. Each
SiV particle consisted of vaterite nano-lamellae enclosed within aminopropyl-
siloxane (referred to as Ap-S). Moreover, this Ap-S formed a coordination
bond with vaterite via carbamate groups. This coordination is suggested to
result in the (00l) plane-preferred crystallization of vaterite. When SiV was
placed into buffer solution at physiological pH, the particles immediately
release either calcium or soluble silicate ions. Overall dissolution rate of the
particles can be reduced by enhancing chemical stability of Ap-S. Besides, the
amount of Ca2+ ion released could be independently reduced by improving the
(00l) plane-orientation of vaterite. Magnesium is known to stimulate the
spread and mineralization of osteoblast cells. SiV particles doped with
magnesium were specifically prepared (referred to as MgSiV) by the bubbling
method, although the doping results in the formation of aragonite or calcite in
Preface ix
the conventional crystallization pathway that includes a solution mixing

process. MgSiV caused the simultaneous release of calcium, magnesium, and
soluble silicate ions within 1 d of soaking in buffer solution. A
cytocompatibility test using mouse osteoblast-like cells in a culture medium
containing ions extracted from MgSiV showed that the cells had excellent
adhesion ability at the initial stage compared with those in the conventional
culture medium, and that the differentiation of cells was also promoted.
This short review highlights the general criteria for application of vaterite
for biomedical applications and the science of its structural modification
towards achieving tunable solubility.
Chapter 4 – One of metastable polymorphic modifications of calcium
carbonate (vaterite) has been successfully used for more than ten years in the
formation of drug delivery systems (DDS). Porous calcium carbonate systems
containing biologically active compounds serve as templates for layer-by-layer
polyelectrolyte assembly and formation of a multilayer bi-polymer shell. After
dissolving carbonate cores with ethylenediamine-tetraacetic acid (EDTA), a
biologically active compound remains in the polymeric microcapsule; the
main disadvantage of this capsule is its low mechanical strength.
If DDS are used for peroral administration, it is necessary to protect
biologically active load from the action of acidic gastric juice and provide its
gradual release in weakly alkaline intestinal medium. Sodium alginate
polyanion is used in the formation of polymeric shells, among other polymers,
and this compound meets all the necessary requirements. It does not dissolve
in acidic medium and swells in weakly alkaline liquids; besides, it is
biocompatible, biodegradable and does not cause any side effects. In the
studies of DDS behavior in vitro in simulated gastrointestinal fluids, 0.05 –
0.10 M HCl solutions are used (acidic gastric medium), as well as various
weakly alkaline buffers. Phosphate buffer or Tris-HCl buffer (with pH varying
from 7.4 tо 8.2) are most frequently used as models for intestinal medium.
Carbonate cores are used as “half-finished product” for preparation of
different variants DDS. The templates without protective coating dissolve in
acidic gastric medium.The authors have studied the influence of ionic
composition of intestinal medium on the structure and morphology of
carbonate cores and release profiles of model and therapeutic proteins. When
phosphate buffer is used, ionic exchange between phosphate and calcium
carbonate takes place; this process results in considerable changes in core
structure and leads to fast protein release. Prolonged release was observed in
the experiments with other buffer systems (e.g., duodenal juice which
reproduces natural intestinal fluid as accurately as possible). Scanning electron
x Alberta Cohen
microscopy allows visualizing morpho-logical changes in carbonate cores

which correlate with release profiles of proteins. The EDS data allow
determining atomic composition of the structures formed from carbonate
vaterites during prolonged exposure to various ionic media.
The obtained results give the ability to make an expert choice of the
medium for controlling quality of DDS in vitro. Besides, the protocol of DDS
formation by polyelectrolyte assembly on carbonate cores can be simplified.
Elimination of the stage involving dissolution of carbonate cores with EDTA
will decrease losses of the encapsulated object during DDS formation and
strengthen the structure of delivery systems.
In: Calcium Carbonate ISBN: 978-1-63483-540-4
Editor: Alberta Cohen © 2016 Nova Science Publishers, Inc.
Chapter 1
CHARACTERIZATION OF SURFACE
PROPERTIES OF CALCIUM CARBONATE
Izabela Polowczyk*, Anna Bastrzyk and Tomasz Koźlecki

Division of Chemical Engineering, Faculty of Chemistry,
Wroclaw University of Technology, Wrocław, Poland
ABSTRACT
In this chapter the Washburn method was described in detail. This
method was compared with other techniques, and eventually used to
measure the contact angle of precipitated calcium carbonate treated with a
cationic surfactant. The values of contact angle for various wetting
liquids were calculated using a modified Washburn equation. These data
were used to estimate the surface free energy components using the van
Oss equation. To confirm the obtained data, the flotation experiments of
modified and untreated calcium carbonate particles were performed in a
single bubble Hallimond tube. The flotation recovery was calculated as a
ratio of a mass of floating particles to a mass of feed. In addition, the
adsorption isotherm of dodecylammonium hydrochloride onto
precipitated calcium carbonate was determined. The zeta potential of non-
modified and surfactant-modified particles was measured to confirm the
mechanism of dodecylammonium hydrochloride adsorption on the
calcium carbonate surface. The obtained data revealed that capillary rise
method (Washburn method) can be used to determine the contact angle
*
E-mail address: izabela.polowczyk@pwr.edu.pl.
2 Izabela Polowczyk, Anna Bastrzyk and Tomasz Koźlecki
and surface energy of non-modified calcium carbonate as well as after its

surface modification.
INTRODUCTION
Either natural or precipitated calcium carbonate (CaCO3) is commonly
used as a filler mineral in papermaking, coating, composites, etc. [1, 2].
However, the raw calcium carbonate particles are incompatible with polymer
and are not well dispersed in the polymer matrix [3, 4]. The improvements in
filled materials depend critically on their surface properties. Calcium
carbonate belongs to sparsely soluble salt mineral, which possesses the
hydrophilic surface [5]. So, the surface properties of mineral usually need to
be modified before it can be successfully incorporated into the hydrophobic
polymeric matrix [6, 7]. Hydrophobicity characterizes the ability of material to
be wet with a liquid in the presence of gas phase. Solids, which can be easily
wet with water, are hydrophilic, while those having limited tendency to be wet
are hydrophobic [5].
In order to improve dispersibility of calcium carbonate in polymer the
mineral surface is often treated with modifiers such as silanes, phosphates,
titanates, fatty acid, etc. [1, 3, 8-12]. Among these modifiers, either fatty acids
or their salts are the most widely used as coating agents [3, 4]. Also, in mineral
processing, to modify the surface of hydrophilic particles such as calcite,
dolomite, magnesite, barite etc., surfactants molecules are used [13-17].
One group of modifiers are either primary long-chain alkyl amines or
alkyl ammonium salts, which are commonly used as flotation collectors.
Among all long-chain alkyl amines, dodecylamine is one of the most applied
collectors used in mineral processing [18-20].
Adsorption of amines on various types of minerals, e.g., silicates, oxides,
carbonates, has been mainly described by the Gaudin-Fuerstenau-
Somasundaran hemimicelle model [21, 22]. In this model, due to electrostatic
interaction with negatively charged surface and hydrophobic interaction
(physical adsorption), amine cations are adsorbed in the outer Stern layer.
With increasing concentration of amine, both surfactant aggregates at the
surface and adsorption become considerable above the so-called critical
hemimicelle concentration (CHC). The hemimicelle are suggested to render
the hydrophobicity of mineral surface as a consequence of orientation of the
alkyl chains toward the bulk solution.
Characterization of Surface Properties of Calcium Carbonate 3
In another proposed adsorption model, a condensation theory (CT) [23],

which is the origin of admicelle model, it is assumed that sudden increase in
the adsorption isotherm at CHC stands for 2D condensation of surfactant at the
interface. The further increase in the surfactant concentration in the bulk may
lead to the bilayer formation due to tail-tail lateral interaction and surface
charge reversal [24]. Finally, the critical micelle concentration (CMC) of
surfactant is reached in the dispersion (3D condensation). The model of
successive 2D and 3D precipitation of long-chain amines adsorption by
spectroscopic investigations was proposed by another group of researchers
[25, 26]. The authors suggested the orientation and packing of
dodecylammonium acetate and chloride molecules adsorbed at the different
regions found in the adsorption isotherms. According to this model, in the
concentration range before increasing adsorption region, the ammonium head-
groups are H-bonded to the negatively charged surface groups. Next, the
neutral amine is precipitated when the concentration of surfactant increases
near a critical value and the adsorbed layer transforms into a crystalline state.
As a consequence, head-groups electrostatic repulsion is screened and the
density of monolayer increases, enhancing adsorption and rendering the
surface highly hydrophobic. 3D precipitation and second phase conversion
occur when the bulk solubility of amine is achieved at the interface [26].
In view of the above, it is obvious that as a result of surfactant adsorption,
under proper conditions the monolayer of hydrophobic alkyl chains at the
surface is formed changing the wettability and surface free energy [17, 27-29].
Thus, it is important to find the optimum concentration of molecules in the
medium because an excessive amount of surfactants leads to the processing
problem. It can be controlled by measurements of contact angle of powders
after modification [17, 29]. Therefore, an understanding of wetting
characteristics of mineral after surfactants treatment is crucial [30].
The wettability of each substances can be expressed by the so-called
contact angle. The contact angle can be determined between the solid surface
and straight line drawn from the contact point between the solid surface and
liquid drop situated on the surface, both immersed by a gas phase. The line is
tangent to the liquid drop and the contact angle is measured through the liquid
droplet. For perfectly wettable (hydrophilic) solids the contact angle measured
by the water phase is zero, and for nonwettable (hydrophobic) substances more
than 90o [31]. However, in mineral processing it is postulated that
hydrophobicity starts when the contact angle measured through the aqueous
phase, is greater than zero [32]. Therefore, the definitions of hydrophobicity
and hydrophilicity, when characterized by the contact angle, should be

distinctly indicated in scientific papers [33].
There are many equations combining the contact angle (hydrophobicity)
with physicochemical properties of multiphase systems. One of them is the
Young expression, which correlates the contact angle (θ ) with the interfacial
energies (γ) of three-phase system of solid (s), gas/vapor (g), and liquid (l)
[34]:
 sg   sl   lg cos (1)
where:  - equilibrium contact angle,

γsg, γsl, γlg – interfacial energies for solid-gas, solid-liquid and liquid-
gas phases, respectively.
There are many methods of contact angle measurements, but three

techniques are most applicable for powders, such as (a) inverse gas
chromatography, (b) sessile drop method, and (c) capillary penetration
method, called as the Washburn method [17, 29, 35-38]. Among these
methods, the capillary penetration technique is very often used for
determination of contact angle of powders. In this method, the liquid
penetration distance and liquid mass gain in bed packed in a glass tube are
measured [37]. This method is based on the Poiseuille equation with the
driving force for rises described by the Laplace equation for the pressure
difference across the invading liquid meniscus [39]. Assuming that, the
powder in a tube behaves like a bundle of n-capillaries, the fluid flow in the
capillaries can be described by equation [36]:
r   l  cos 
h2  t (2)
2 
where h is the length wetted by the liquid (wetting perimeter), r capillary

radius (which is equal to the mean or equivalent pore radius), η viscosity of
liquid, t flow time, γl surface tension of the liquid, and θ is the advancing
angle. Additionally, several restrictions are applied in the capillary rise
technique, such as steady-state laminar flow, zero velocity of liquid at the
solid/liquid interface, no external pressure and negligible gravitation
differences [37, 40]. Most studies concerned the application of this technique
to characterize non-modified powders. Only a few papers deal with
measurements of contact angle of modified particles [17, 29]. However, there

is a lack of information about possibility of characterization of precipitated
calcium carbonate treated with surfactants widely used in the industry.
Therefore, the aim of current study was to determine the value of contact
angle, calculate components of the surface free energy of precipitated calcium
carbonate treated with the cationic surfactant (dodecylammonium
hydrochloride), and to compare the obtained data with the flotation test results.
MATERIALS AND METHODS

Precipitated calcium carbonate was purchased from POCh (Poland). The
calcium carbonate size analysis was carried out by using a Mastersizer 2000
laser diffractometer (Malvern) equipped with a HydroMu dispersion unit
(Malvern). The particle size distribution as frequency and undersize curves is
shown in Figure 1. The particle size analysis indicated the volume median
diameter d50 of about 50 µm, while d10 and d90 were 29 and 83 µm,
respectively.
Figure 1. Particle size distribution of precipitated calcium carbonate.
A surface area of precipitated calcium carbonate was measured by the

Brunauer–Emmett–Teller (BET) method for the helium/nitrogen mixture by
using a FlowSorbII apparatus (Micromeritics). The surface area of CaCO3 was
found to be 0.45 m2/g.
The microstructure of calcium carbonate was observed using a JSM-
6610LVnx scanning electron microscope (JEOL). A SEM image of non-
modified CaCO3 was shown in Figure 2. It can be seen that precipitated
calcium carbonates formed aggregated rhombohedral calcite crystals with

damaged surface edges.
Figure 2. SEM image of precipitated calcium carbonate particles.
Figure 3. XRD pattern of precipitated calcium carbonate.

The crystallographic structure of calcium carbonates was determined by

using a D8 Advance (Bruker) X-ray powder diffractometer with CuKα
radiation. The quantitative analysis of the powder diffraction data was
performed by using the ReX ver. 0.7.0 software [41]. The software is free for
personal and non-commercial use and along with a short tutorial and is
available at http://www.rexpd.com. The XRD pattern of precipitated calcium
carbonate is shown in Figure 3.
Fourier transform infrared spectroscopy (FTIR) was carried out using a
VERTEX 70v spectrometer (Bruker). The investigated samples were mixed
with a KBr powder. The spectrum was recorded in a reflection mode from
4000 to 400 cm-1 at a resolution of 2 cm-1. The FTIR spectrum of calcium
carbonate is shown in Figure 4.
Figure. 4. FTIR spectrum of precipitated calcium carbonate.
The XRD pattern (Figure 3) and FTIR spectrum (Figure 4) of precipitated

calcium carbonate showed only picks and bands characteristic for calcite,
respectively.
The surface of calcium carbonate particles was modified by immersion in
surfactant solutions, using dodecylammonium hydrochloride, DDAHCl
(purchased Alfa Aesar), a cationic surfactant. The concentration of surfactant
corresponded to 0.01, 0.05, 0.1, 0.5 and 1.0 mg of surfactant per gram of solid
(mg/gsolid). After 24 hours the calcium carbonate particles were separated from
the suspension and dried.
The adsorption isotherm of DDAHCl onto the CaCO3 surface was
determined by measuring a surfactant concentration before and after 24 h of
contacting calcium carbonate particles with surfactant solution of initial
concentration in the range of 5-1000 mg/L and with the adsorbent dosage of
500 g/L. The cationic surfactant concentration was determined by using a
standard two-phase titration method with dimidium bromide and disulphine
blue indicator.
The calcium ions release of calcium carbonate particles into the aqueous
medium was estimated after one day of contacting solid particles (2 g) with
deionized water (50 mL) using a standard complexometric titration method
with an eriochrome black T indicator.
The zeta potential measurements were performed by using a Zetameter
2000 (Malvern) apparatus for non-modified and DDAHCl-treated calcium
carbonate particles at natural pH, i.e., imposed by 20 mg of calcium carbonate
in 20 mL of deionized water, pH 9.45 – at the beginning of the measurement
and pH 8.85 – within one hour.
The contact angle of calcium carbonate before and after surface
modification was determined with the capillary rise method [37] for liquids
such as water, formamide and 1-bromonaphtalene. As a reference liquid n-
heptane was used. The properties of liquids used are presented in Table 1.
The experiments were performed by using the experimental set-up
reported previously [17]. In this method the powder (2 g) was placed manually
in a small glass column (inner diameter of 8.0 mm and length 70.0 mm)
plugged at the bottom by a supporting non-woven fabric. To obtain repeatable
packing density, the powder bed was compressed by tapping several times to
the given column height. The column was hung up to a special arm of the
balance (PS 1000/C/2, Radwag) above a beaker containing a given liquid. An
increase in weight of the glass column due to the liquid penetration in the
powder bed was recorded automatically every second by the balance dedicated
software (PomiarWin ver. 5.2.0.2, Radwag).
The values of contact angle were calculated using a modified Washburn
equation [42]:
 2 l cos 
m2  C t (3)

where; γl – surface tension of liquid,

ρ – density of liquid,
η – viscosity of liquid,
m – penetrating liquid mass,
t – time,
θ – contact angle,
C – geometric parameter:
 
 r R 2 2  2 
C k  (4)
 2 
 
where; r – capillary radius,
Rk – glass tube radius,
ε – porosity.
The value of C must be determined from a slope of m2= f(t). It is possible

by using a low-energy apolar liquid (so-called reference liquid) such as n-
alkane, which spread over the particle surface without forming a finite contact
angle. For such spreading liquid, the contact angle θ remains equal to zero
(cos θ = 1). In this work, n-heptane was used as the reference liquid. The
samples of powder packed in the glass columns were kept under n-heptane
vapor for a few days to create a film on the calcium carbonate surface ahead of
liquid front. Under such condition, it can be assumed that the surface of
particle is completely wet by the reference liquid (θ = 0◦) [43, 44]. For the
know value of C value, the contact angle between the solid and test liquids can
be calculated.
The value of the contact angle for two polar and one apolar liquids made
possible to calculate the components of surface free energy using the van Oss
equation [45]:
 l 1  cos    2  sLW  lLW  2  s l  2  s l (5)
 sAB  2  s s (6)
 s   sAB   sLW (7)
In Equation 7 the surface free energy is regarded as a sum of Lifshitz-van

der Waals γsLW and Lewis acid-base γsAB components. The latter one consists of
two non-additive parameters: the electron donor (basic) (γs-) and acceptor
(acidic) (γs+).
The capillary rise tests were carried out using a group of liquids for which
properties were collected in Table 1.
Table 1. Properties of investigated liquids [46]
Liquid ρ η γl γlLW γl- γl+

[kg/m3] [mPa·s] [mJ/m2] [mJ/m2] [mJ/m2] [mJ/m2]
n-heptane 680 0.41 20.6 20.6 0 0
Water 997 1.01 72.8 21.8 25.5 25.5
Formamide 1133 3.81 58.2 39.0 39.6 2.28
1- 1483 5.11 45.9 45.9 0 0
bromonaphthalene
Figure 5. Extended Hallimond tube.

The flotation tests of modified and unmodified calcium carbonate

particles were performed in a single bubble Hallimond microflotation cell
[47]. To avoid the unwanted effect of hydrophilic particles entrainment, the
extended Hallimond tube was used (Figure 5). In each experiment, a sample of
0.5 g of calcium carbonate was dispersed in water and then the suspension was
transferred to the Hallimond tube and floated by passing air bubbles with a
constant rate through the tapering (capillary) at the bottom of the tube. The
particles which attached to the air bubbles and lifted up to water level were
collected in a special graduated reservoir. The length of the Hallimond tube
that is the distance from the capillary to the water level was about 32 cm.
The flotation kinetics was shown as a height of particles collected in the
reservoir in time and the flotation recovery was calculated as a ratio of the
mass of floating particles to mass of feed [29].
RESULTS AND DISCUSSION

Surface Modification by Surfactant Adsorption
The adsorption isotherm of dodecylammonium hydrochloride onto

precipitated calcium carbonate is shown in Figure 6. Two significant regions
can be seen. The first one within the initial concentration of surfactant 5-100
mg/L and the second one in the range 100-1000 mg/L. In the range of
surfactant used per gram of calcium carbonate, i.e., 0.01-1.0 mg/g, the
surfactant adsorbed amount increased from 0.06 to 0.79 mg/g.
The initial concentrations of surfactant, during the modification of calcium
carbonate surface for further experiments with dry modified particles, that is
contact angle, flotation, zeta potential, were 5, 25, 50, 250 and 500 mg/L.
These concentrations are within the range of the adsorption isotherm.
However, the highest bulk concentration of DDAHCl in the suspension, also
in the isotherm determination experiment, was below the value of the critical
micelle concentration CMC. The CMC of DDAHCl is 1.2·10-2 mol/L (~2660
mg/L) [48]. By adsorption of ionic surfactant on mineral surfaces,
hemimicelles and admicelles are formed [49]. In the hemimicelles, the
surfactant molecules are adsorbed with head-groups towards the solid particles
surface and chain groups towards bulk solution. The admicelles are formed by
hydrophobic interactions of surfactant layer and chain groups of molecules
from the bulk, so the outer surface of admicelle is ionic [24, 50]. This typically
occurs at a concentration of about 60% of the critical micelle concentration
(CMC) and is called the critical surface aggregation concentration (CSAC)

[49, 51]. According to this, in our system the CSAC can be reached at about
1460 mg/L for DDAHCl.
Figure 6. Adsorption isotherm of dodecylammonium hydrochloride onto precipitated

calcium carbonate. pH ~8.4.
Since the adsorption isotherm was determined for the surfactant initial
concentration between 5 and 1000 mg/L and two well-defined regions can be
seen, it could be concluded that under this conditions the distinct forms of
single molecules and hemimicelle of dodecylammonium hydrochloride exist.
For modified particles, two the highest initial concentrations of amine (250
and 500 mg/L, i.e., 1.1·10-3 and 2.2·10-3 mol/L, respectively) are within the
second region and the hemimicelles aggregates are expected to exist and
hydrophobization of the calcium carbonate surface was observed.
Dissolution of Calcium Carbonate
Dissolution of calcium carbonate sustained in deionized water was

observed as a result of exchange of protons from the aqueous medium with the
surface calcium ions. As a consequence, pH of the suspension rapidly
increased. According to Somasundaran et al. [52, 53], calcium carbonate
belongs to the so-called sparingly soluble salts. Many useful minerals belong
to this group, including calcite (CaCO3), magnesite (MgCO3), and dolomite
(CaMg(CO3)2). These salts float with many different ionic collectors, in wide
pH and collector concentration ranges [5]. Comprehension of the mechanism

of interaction between polar collector and sparingly soluble salt is quite
difficult because the solubility products are involved in the flotation system,
such as anions and cations concentrations. The solubility product (Ksp) for
[Ca2+][CO32-] at 25oC is reported to be 3.36 to 8.7·10-9, depending on the data
source [54]. It means that the product of molar concentration of calcium ions
(moles of dissolved Ca2+ per liter of solution) with the molar concentration of
dissolved CO32− cannot exceed the value of Ksp. However, in this simple
solubility equation more complicated correlation of carbon dioxide with water
should be taken into account, and therefore, pH of the solution [55]. In the
current study, the concentration of Ca2+ ions in the solution was found at 0.5
mM in the suspension at room temperature and pH about 8.4, which
corresponds to about 20 mg/L of dissolved Ca2+. These data are in a good
agreement with the literature values 3.16·10−4 and 4.70·10−4 mol/L at pH 8.62
and 8.27, respectively, for Ksp=4.47·10−9 [56]. Most reports suggested that any
additives, which consist of –NH or –NH2 groups, could trap HCO3- by forming
carbamate intermediate molecule. Furthermore, the addition of amine or
diamine buffered the decreasing pH by reacting with CO2 to keep higher pH
that should prevent the dissolving CaCO3 [57-59]. According to Gao and Hu
[60] adsorption of dodecylamine onto the calcite surface is possible by neutral
species RNH2 through Ca-N bonding and hydrogen bonding between
hydrogens of –NH2 group and surface oxygens. In addition, cationic species
RNH3+ adsorb on the surface CO32- sites through the electrostatic attraction
and hydrogen bonding. Another important mechanism involves the main
anionic species (HCO3- and CO32-) released by the mineral surface. The
cationic species RNH3+could interact with the released anionic species to form
complex precipitates, which could adsorb through physical adsorption on the
calcite surface to increase hydrophobicity and enhance flotation recovery.
Zeta Potential
The zeta potential values of non-modified calcium carbonate particles as

well as surfactant-modified ones at pH 9.45 and 8.85 were shown in Figure 7.
The isoelectric point of calcite (iep), that is the pH value at which the surface
assumes zero potential, is reported in literature to depend on the origin and
purity of calcium carbonate, measurement method, type of electrolyte used as
well as concentration of suspension, and varied from pHiep 8 to pHiep 12 [61,
62]. In this study, the pHiep for precipitated calcium carbonate was not
observed in the investigated pH range and collector concentration.
Figure 7. Zeta potential of CaCO3 modified with dodecylammonium hydrochloride in

aqueous solution at natural pH (9.45 – at the beginning of the measurement and 8.85 –
within one hour).
When the calcium carbonate particles were dispersed in deionized water,

pH of the suspension increased rapidly and reached pH value at 9.5. Due to the
complicated equilibrium between CO32− and water as well as atmospheric
CO2, pH gradually decreased in time. Within one hour, pH attained 8.85. From
the adsorption equilibrium study, it was found that continual mixing for 24 h
and proceeding dissolution resulted in the final pH about 8.4 of the suspension
of precipitated calcium carbonate. In addition, the presence of dodecyl-
ammonium hydrochloride in the solution, slightly affected pH of calcium
carbonate suspensions. The final pH (after 24h) of suspensions with two
highest initial concentrations of amine at the immersion modification of
CaCO3 stage (250 and 500 mg/L) was found to be 8.3 and 8.2, respectively.
According to the chemistry of amine solution, the formation of ammonium
ions from hydrolysis of amine in water as well as association of neutral
molecules (RNH2) and ions RNH3+ results in dimers (RNH3)22+ and iono-
molecular species (RNH2RNH3)+. The distribution of these species depends on
pH of solution and adsorption of amine [63-65].
The electrokinetic measurements [19, 62] proved that the increasing

positive value of the zeta potential at higher amine concentration is not due to
adsorption of amine cations but is a consequence of precipitation of molecular
amine. The zeta potential of amine colloidal precipitates is positive and
increases with increasing amine concentration [19, 66].
From Figure 7 it can be also seen that the zeta potential for both non-
modified and DDAHCl-treated calcium carbonate is higher for the initial pH
of suspension (9.45) For non-modified precipitated calcium carbonate the
obtained zeta potential values were -26.3 ±1. 7 and -9.8 ± 0.4 mV at pH 9.45
and 8.85, respectively. With increasing amount of dodecylammonium
hydrochloride, the zeta potential values diminished, i.e., the absolute value
decreased. For samples with two highest concentrations of amine (0.5 and 1.0
mg/gsolid), within one hour, the zeta potential was below -5 mV. It was
probably due to amine colloidal precipitation [25, 66]. Amine does not
precipitate when the concentration is below 2·10-5 mol/L. For higher
concentrations pH for amine precipitation changes with its concentration. For
example, dodecylamine precipitates at pH around 8, 9 and 10 with the
concentration above 10-2, 10-3 and 10-4 mol/L, respectively [64].
Contact Angle
The use of the Washburn equation requires the reference liquid

application. Under condition of saturation, the reference liquid should provide
the wetting contact angle equal to zero, and then the geometric parameter C
can be determined (Eq. 4). The C parameter values for n-heptane as a
reference liquid as well as the results of calculated contact angles for water,
formamide and 1-bromonaphtalene are shown in Table 2.
According to Eq. 3, the geometric parameter C is directly proportional to
the mean radius of capillaries created in a packed powder bed and proportional
to the square of its porosity. From Table 2, the values of C decreased with
increasing concentration of surfactant. This effect may be due to dissolution of
calcium carbonate surface in the presence of DDAHCl solution; particularly
the edges of calcite crystals were found to be more irregular and porous, as can
be seen in Figure 8.
Table 2. Calculated values of contact angle of precipitated calcium

carbonate particles modified with DDAHCl. Geometric parameter
C values were determined for n-heptane as the reference liquid
Amount of C [m5] Contact angle

DDAHCl
Water Formamide 1-bromonaphthalene
[mg/gsolid]
-15
0 0.9810 27 20 34
0.01 1.0310-15 28 21 23
0.05 1.0510-15 30 25 22
0.10 1.1110-15 57 31 16
0.50 0.5110-15 82 75 50
1.00 0.6410-15 85 77 84
Figure 8. SEM image of precipitated calcium carbonate particles after contacting with
DDAHCl solution.
The calculated values of contact angle increased from 27o and 20o for
unmodified CaCO3 to 85o and 77o after surfactant adsorption (1.00 mg/gsolid),
for water and formamide, respectively. Thus, modification of calcium
carbonate particles with DDAHCl resulted in rendering the surface of CaCO3.
The increase in the surfactant concentration up to 0.1 mg/g resulted in slight
decrease in the contact angle for 1-bromonaphtalene. Subsequent increase in
the contact angle is probably due to surfactant aggregates formation with the
head groups facing both the solid surface and solution.
In literature, the different values of contact angle for natural and
precipitated calcium carbonate can be found. The values of contact angle
depend on the method used. Costanzo et al. [67] showed that for ground
calcite using a TLW method the contact angle of water, formamide,
diiodomethane, and 1-bromonaphtalene was found to be 56, 54, 61, and 48o,
respectively. For the same material, the contact angle determined from the
direct contact angle measurement on smooth surface, were 6, 8, 39, and 26o,
respectively [67]. For limestone (sedimentary rock composed mainly of
calcite) the contact angle values measured by the sessile drop method was 51,
29 and 29o for water, formamide and diiodomethane, respectively [68]. The
differences may rise from the origin of material as well as the method of
sample preparation, i.e., polishing or grounding, which may affect the crystal
planes.
Surface Free Energy
For the known values of contact angles for various testing liquids, the
surface free energy components of non-modified and surfactant-modified
precipitated calcium carbonate were calculated (Eq. 5-7) and shown in
Table 3.
Table 3. Surface free energy components of non-modified and surfactant

modified particles of precipitated calcium carbonate
Amount of
γsLW γs- γs+ γsAB γs
DDAHCl
[mJ/m2] [mJ/m2] [mJ/m2] [mJ/m2] [mJ/m2]
[mg/gsolid]
0 37.2 44.1 1.8 17.7 54.9
0.01 40.9 44.8 0.9 13.1 54.0
0.05 41.1 44.1 0.7 11.5 52.7
0.10 32.4 31.7 2.7 18.4 50.9
0.50 29.8 13.1 0.2 3.4 33.3
1.00 13.5 10.4 1.5 8.0 21.5
Based on the results shown in Table 3, the decrease in the value of

electron donor (basic, γs-) component of free energy can be observed with
increasing concentration of DDAHCl. For non-modified calcium carbonate
this value was 44.1 mJ/m2 and slightly changed to 44.8, 44.1, and 31.7 mJ/m2
for 0.01, 0.05 and 0.10 mg/gsolid, respectively. According to [69] this
component reflects the hydrophobicity of surface. For materials to be neither
hydrophilic nor hydrophobic, usually γs- has to be around 28 mJ/m2, taking
slight Lifshitz-van der Waals attraction into account. Hydrophobic substances,
which tend to aggregate in water, have the electron donor component below
this critical value of γs-. On the other hand, hydrophilic materials, which
repulse each other when immersed in water, tend to have values of γs- > 28
mJ/m2 [69, 70]. In the current study, the concentrations of 0.5 and 1.0 mg/gsolid
DDAHCl provided the successful modification and the hydrophobicity of the
calcium carbonate surface was confirmed by the electron donor component
values below the critical 28 mJ/m2 value. Wu et al. [71] reported that for
natural calcite the values of γs- were 54.4 and 31.6 mJ/m2. It was determined
from the direct contact angle measurement on the smooth surface as well as
TLW technique for ground material, respectively. This surface free energy
component for limestone also differs depending on the method used. From the
direct contact angle measurements, γs- was found to be 21.8 mJ/m2, but the
TLW method yielded in 24.6 and 24.8 mJ/m2 for diiodomethane-water-
formamide and dodecane-water-formamide sets of liquids applied,
respectively [68].
Collectors, such as dodecylammonium hydrochloride, are reported to
increase the hydrophobicity of mineral surface and contact angle mainly by the
decrease in surface energy [72]. In general, high values of contact angle can be
obtained with increasing concentration of collector. In this work, the
adsorption of dodecylammonium hydrochloride changed the total surface
energy of precipitated calcite from 54.9 to 21.5 mJ/m2, for non-modified and
modified with 1.0 mg/gsolid DDAHCl, respectively.
The surface energy of solids, γs, is difficult to determine, therefore the data
found in literature may be different and have the substantial error [5]. In
literature, data of surface energy of solids, such as calcium carbonate,
estimated using different methods, can be found [2]. Generally, the surface
tension of solid particles in contact with a liquid decreases either linearly or
exponentially with increasing temperature. In addition, the value of γs depends
on the size of solid particles. The study on the size dependence of surface
energy revealed that γs first decreased with decreasing diameter, but in the
region of small sizes (during nucleation) began to increase monotonically [2].
The interfacial solid-water energy for CaCO3 at room temperature using a
method based on the measurement of conductivity changes in the
supersaturated solution was found to be 83 and 98 mJ/m2 [73, 74]. The values
of the surface free energy for precipitating calcium carbonate from the kinetics
data yielded values 58 and 68 mJ/m2, typical for sparingly soluble salts,
however, lower than estimated from solubility data [75, 76].
To compare the data of contact angle using the capillary rise method, the
sessile drop method was tried to use. However, it was not possible to produce
a compressed disk of calcium carbonate, on which the drop of investigated
liquid could not permeate, i.e., the water drop lied down on the disk surface
immediately soaked into it. For some powders, the surface of the compressed
disk is rough and porous and the sessile drop method could not be applicable
[77]. The only method which can be applied is a wicking of appropriate liquid
into the packed bad or deposited layer of powder, such as the capillary rise or
thin-layer wicking methods.
Flotation
To confirm the obtained data, the flotation experiments of modified and

unmodified calcium carbonate particles were performed in a single bubble
Hallimond tube. In this microflotation cell, flotation of a single particle can be
accomplished and the hydrophobicity of solids can be determined [78, 79].
The kinetics of flotation, represented as the height of collected floating
particles in time, is shown in Figure 9. It is worth to emphasize that pre-
modified by immersion in the DDAHCl solution and subsequently separated
and dried calcium carbonate particles were used in the zeta potential, capillary
rise method and microflotation measurements, as well. The flotation
experiments were performed by dispersing CaCO3 particles in deionized
water, without any additives.
It can be seen that the flotation performance increased with increasing
amount of dodecylammonium hydrochloride, during a particular period of
time. In addition, the flotation kinetics was the fastest for particles modified
with 0.5 and 1.0 mg/gsolid DDAHCl.
The flotation maximum recovery as a percentage of the mass of collected
floating particles to mass of feed is shown in Figure 10. It can be seen that the
recovery increased with increasing dodecylammonium hydrochloride
concentration and changed from about 32% to above 90% for untreated and
DDAHCl-modified particles (1.0 mg/gsolid), respectively.
Figure 9. The kinetics of flotation of the precipitated calcium carbonate particles.
By comparing the data in Figure 9 and 10 it can be seen that higher

recovery of particles corresponded to higher particle deposit as well as faster
flotation kinetics.
Figure 10. The flotation recovery of calcium carbonate particles.

The flotation data are in a good agreement with the data obtained from the
adsorption isotherm (Figure 6) and capillary rise experiments (Table 2 and 3)
and indicated that the precipitated calcium carbonate could be modified with
dodecylammonium hydrochloride to obtain the hydrophobic surface. In
addition, floatability of calcium carbonate particles increased with increasing
amount of cationic surfactant and the calcium carbonate surface could be
regarded as sufficiently hydrophobic for two highest concentration of
DDAHCl. According to the adsorption isotherm, these two surfactant
concentrations are within the hemimicelle formation region. However, the
flotation process often diminishes at high concentration of the collector [64].
The activity of collector due to micelles formation in the solution does not
reflect the concentration increase above the critical micelle concentration
CMC. Thus, the most interesting concentration in flotation is in the
pre-micellar region [64]. In this study, the concentration of dodecylammonium
hydrochloride reached the adsorption isotherm region.
CONCLUSION
The adsorption isotherm of dodecylammonium hydrochloride on
precipitated calcium carbonate revealed two well-defined regions in the
investigated bulk concentration range. It could be concluded that under this
condition the DDAHCl exists in the distinct forms of single molecules and
hemimicelles on the surface. Within the second region, the hemimicelles
aggregates were expected to render the surface of calcium carbonate to be
more hydrophobic.
Since CaCO3 belongs to sparingly soluble salts, slight dissolution of
calcium carbonate dispersed in water was observed. As a consequence, pH of
the suspension rapidly increased. In addition, more complicated correlation of
CO2 with water and calcium carbonate solubility products resulted in gradual
pH decrease. Amine cationic species RNH3+ could interact with surface CO32-
sites. Also, complex precipitates formed by cationic RNH3+ with anionic
species realized by the mineral surface (HCO3- and CO32-) can be formed.
These precipitates could enhance the hydrophobicity when adsorbed on the
calcite surface.
The zeta potential measurements results for both non-modified and
DDAHCl-modified particles showed that the absolute zeta potential was
higher for the initial pH of suspension and decreased in time. With increasing
amount of dodecylammonium hydrochloride, the zeta potential of calcite

decreased.
A using of cationic surfactant such as dodecylammonium hydrochloride
(DDAHCl) changes the wetting properties of calcium carbonate surface for
both polar and apolar liquids. The calculated values of contact angle for water
increased from 27o to 85o after surfactant adsorption. Thus, modification of
calcium carbonate particles with DDAHCl resulted in less hydrophilic surface
of CaCO3. In addition, the geometric parameter C decreased for two higher
DDAHCl concentrations. The SEM images confirmed more irregular and
porous edges of calcite crystals after contacting with the DDAHCl aqueous
solution.
From the components of a free energy, especially the most significant
electron donor (γs-) parameter, it can be seen that non-modified precipitated
calcium carbonate particles has the hydrophilic surface, while the surfactant-
modified ones changed to be more hydrophobic as the critical value of γs- was
suggested to be 28 mJ/m2. The surface free energy was found to be dependent
on the concentration of DDAHCl.
The flotation experiments confirmed the effect of dodecylammonium
hydrochloride on the wetting properties of precipitated calcium carbonate
particles. The flotation data are in a good agreement with the data obtained
from the adsorption isotherm and capillary rise measurements and indicated
that the precipitated calcium carbonate could be modified with
dodecylammonium hydrochloride to obtain the hydrophobic surface. In
addition, floatability of calcium carbonate particles increased with increasing
amount of cationic surfactant and the calcium carbonate surface could be
regarded as sufficiently hydrophobic for two the highest concentration of
DDAHCl. According to the adsorption isotherm, these two surfactant
concentrations are within the hemimicelle formation region.
It is worth pointing out that the microflotation tests are the fastest and the
easiest way to characterize the floatability and hence the hydrophobicity of the
mineral particle surface. However, for dodecylamine-modified calcium
carbonate we have encountered difficulties in carrying out the flotation
experiments. With increasing amount of surfactant, dispersion of calcium
carbonate particles in water has been becoming more and more complex as the
particles were prone to float on the water surface and not to sink. Using of an
ultrasound generator only partially helped to address the problem. In addition,
the particles stuck to the glass walls of the Hallimond tube and the collection
of floating particles was difficult.
On the other hand, the production of non-porous, uniform compressed

disk of calcium carbonate, for the application of the pendant drop method, was
not successful. In view of these considerations, the capillary rise method is
likely to be an efficient and simple method of characterizing the surface
properties of mineral particles, especially the hydrophobicity of modified
calcium carbonate. However, it is very important to ensure a uniform and
reproducible packing of the particles bed as well as the choice of the most
appropriate testing liquids when using the capillary rise method for
characterization of surface properties.
The obtained data revealed that the capillary rise method can be used to
determine the contact angle and surface energy of non-modified calcium
carbonate and after surface modification.
ACKNOWLEDGMENTS
This work was financially supported by the National Science Centre
Poland, grant No. 2011/01/B/ST8/02928.
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Reviewed by Przemyslaw B. Kowalczuk, Ph.D.; Mineral Processing Division,

Wroclaw University of Technology, Poland.
Chapter 2
EFFECT OF MACROMOLECULES ON
THE STRUCTURES OF CALCIUM CARBONATE
Zygmunt Sadowski, Anna Bastrzyk

and Izabela Polowczyk
Faculty of Chemistry, Department of Chemical Engineering,
Wroclaw University of Technology, Norwida, Poland
ABSTRACT
Calcium carbonate is one of the most abundant materials present in
nature. In this review chapter, the effect of natural and synthetic
macromolecules on the structure of calcium carbonate will be described.
Also, an influence of concentration of additives on the morphology of
precipitate will be considered. The several mechanisms of molecule
interaction with ions and mineral surface, leading to creation of unique
structure, will be also discussed.

Corresponding author: Zygmunt Sadowski. Faculty of Chemistry, Department of Chemical
Engineering, Wroclaw University of Technology, Norwida 4/6, 50-373, Poland. Tel.:
+48713202402, e-mail address: zygmunt.sadowski@pwr.edu.pl.
30 Zygmunt Sadowski, Anna Bastrzyk and Izabela Polowczyk
INTRODUCTION
Nature is the most creative designer and constructor of biomaterials with
extremely sophisticated shapes, size, crystallinity, isotopic and trace element
composition and highly organized microstructures. Because of the unique
properties, the complicated naturally occurring ordered structures have been a
motivation for humans to copy Nature and to adapt ideas from Nature to
achieve analogous hierarchically ordered materials. During the last decades,
many researchers tried to mimic the synthesis of inorganic materials with
unique properties and morphology present in living organism (Liu et al. 2010).
Among all of biomaterials, calcium carbonate is most common mineral
not only in living organism but also in industrial application. Pure calcium
carbonate has three crystalline forms, calcite, aragonite, and vaterite. The
conventional morphologies of them are rhombohedral, needle-like and
spheroidal, respectively. Most of the calcium carbonates formed in biological
systems have structures of calcite or aragonite, e.g., avian eggshells, seashells,
cocoliths, otoliths, etc. But also, some organisms can deposit vaterite or
amorphous phase. Vaterite occurs as elaborately shaped spicules in marine
creatures, for example otoliths in the ears of some fish (Tomás et al. 2004).
Amorphous calcium carbonate is formed in the leaves of many plants as
spindle-shaped deposits (Wu et al. 2006). Also, numerous bacteria are able to
produce calcium carbonate particle by ionic exchange through the cell
membrane via activation calcium pumps (Ferrer et al. 1988; Castanier et al.
2000). Comparing calcite with vaterite and amorphous phase, the latter forms
have higher solubility, porosity and surface area, and therefore they are more
favorable structures of calcium carbonate used in industrial application (filler
material or as templates). Ability to control the growth of ordered structures of
calcium carbonate in vitro will lead to significant advance in materials science.
It is known that the formation of biomaterials in organisms is controlled by
different processes, sometimes synergic, like the regulation of concentration of
respective ions, formation of initial amorphous, and the other metastable
precursor phases and the presence of specific biomolecular additives.
Thus, a wide range of macromolecules additives or templates has been
used in the biomimetic mineralization of calcium carbonate. It was discovered
that many proteins, polysaccharides, and other biopolymers can affect calcium
carbonate polymorph. In literature, it can be seen that not only the natural
biopolymer can be used to control the properties of CaCO3, but also the
synthetic ones (polyelectrolytes and copolymers).
Effect of Macromolecules on the Structures of Calcium Carbonate 31
THE ROLE OF ORGANIC BIOPOLYMERS IN CALCIUM

CARBONATE PRECIPITATION BY LIVING ORGANISMS
A variety of biopolymers, such as polypeptide, proteins, and
polysaccharides, has a huge impact on the crystallization of calcium carbonate.
The very important controlling biopolymers in calcium carbonate
crystallization are extracellular proteins and polysaccharides. Biomimetic
investigation indicates that biopolymers enriched in carboxylate groups can
interact with strong polar {001} faces of CaCO3 crystal. The preferential
binding of biopolymers to the prismatic faces of calcite causes lowering their
face energy (Zhou et al. 2010).
Literature data showed that collagen could be an example of biopolymer,
which has a morphological effect on the calcite growth (Shen et al. 2002).
Calcium carbonate crystallization inhibited by collagen causes a development
of new planes on the crystal. The preferential direction of the planes is a result
of collagen adsorption onto some sites of crystal. The crystals grown in the
absence of protein are always perfect rhombohedra. At collagen concentration
equaling 0.1-5g/l, the edges of {104} planes are inhibited, and new planes
{110} appeared. At the higher collagen concentrations (> 10g/l) aggregates of
calcium carbonate are formed (Shen et al. 2002). It can be seen that both the
number and size of the calcite crystals vary with the change of collagen
concentration. For the explanation of the effect of collagen on the crystal
growth the simple crystal model was used. According to the crystal model
three groups of planes can be classified. It was considered: flat (F), stepped
(S), and kinked (K) planes. In the presence of collagen, new planes started to
appear at the edges of rhombohedral crystals. New planes were developed at
the corners between (K) and (S) planes of calcite crystal ({104} planes).
The collagen also has a strong effect on the polymorph of calcium
carbonate crystals in the presence of magnesium ions during CaCO3
precipitation. Scanning electron microscopy (SEM) and X-ray diffraction
analyses showed that in the presence of both collagen and magnesium ions,
aragonite and vaterite were precipitate at low Mg/Ca ratio.
In this case, with the high Mg/Ca ions concentration ratio, only aragonite
was formed (Jiao et al. 2006). The experimental results showed that the
addition of collagen to the reaction mixture resulted in the formation of
irregular rhombohedral calcite with lamellar structure.
Whereas in the presence of biopolymer and magnesium ions, the
morphology of calcium carbonate was completely changed.
Depending on the concentration of Mg2+ irregular lumpish, discoid and

dumbbell crystals were produced. In this case, when the concentration of
magnesium was high the spherical aragonite crystals were precipitated (Jiao et
al. 2006).
The biomineralization process of calcium carbonates most often occurs in
seawater where the concentration of magnesium ions is up to 5 mM. The
influence of magnesium ions on the calcite growth crystals was investigated
by Cheng and co-workers (2007). At low concentration (< 24 mM) of Mg2+
ions the modification of calcite to the prismatic shape was observed. At high
concentration (> 24 mM) the metastable CaCO3 phase aragonite was formed.
Magnesium calcites with magnesium contents varying from 4 to 45% mol.
exist in many marine organisms (Long et al. 2014). Several biopolymers play a
crucial role in the crystallization of the thermodynamically unstable Mg-
containing calcite. Aspartic acid (Asp) was localized at the regions with very
high magnesium content. Therefore, many polymer molecules, including those
extracted from biominerals and synthetic, were used as soluble additives to the
synthesis of Mg-containing calcites. The soluble polymers are stabilized ACC
(amorphous calcium carbonate) and they induce the formation of calcite under
ambient conditions. However, the detailed role of organic polymers on the
formation of synthetic high-Mg calcite with determined crystalline
orientations, morphologies, and sizes are still extremely difficult to access.
Protein isolated from mollusc shell and sea urchin species have shown a
strong effect on the morphology and the polymorph of calcium carbonate. A
bovine serum albumin (BSA) monolayer can form a regular molecular
template in the nucleation and crystallization of calcium carbonate (Xue et al.
2009). The process of preparation of calcium carbonate was investigated when
water was substituted by the super-saturated Ca(HCO3)2 solution. It was
shown that the reaction time has a significant effect on the crystallization
behavior of the calcium carbonate polymorph. XRD patterns of the CaCO3
crystals showed that at a surface tension of 15 mNm-1 and crystallization
duration of 30 min, the polymorphic calcium carbonate (ACC) phase was
composed. The ACC phase is the most thermodynamically unstable phase, and
will be transformed into a more stable phase. The increase in the
crystallization time to 60 min and 2 h promoted the transformation.
In reaction conditions, the BSA molecules have negatively charged
headgroups, which can preferentially attract calcium ions at the film/subphase
interface as a crystallization site. Then nucleation, ACC formation and
transformation into more stable phases take place.
BSA Langmuir monolayer with good structural flexibility can provide

enough room for CaCO3 to modulate and self-aggregate resulting in
nanoparticles film creation.
It was shown that O-carboxymethylchitosan (CMCS) has an influence on
the crystallization of CaCO3 through the strong electrostatic interaction (Yang
et al. 2010). This electrostatic interaction has a place between Ca2+ ions and –
COO- groups. The precipitated CaCO3 in the presence of chitosan has a form
of aggregates consisting of nanocrystals.
The side distribution of these aggregates was from 1.5µm to 17µm. With
the increase of polymer concentration, the average size of CaCO3 nanoparticles
increases from 15nm to 25nm. The XRD analysis showed that particles
obtained in solution after the calcium carbonate synthesis with and without
chitosan are mixtures of vaterite and calcite. When, the chitosan concentration
increases the content of vaterite in the mixture decrease.
Polysaccharides of various classes such as: hydroxylated, carboxylated or
sulphated are associated with calcium carbonate biomineralization. To
understand the role of polysaccharides in CaCO3, biomineralization
carrageenans were used as additives (Fried and Mastai, 2012). Carrageenans
are linear biopolymers of galactose derivatives.
It was shown that the presence of different carrageenans in solution
strongly affected the calcium carbonate morphology and the induction time for
crystallization. For pure CaCO3 the induction time was 300s and for Kappa
carrageenan the induction time increases to 1100s.
Calcium carbonate crystals grown in the absence of the polysaccharides
were always perfect rhombohedra of 3 to 4 µm in size. The effect of the
polysaccharides on the calcite crystals’ morphology was different depending
on the type of polysaccharides used. The rhombohedral calcite structure was
formed when Kappa carrageenan was used. The rectangular shape and size of
8µm showed the calcium crystals, which were obtained in the presence of both
Iota and Lamba carrageenans (Fried and Mastai, 2012).
Carrageenan, as a polyelectrolyte, can strongly bind free Ca2+ ions,
decrease the free Ca2+ ion concentration, and thus slow down the related speed
of crystals nucleation. In such system formation polysaccharide-Ca complexes
can be observed. The initial calcium carbonate crystal nucleation takes place
on the molecules backbone.
It was also proven that these molecules can absorb onto surfaces of crystal
and therefore can effectively modify calcite crystals’ morphology.
AVIAN EGGSHELL AS A MODEL

OF CALCITE PRECIPITATION
Avian eggshell formation is interesting as it is a relatively simple process

of biomineralization (Ehrlich 2010). Around 49 proteins are associated with
the calcite biomineralization leading to the eggshell formation in birds (Rose-
Martel et al. 2015).
Eggshell formation is one of the fastest known processes; 6 g of calcium
carbonate is precipitated over a 17-hour period within the uterus. Calcite
crystal formation involves two major steps: nucleation and growth. The
nucleation of calcium carbonate begins in the uterine portion of the oviduct.
Next, mammillary cone (~ 100 µm) and palisade (~ 300 µm) layers are
deposited. Organisms can produce some functional macromolecules to
manufacture crystals with special morphologies at particular tissue sites. The
eggshell microstructure is the result of control exerted by organic
macromolecules affecting the morphology, size, and structure of individual
growing calcium carbonate crystals.
The protein composition of the palisade and cuticle layers of the chicken
eggshell was analyzed (Rose-Martel et al. 2015). In the unfertilized egg
procedure, 80 unique proteins were identified, while 317 proteins were
identified in the shell sample from fertilized eggs incubated for 15 days.
Proteins from both identification procedures were compared and 49 proteins
were common for the both procedures. 10 proteins belong to the “egg white”
proteins group. This group contains: ovalbumin, lysozyme, ovotransferrin,
ovomucoid, riboflavin-binding protein, cystatin, ovoinhibitor, ovostatin,
avidin, and TENP.
The influence of proteins on biomineralization results not only from the
interaction of proteins with calcium carbonate crystal surface, but also from
their subsequent occlusion within the mineral structure. Osteopontin (OPN)
was identified as a prominent protein constituent of the eggshell. The effect of
OPN protein on the calcite crystal growth in vitro was investigated (Chien et
al. 2008). Without protein, the calcite crystals were observed after 1 h of
crystallization time. With OPN protein added to the CaCl2 solution, crystal
nucleation and growth were delayed by approximately 30 min. The crystals of
calcite were forming at about 1.5 h of reaction time. At low concentration of
OPN (0.15µM), the morphology of calcite crystal appears like {104}
rhombohedra.
A significant morphological change of calcite crystal was observed at the

higher concentration (0.78 µm). In summary, in the eggshell, OPN protein
functioned as a guide to the calcite crystallization.
Ovalbumin and lysozyme are major egg white proteins. They represent
54% and 3.5% of all proteins at the cone layer of the eggshell (Zhao et al.
2013). These two proteins participate at the mammillary cone layer formation.
The presence of lysozyme during the CaCO3 precipitation process led to
the formation of spherical particles. The particle size depends on the lysozyme
concentration. The mean diameter of the particles for the lysozyme
concentration of 0.4 and 0.1 g/l are 230 and 123 nm, respectively (Voinescu et
al. 2007).
SHELL STRUCTURE AS A
RESULT OF BIOMINERALIZATION
The mollusc shell is one of the most fascinating biominerals. Chitin is
known to be a main organic component in mollusc shells. The crystallographic
examinations show that calcium carbonate crystals are aligned with chitin
fibers in extracellular composites. For this reason, chitin plays a major role in
calcium carbonate biomineralization.
The major inorganic component of the shell is calcium carbonate, which
ordinarily exists as a crystalline polymorph, either calcite or aragonite. The
shell consists of the nacreous layer and the prismatic layer on the outer side.
The nacreous layer consists of aragonite tablets and organic membrane
(“bricks and mortar”). In contrast, the prismatic layer consists of calcite prisms
surrounded by organic walls (Suzuki et al. 2011).
The molluscan shell proteins can be categorized into three groups
according to their theoretical isoelectric point (pI) (Cusack and Freer, 2008).
These three groups are as follows: extremely acidic (pI below 4.5), moderately
acidic (pI between 4.5 and 7.0), and basic shell proteins (pI greater than 7.0).
All molluscan proteins are characterized by aspartic acid.
Structural analysis of the shell of Tetraclita rufoticta shows three
structural components: an outer layer, a honeycombed interior mass and an
inner layer (Astachov et al. 2011). The outer layer is constructed with
elongated crystals, which are created the walls of the honey combed ulterior
layer. The inner layer is composed of sub-layered sheets of the insoluble
organic sub-layer and calcitic prismatic microcrystal.
New biomineralization mechanisms were observed in brachiopod shells

(Perez-Huerta et al. 2013). High-resolution AFM pictures showed protein
envelopes surrounding calcite fibers. These organic envelopes are enriched in
sulphated polysaccharides and proteins, which have a direct involvement in
the biomineral processes forming these fibers. An additional function of these
proteins is participation at the genesis of granules inside the sheaths. These
particles are about 100 nm in size and it is evident that they are composed of
smaller granules surrounded by organic molecules.
SYNTHETIC MACROMOLECULES AS MODIFIERS

IN CALCIUM CARBONATE SYNTHESIS
Synthetic polymers added to precipitation system can induce the

formation of various structure of calcium carbonate. Studying the literature, it
can be seen that several types of synthetic macromolecules were used to
control the synthesis of calcium carbonate, such as polyelectrolyte, double
hydrophilic block and triblock copolymers and non-ionic polymers.
Most of these molecules possess the functional group similar to that
present in natural biopolymers, hydroxyl, carboxylate, phosphate, sulphate and
amines group (Auschauer et al. 2010). The method of calcium carbonate
synthesis in the presence of synthetic macromolecules can be divided into
biomimetic synthesis and the CO2 bubbling method (Boyoo et al. 2014).
POLYELECTROLYTE-MEDIATED
SYNTHESIS OF CALCIUM CARBONATE
Most research has focused on the influence of anionic polyelectrolytes on
the precipitate properties. The polyelectrolytes have been shown to inhibit the
growth of crystal, and change the polymorphs and structure of precipitate.
It was observed that polyacrylic acid (PAA) could influence the shape,
size and polymorph type of crystals. This polymer was used as an inhibitor to
produce amorphous calcium carbonate (ACC) films deposited over a period of
4 hours on a silicon wafer from a CaCl2 solution using the diffusion method
(Xu et al. 2004; Han et al. 2007). With increasing times of precipitation, the
amorphous particle was transformed into polycrystalline spherulites, vaterite.
In the solution reaction containing dimethyl carbonate calcium chloride

and PAA (Mw = 2000) ACC was also produced in a form of nanoparticles and
some gel-like aggregates (Xu et al. 2008). Additionally, PAA inhibited the
crystallization of the ACC phase to stable form under thermal and water
treatment. The other structure of calcium carbonate was observed when PAA
(Mw = 5000) was added to the reaction mixture at high temperature. The
reaction was performed through crystallization in a single-jet system. In such
condition calcite was developed in various shapes (rod or diamond box shape)
(Watamatura et al. 2014). This morphology was controlled by the
supersaturation rate of calcium cation up to 30 mL min-1. Investigating the
effect of molecular weight of PAA on the calcium carbonate formation at high
temperature it was observed that at PAA with low molecular weight only
calcite was precipitated. With increased molecular weight to 25 000 and 250
000 the polymorph of crystals changed to aragonite and vaterite, respectively.
Not only does molecular weight have an effect on the crystal structure but also
the pH of the solution (Yu et al. 2004).
It was shown that at low pH, there was only slight influence of PAA on
the morphology of CaCO3. While at higher pH the chaotic structure of crystals
appeared. In the presence of PAA (Mw = 240 000) the unusual morphology of
the calcium carbonate was synthesized using precipitation method at high
temperature when CaCl2 and K2CO3 were used as a source of ions (Ouhenia et
al. 2008). At 50°C, a mixture of three types of particles shaped were observed;
flower-like conglomerates of vaterite, dendric particles of aragonite and
rhombic porous calcite. The volume fractions were 10.2, 79.0 and 10.8% of
calcite, vaterite and aragonite, respectively.
PAA carries carboxylic group, and the degree of deprotonation strongly
depends on the pH of solution (Yu et al. 2004). The changes in the structure of
calcium carbonate in the presence of PAA can arise from that; at proper pH the
calcium ions can form a complex with PAA by carboxyl groups via the
following reaction:
PAA(COO-)+Ca2+→PAA(COO)Ca2+ (1)
The adsorption of these complexes’ on the highly energetic sites of

crystals, and oxygen sites, may result in inhibition of rapid crystal growth and
transformation into calcite (Yu et al. 2004; Ouhenia et al. 2008; Watamatura et
al. 2014). The roughness of calcite in the presence of PAA can be explained by
the fact that formation of complexes’ PAA-Ca2+ lead to a decrease in Ca2+ ions
in the solution (Ouhemia et al. 2008).
It was also observed that a small amount of PAA present in the system
may induce the aggregation of ACC nanoparticles, because the PAA can
adsorb on the crystal surface via the carboxylic group (Han et al. 2007). For
example, the negatively charged PAA can adsorb on a vaterite surface on the
{100}, {101} and {110} planes (Matahwa et al. 2008).
The crystallization of calcium carbonate with anionic polyacrylamide
(PAM) using the carbonization method resulted in the formation of irregular
shapes of calcite and rod-like aragonite depending on the reaction condition
(Lee et al. 2015). The aragonite appeared when the reaction temperature
increased. It was also observed that the addition of PAM to the reaction
mixture resulted in aggregation of particles. The presence of aggregates of
CaCO3 in the system is due to conformational changes of anionic PAM.
In the system containing ionic species from calcium hydroxide and carbon
dioxide, the electrostatic attraction and repulsion interaction between anionic
PAM and ionic substrate take place. In the presence of high concentrations of
calcium ions, the polymer is coiled due to screening of electrostatic forces
between the segments. Calcium ions aggregated with the anionic functional
group of polymers enhancing the nucleation rate.
In literature it was shown that poly(sodium 4-styrene-sulfonate) (PSS) can
be used as an effective modifier to control the morphology of calcium
carbonate (Lei et al. 2006). Crystals were prepared by simple precipitation
reaction of calcium chloride and sodium carbonate at room temperature and
pH 10. The presence of PSS in that reaction mixture resulted in a formation of
monodispersed microsized spheres and microspheres with zigzag surface
depending on the molecular weight of polymer.
Also, hollow vaterite nanospheres were achieved by water-induced phase
transformation of poly(4-sodium styrene sulfonate)-stabilized amorphous
calcium carbonate in water-ethanol solution at room temperature (Cai et al.
2008). It was found that the size of these structures could be regulated by the
content of PSS.
PSS possesses high density of sulfonate group, which at pH 10 are
completely charged and form a polyanionic chain to which Ca2+ ions are
bound. Based on this it can be supposed that facile nucleation near the region
of chains of polymer takes place. The polymer-stabilized ACC is formed,
which then aggregates each other and transmits into nanocrystals (Colfen et al.
2001; Cai et al. 2008). During crystallization, PSS can adsorb on the surface of
crystals and block growth of specific planes resulting in spherical morphology
(Lei et al. 2006).
It was demonstrated that the positively charged additive poly(allylamine

hydrochloride) (PAH) can also cause dramatic changes in the calcite
morphology. PAH induces the formation of continuous and uniform calcite
thin film and fibers using the diffusion method (Cantaert et al. 2012; Cantaert
et al. 2013).
In the absence of PAH the rhombohedra calcite and small amounts of
vaterite were distributed over the glass substrate. It showed that the
concentration of PAH has an effect on the morphology of calcium carbonate,
which was shown in Table 1.
The creation of a thin film in the presence of PAH can be explained by the
fact that PAH undergoes a microphase separation driven by carbonate ions
(Cantaert et al. 2013). During precipitation, exposing the solution of PAH and
Ca2+ to ammonium carbonate vapor leads to the separation of droplets of the
hydrated Ca2+/PAH/CO32- phase. These droplets then merge to generate the
thin films and fibers that provide an immediate fingerprint of this
crystallization (Cantaert et al. 2012; Cantaert et al. 2013).
Polyacrylamide (PAM) plays a crucial role in the formation of calcium
carbonate. Yu and co-workers (2006) observed that polyacrylamide (PAM)-
controlled crystallization lead to synthesis of nanorods of aragonite with the
diameter ca. 50 nm and length ca. 1µm. The nanorods were obtained at 80°C,
pH 7.0 for 12 hours. It was noticed that with the longer time of reaction up to
48 hours, the obtained CaCO3 was in a form of nanorods and hollow
hexagonal disks. The XRD analysis demonstrated that the hexagonal hollow
disks were vaterite. The hexagonal vaterite discs were also obtained when pH
was increased. At 25°C the CaCO3 obtained in the presence of PAM, CaCl2
and Na2CO3 was a mixture of flat rhombohedra and single cubical crystals.
The reaction condition was as follows; pH 8.5 and 24 hours of reaction time
(Matahawa et al. 2008).
Table 1. Effect of PAH concentration on the structure of obtained calcium

carbonate
Concentration of PAH [µg mL-1] Morphology of calcium carbonate

5 Rhombohedra calcite
Calcite with rounded surfaces and fibrous
80
structures originated from the surface
500 Fibers growing from a central core
Prepared based on data published by Cantaert et al. 2013.
Here, it is interesting that relatively stable aragonite can be transformed

into unstable vaterite when the reaction time is increased. This transition of
stable phase into a metastable one is a result of the presence of
macromolecules. The PAM may stabilize the vaterite phase (Yu et al. 2006).
Flat structures of calcite can result from selective adsorption of PAM
chains on some faces forcing them to grow in two dimensions. Then these
structures form polycrystals to lower their interface energy (Matahwa et al.
2008).
Literature demonstrated that the effect of polyamines on the morphology
of CaCO3 depends on the nature of the amine group, the polymer molecular
weight and chemical structures (Schenk et al. 2014). It was shown by the
authors that with poly(vinylamine) (PVAm), a primary amine functionalized
polymer, thistle-like calcite particles with pronounced fibrous outgrowths were
formed. The other primary amine, poly(2-aminoethyl methacrylate
hydrochloride) (PAMA) lead to synthesis of spherical vaterite and elongated,
cigar-shape calcite crystals mixture. It was observed that PAMA with lower
molecular weight has little effect on the morphology of calcium carbonate,
yielding only rhombohedral calcite. Vaterite become predominant at low
concentration of a linear poly(ethyleneimine) (PEI). Addition of 1gL-1 PEI to
the reactant solution led to formation of aggregated calcite rhombohedra and
agglomerated vaterite platelets. Whereas in the presence of branched PEI only
calcite rhombohedra were formed. Polymers possessing tertiary amine groups
exert a minor effect on the fundamental calcite morphology. Schenk and co-
workers (2014) suggested that the crucial factor determining the efficacy of
the polyamines additive is the capacity of the polymer for ion complexation.
This is associated with the degree deprotonation of amine groups and the steric
accessibility of the primary amine groups.
EFFECT OF NONIONIC POLYMERS ON THE

MORPHOLOGY OF CALCIUM CARBONATE
Not only ionic polymers but also nonionic ones can influence the growth
of calcium carbonate (Xie et al. 2006; Polowczyk et al. 2013; Polowczyk et al.
2015). Poly(ethylene glycol) (PEG) is one of these polymers, which can
change the nucleation and crystal growth of CaCO3 with the use of 0.02 mole
of CaCl2 and Na2CO3 (Xie et al. 2006). The presence of 0.1% PEG in the
reaction solution resulted in the cubic particles precipitation.
At condition of 0.5% PEG (Mw = 6000) concentration the irregular-shaped

aggregates consisting of ellipsoid and cubic particles appeared. Layered
structure particles were produced with the polymer at a concentration of 0.7%
ellipsoid. XRD analysis indicated that low concentration of PEG favored
calcite precipitation, while with a higher amount of PEG the vaterite and
calcite products were formed.
Additionally, using different molecular weights of PEG, it was seen that
high molecular weight PEG in the reaction solution led to formation of crystal
structures composed of calcite particles. Similar results were also observed by
Polowczyk and co-workers (2013; 2015).
Authors also observed that adding PEG to the reaction could result in
slight changes in diameter of precipitate obtained after 5 minutes of incubation
(Polowczyk et al. 2013). The slight increase in the particle size observed at a
high polymer concentration may be caused by the flocculation effect. The
particle formed agglomerates of crystals linked by the polymer molecules.
Poly(ethylene glycol) is an innoxious, non-ionic surfactant with the
hydrophilic groups (-OH and –O–). The groups (–O–) of PEG exert the ability
to bind calcium as a nucleation site of CaCO3 and induce the formation of
CaCO3 crystals to adsorption on the special face, and inhabitation of the
crystallization on the orientation (Xie et al. 2006).
CALCIUM CARBONATE CRYSTAL

DESIGN WITH THE USE OF COPOLYMERS
Other series of crystal modifiers, the double-hydrophilic block
copolymers, were found to be effective in controlling calcium carbonate
crystal morphology (Cölfen and Qi, 2001; Lei et al. 2005; Wei et al. 2007).
One of the examples of that copolymer is poly(ethylene glycol)-block-
poly(methacrylic acid)(PEG-b-PMAA) (Cölfen and Qi, 2001).
Addition of PEG-b-PMAA to the reaction system containing Na2CO3 and
CaCl2 at pH 10 resulted in crystals that exhibited a twinned morphology with
rough surfaces. The XRD pattern indicated that the structure is composed of
calcite. By decreasing the pH in such system the rod-like structures appeared,
while increasing pH up to 11 led to the formation of a mixture of ellipsoidal
particles and irregular aggregates.
Authors observed that concentration of the copolymer also has an effect
on the morphology of calcium carbonate.
Increasing the concentration of the copolymer resulted in the formation of

spherical, rod-like and dumbbell-like particles at 0.05, 0.1 and 0.5 gL-1,
respectively.
The effect of this copolymer on the morphology of calcium carbonate can
be explained by the fact that this polymer consists of one hydrophilic block,
PMAA, designed to interact strongly with the appropriate inorganic minerals
and surfaces, and another hydrophilic block, PEG that promotes solubilization
in water (Cölfen and Qi 2001). The PMAA block possesses carboxylic groups,
which can be deprotonated depending on the pH of the solution. This group
has strong affinity to bind Ca2+ ions and specific sites on the surface of created
crystals.
Poly(acrylic acid)-block-(acrylic hydroxyl lactide) (PAAL) (Lei et al.
2005) are other types of molecules that affect crystallization of CaCO3.
Depending on the experimental condition (pH and polymer concentration),
various morphologies of calcium carbonate, such as plate-like aggregates,
poly-nucleated spheres, ellipsoids, mono-dispersed spheres and rhombohedra
can be obtained. XRD analysis showed that at higher concentration of PAAL
the vaterite phase was stabilized during precipitation. This happened because
block copolymer PAAL consists of –COOH block that interacts strongly with
inorganic minerals and a non-ionic OH block mainly promoting solubilization.
The other group of polymers used as templates for synthesis of calcium
carbonate are asymmetric triblock copolymers. An example of that copolymer
is poly(styrene-b-acrylic acid-b-ethylene glycol) (PS-b-PAA-b-PEG), which
was used to form calcite hollow nanospheres (Bastakoti et al. 2011). The
addition of Ca2+ ions to the copolymer solution induces the formation of the
Ca2+ complex with PS-b-PAA-b-PEG micelles under basic conditions. This
binding lead to conformational changes in the PAA block from an extended to
a shrunken form due to electrostatic interaction between metal cation and
anionic PAA block, resulting in colloidal droplets of Ca2+ chelated particles.
Adding carbonate ions to the suspension of Ca2+ chelated particles started
CaCO3 formation.
CONCLUSION
To sum up, it can be said that during recent decades much effort has been
made to mimic the synthesis of calcium carbonate with unique properties and
morphology present in living organisms. However, it is not yet possible to
rebuild any of these biomaterials in vitro.
Organisms can control the mineralization process, but there is still a need
to find a way to obtain ordered inorganic structures with desired properties. It
was found that the main cause of the influence of organic additives on the
nucleation and crystal growth rates of CaCO3 is the specific adsorption of
polymers on the forming faces of calcium carbonate crystals. The differences
in the strength of this adsorption interaction is because of the nature of
polymers (the number and nature of functional groups, including polar ones,
and the molecular weight), which determine the final form of the calcium
carbonate precipitate. Despite many studies, there is still a need to find new
molecules to control the synthesis of calcium carbonate with desired
properties, which can be useful in industrial applications.
ACKNOWLEDGMENTS
This work was financially supported by a statutory activity subsidy from
the Polish Ministry of Science and Higher Education for the Faculty of
Chemistry of Wroclaw University of Technology.
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Reviewed by Aleksandra Szcześ, Ph.D., Maria Curie-Sklodowska

University, Lublin, Poland.
Chapter 3
STRUCTURAL DESIGN OF SILOXANE-

CONTAINING VATERITE FOR APPLICATION
IN BONE RECONSTRUCTION REMEDIES
Jin Nakamura1, Shinya Yamada2, Yoshio Ota3,

Yoshio Sakka1 and Toshihiro Kasuga2,*
1
Advanced Key Technologies Division, Materials Processing Unit,
National Institute for Materials Science, Tsukuba, Ibaraki, Japan
2
Department of Frontier Materials, Graduate School of Engineering,
Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya, Japan
3
Yabashi Industries Co., Ltd., Ogaki, Gifu, Japan
ABSTRACT
Vaterite, an artificially prepared compound, has the least
thermodynamic stability among the three crystalline polymorphs of
calcium carbonate (CaCO3). Its structure comprises of a hexagonal unit,
consisting of alternatively stacked Ca2+/CO32- uni-ionic planes along its c-
axis. This crystal exhibits a wide range of structural modifications by
incorporating different ionic compounds within the uni-ionic planes.
Here, we describe the preparation of vaterite micro-particles doped with
aminopropyl-siloxane (referred to as SiV) using a CO2 gas bubbling
method, with the purpose of using this as a biomaterial in bone
reconstruction remedies.
On contact with body fluids, vaterite immediately releases Ca2+ ions,
which is an essential raw material for bone formation by osteoblast cells,
50 Jin Nakamura, Shinya Yamada, Yoshio Ota et al.
while a trace amount of soluble silicate ion enhances cellular activities to

accelerate bone formation. These effects are known to be dose-dependent.
Therefore, the solubility tuning of SiV is particularly important, achieved
via design of coordination structures between vaterite and siloxane. In the
bubbling method, a precursor gel of amorphous CaCO3 with silane
monomer was produced, which spontaneously crystallized into spherical
SiV particles with average diameter of 1.5 μm. Each SiV particle
consisted of vaterite nano-lamellae enclosed within aminopropyl-siloxane
(referred to as Ap-S). Moreover, this Ap-S formed a coordination bond
with vaterite via carbamate groups. This coordination is suggested to
result in the (00l) plane-preferred crystallization of vaterite. When SiV
was placed into buffer solution at physiological pH, the particles
immediately release either calcium or soluble silicate ions. Overall
dissolution rate of the particles can be reduced by enhancing chemical
stability of Ap-S. Besides, the amount of Ca2+ ion released could be
independently reduced by improving the (00l) plane-orientation of
vaterite.
Magnesium is known to stimulate the spread and mineralization of
osteoblast cells. SiV particles doped with magnesium were specifically
prepared (referred to as MgSiV) by the bubbling method, although the
doping results in the formation of aragonite or calcite in the conventional
crystallization pathway that includes a solution mixing process. MgSiV
caused the simultaneous release of calcium, magnesium, and soluble
silicate ions within 1 d of soaking in buffer solution. A cytocompatibility
test using mouse osteoblast-like cells in a culture medium containing ions
extracted from MgSiV showed that the cells had excellent adhesion
ability at the initial stage compared with those in the conventional culture
medium, and that the differentiation of cells was also promoted.
This short review highlights the general criteria for application of
vaterite for biomedical applications and the science of its structural
modification towards achieving tunable solubility.
1. INTRODUCTION
Human skeletal tissue has vital functions including structural support for
the body, protection of organs, and being a reservoir of minerals [1]. Bone is a
vascularized and dynamic tissue, which enables self-restoration when
fractured [2]. Sometimes these functions are affected by pathology.
Osteoporosis has been considered to be a serious condition, due to its strong
association with morbidity and mortality in the elderly [3]. Imbalanced bone
resorption by osteoclasts in osteoporotic patients results in a lower mineral
density of bone, with accumulated micro-fractures. This raises the risk of bone
Structural Design of Siloxane-Containing Vaterite … 51
fracture and increases the difficulty associated with daily activities [3-7].
Bone-filling materials such as hydroxyapatite (HA) (Ca10(PO4)6(OH)2) and β-
tricalcium phosphate (β-TCP) (Ca3(PO4)2) are most frequently used in
mechanical support fixation, in addition to plates and screws made of metal
and biodegradable polymers [8]. In today’s aging society, there is an ever-
increasing need for the development of novel materials that can efficiently
support bone reconstruction while reducing the need for invasive medical
treatments.
A closer look at bone metabolism reveals a vast amount of signal
molecule transactions between osteoblast cells. The cells constantly interact
with growth factors (GFs) such as insulin-like growth factor (IGF),
transforming growth factor β (TGF-β), bone morphogenic proteins (BMPs),
and vascular endothelial growth factor (VEGF). These GFs play a vital role in
the regulation of cell proliferation, differentiation, adhesion, and gene
expression at the remodeling site. Calcium is an essential inorganic element
for bone formation. Recently, calcium in extracellular fluid has been revealed
to provide a dose-dependent stimulating effect on osteoblasts. During in vitro
osteoblast cell culture in Ca2+ ion-supplemented culture medium, extracellular
Ca2+ ion concentration ([Ca2+]o) at levels as high as 40 mM was found to
enhance chemotaxis and proliferation of osteoblasts [9-11]. The enhancement
of proliferation and differentiation were observed at a concentration ranging
between 1.8 and 2.5 mM, while production of type I collagen, the main
organic component in bone, was observed at approximately 3 mM [12].
In addition to calcium, inorganic ions such as soluble silica and
magnesium have also been implicated as osteoblast stimulants. In 1971, Hench
et al. reported that Bioglass® 45S5, a glass composed of 46.1 mol% SiO2, 24.4
mol% Na2O, 26.9 mol% CaO, and 2.6 mol% P2O5, formed strong bonding
with native bone [13]. They later reported that soluble silica and Ca2+ ions
from the glass promoted bone formation by the enhancement of IGF-II gene
expression by osteoblasts [14-17]. Recently, Saboori et al. reported enhanced
expression of alkaline phosphatase (ALP), a differentiation marker expressed
during early stages within the osteoblasts when they were cultured on a
bioactive SiO2-CaO-P2O5-MgO glass surface. The origin of this stimulatory
effect is suggested as being contact of cells with Mg2+ ions in the culture
medium, derived from the bioactive glass [18]. The incorporation of these
inorganic ions is one of the promising approaches for providing stimulatory
effects to polymer-based reconstruction materials because of their potential for
long shelf lives and processing that involves organic solvents and heating
[19, 20].
Calcium carbonates (CaCO3) is a mineral found abundantly in nature,

such as in mollusk shells and corals. CaCO3 has three anhydrous
crystallographic polymorphs: thermodynamically least stable, vaterite;
intermediate, aragonite; and the most stable, calcite; with solubility product
constants (log Ksp) -7.7, -8.1, and -8.3, respectively [21]. Among them, vaterite
shows highest solubility and dissolves immediately, releasing Ca2+ ions on
contact with aqueous media. This property is suitable for its use as a reservoir
of Ca2+ ions. Simultaneously released, CO32- ions are expected to neutralize
the drop in pH at the site of reconstruction of bone, damaged due to
inflammation. However, Ca2+ ion solubility is required to be adjusted in order
to allow specific targeting of stimulatory effects.
The hexagonal crystalline structure of vaterite consists of alternatively-
stacked uni-ionic planes of Ca2+ or CO32- ions towards its [001] direction,
which is known to result in high surface energy and de-stabilize the face [22,
23]. Similar to the biomimetic synthesis route of calcium carbonate-protein
nano complexes in mollusk shells, [24] various anionic and cationic additives
have been explored to induce crystallization and enhance chemical stability of
vaterite. The additives act as chemical stabilizers by coordinating with the uni-
ionic (00l) planes. Sommerdijk et al. observed the crystallization process of
hexagonal-shaped vaterite from an amorphous precursor in the presence of
NH4+ ions by means of cryo transmission electron microscopy (TEM) in
combination with melting ethane vitrification technique [25]. The crystalline
facet of the (001) plane was formed, where NH4+ ions were suggested to serve
as a template, within the amorphous precursor. Xu et al. prepared hexagonal-
shaped vaterite mesocrystals made of (001) plane-oriented crystal units, in the
presence of N-trimethylammonium-functionalized cellulose [23]. Chujo et al.
reported the formation of vaterite microparticles by liquid mixing process
supplemented with hydrophobic sodium trisilanolate [26]. Non-bridging
oxygen molecules in the cyclopentane-functionalized silanorate strongly
bonded with vaterite. These silanorates inhibited the dissolution of vaterite in
aqueous medium for more than a week. Considering applications in the
medical field, however, these additives are required to be non-toxic and ideally
should stimulate bone formation.
In this review, we focus on the development of vaterite micro-particles
stabilized with aminopropylsilane-derived siloxane (siloxane-containing
vaterite; SiV) using a carbonation process, for applications as bioactive
functional fillers. In a single SiV particle, vaterite is included as nano-sized
primary particles and enclosed in the shell of siloxane.
On contact with aqueous media, siloxane hydrolyzes to release soluble

silica, which is followed by the dissolution of vaterite. Subsequently, strategies
for tuning the solubility of SiV is described, namely, controlling the
siloxane/vaterite composition ratio, enhancing the degree of (00l) plane-
orientation in the vaterite and introducing the calcium salt of lactate into nano-
order pores within a SiV particle. By utilizing the carbonation process, Mg2+
ion was successfully incorporated into the SiV, though the addition of
magnesium into a conventional precipitation route of calcium carbonate
preferentially induces either aragonite or calcite formation [27]. The responses
of osteoblast cells to the ions leached from Mg2+- and siloxane-containing
vaterite (MgSiV) are also described.
2. PREPARATION OF SILOXANE-CONTAINING VATERITE

BY CARBONATION PROCESS
In general, vaterite growth requires the (00l) plane to be compact, owing

to its high surface energy [28, 29]. This crystal growth leads to the formation
of particles with a spherical superstructure. In the preparation of anisotropic-
shaped vaterite particles, the addition of various additives as well as
crystallization routes plays an important role. A vaterite mesocrystal consisting
of hexagonal-shaped nano-order units is prepared by the slow decomposition
of dimethyl carbonate in calcium chloride/cetyltrimethylammonium bromide
(CTAB) aqueous solution [30]. During the initial 4 h of reaction, crystallites of
vaterite are gradually formed within amorphous precursor particles. In another
study, hexagonal micro-plates of vaterite were prepared via ammonium
carbonate vapor diffusion into calcium chloride aqueous solution [23]. Thus,
slow crystallization under static conditions is suggested to favor the formation
of anisotropic vaterite.
For the preparation of the SiV, a carbonation process has been used [31].
Figure 1 shows a schematic of the experimental setup. A small volume of
distilled water, calcium hydroxide, and aminopropyltrialkoxysilane was added
into a beaker filled with methanol. Hereafter, this silane was used as silicon
source unless otherwise specified. Carbon dioxide gas was then introduced to
the slurry for 60 min with stirring, resulting in the formation of a translucent
precursor gel consisting of amorphous calcium carbonate (ACC) and silane
monomers. Kept at room temperature for 12 h, the SiV particles precipitated
and sedimented at the bottom of the beaker.
Figure 1. Schematic of experimental setup for the carbonation process.
Figure 2. ATR-FTIR spectra (a) and LR spectra (b) of the SiV precursor gel at the
indicated intervals.
In order to monitor the reaction, the gel was periodically sampled and
analyzed using attenuated total-reflection Fourier-transform infrared (ATR-
FTIR) and laser Raman (LR) spectrometers. Figure 2(a) and (b) shows the
ATR-FTIR and LR spectra of the gel, respectively. After 1 to 2 h of aging, a
new band, originating from the carbonate ion in vaterite, was observed in the
ATR-FTIR spectra at 876 cm–1 next to the band corresponding to ACC at 859
cm–1, thus, vaterite crystallization began at this point. After 5 h of aging,
another band corresponding to vaterite was observed at approximately
740 cm–1. In the LR spectra, the onset of condensation between the silanes was
confirmed after 1 h of aging, in the form of a new peak corresponding to Si–
O–Si bonding at 515 cm–1. At this point, peaks at 654 and 610 cm–1 were also
observed. These are typical peaks attributable to the vibration of R-Si(OH)3
structure in uncondensed silane [32, 33]. These peaks completely disappeared
after 5 h, when most of the silane was assumed to have condensed. Thus, this
carbonation process is a kind of slow crystallization of vaterite from an
amorphous precursor.
Figure 3. SEM image of SiV particles (a) and TEM image of the SiV particle cross-
section. Arrows in (b) indicate typical primary particles of vaterite.
Figure 3(a) shows typical morphologies of the SiV particles. The particles
show spherical morphology with the mean diameter of about 1.4 μm, which is
relatively large in comparison with the pristine vaterite particles prepared with
an identical composition [31]. Figure 3(b) shows TEM images of SiV and the
vaterite particles, sectioned with a focus ion beam (FIB). From the high
magnification images, lamellae of vaterite, approximately 5 to 20 nm long
were observed as regions with dark contrast. An energy dispersive X-ray
(EDX) mapping image and a line scan profile showed the presence of silicon
through the entire particle [31]. These findings suggest that the lamellae are
enclosed in the siloxane. In contrast, pristine vaterite showed spherical primary
particles, with a diameter of approximately 70 nm.
X-ray diffraction pattern of the SiV particles showed the peaks of vaterite
with the P63/mmc space group (ICDD No. 33-268). The peaks at 21° and 25°
originated from diffraction of (004) and (110) planes of the hexagonal unit cell
[31]. Their peak integrated area ratio (I(004)/I(110)) was greater than that of
pristine vaterite. Therefore, the lamellae of vaterite in SiV particles were
slightly orientated to the (00l) plane. The structure of siloxane was determined
using a 29Si-cross polarization magic angle spinning nuclear magnetic
resonance (29Si-CP/MAS NMR). Based on the presence of T2
(R-Si(OH)(OSi)2) and T3 (R-Si(OSi)3) peaks with the fractions of 30 and 70
mol%, the siloxane was confirmed to mostly condensed form, including
almost no monomeric silane [34]. ATR-FTIR spectrometry was also
performed on the SiV and vaterite particles to achieve an insight into the
structure at the siloxane-vaterite interphase. Their differential spectra revealed
the presence of carbamate salt (R-NH-COO-) bands in the SiV particle, which
existed in the same range of asymmetric stretch (ν3) band from carbonate ions
in vaterite. The formation of this group has been reported as occurring during
the chemisorption of CO2 gas by aminopropyl-functionalized mesoporous
silica [35, 36].
In the case of SiV, carbamate was expected to form during carbonation of
the precursor slurry; the amino terminal of the silane formed an amide bond
with a CO2 gas molecule to form the carbamate group. Particularly, the
carbamate group was found in the salt form in the SiV. The carbamate group
in the siloxane is suggested to act as a coordination site with the Ca2+ uni-ionic
face of vaterite.
Based on monitoring results of the precursor gel and backcasted from the
structures, the formation of SiV particle is expected to proceed via the reaction
steps described below (Figure 4). At the onset of reaction (0 h), the precursor
gel contains nascent nuclei of ACC. Uncondensed silane molecules are also
present. Their amino terminals are converted into the carbamate groups by a
reaction with bubbled CO2 gas. During 1 to 7 h of reaction, Si-OH groups in
the silane molecules condense to form siloxane in the vicinity of the ACC.
Simultaneously, the ACC gradually crystallizes to vaterite lamellae. During
this crystallization, the carbamate groups template the formation of (00l)
facets. The siloxane grows to enclose the vaterite particles and aggregate with
each other. After 12 h of this process, their aggregation proceeds and they
precipitate as a 1.4 μm-sized spherical particle.
Figure 4. Prospected steps involved in the formation of an SiV particle. At the

beginning of aging, the precursor gel includes nascent nuclei of amorphous calcium
carbonate (ACC) and silane molecules. During 1 to 7 h of aging, the ACC crystallizes
to vaterite. The silanes coordinate with vaterite and induce (00l)-plane oriented
anisotropic crystal growth. Simultaneously, the silanes condense in the vicinity of the
vaterite. After 7 h of aging, the siloxane encloses vaterite and ACC as well as forms
aggregates with other primary particles to form SiV particles.
3. DISSOLUTION BEHAVIOR OF SIV PARTICLES

IN PHYSIOLOGICAL PH BUFFER MEDIUM
Within the SiV particle, the primary particle of vaterite is enclosed in

siloxane. To monitor the decomposition route of this structure in a
physiological condition, SiV and vaterite particles were soaked into
tris(hydroxymethyl)aminomethane/hydrochloric acid buffer solution (TBS), a
fundamental testing medium to evaluate the decomposition route of
biomaterials in vitro, at a pH of 7.4 at 36.5°C. Vaterite exhibits the highest
solubility among the three polymorphs of calcium carbonate. Even a small
amount of moisture in the atmosphere induces the transformation of vaterite

into the most thermodynamically stable, calcite. In fact, vaterite totally
dissolved and was converted into calcite within the initial 30 min of the test
[31]. Figure 5(a-c) shows the silicate and Ca2+ ion concentrations in the media,
X-ray diffractometry (XRD) patterns, and ATR-FTIR spectra of the samples
after the given period of soaking, respectively. In the case of SiV particles
with silicon content of 2.6 wt% (Si2.6V), the siloxane is suggested to partly
hydrolyze after 1 h of soaking based on the attenuated absorption value, at
1125 cm-1, originating from Si-O-Si bonding, in the ATR-FTIR spectra.
Figure 5. (a) Silicate and Ca2+ ion concentration in Tris buffer solutions after soaking
Si2.6V particles for the indicated period. (b) XRD patterns and (c) ATR-FTIR spectra
of Si2.6V particles after being soaked in Tris buffer solution.
During this period, rapid increase in the soluble silica concentration was
observed, where the soluble silica in the buffer solution was equivalent to
approximately 60 wt% of total siloxane content in the Si2.6V particle.
Simultaneously, vaterite also partly dissolved, to raise the Ca2+ ion
concentration within initial 1 h of soaking. At this soaking period, the
diffraction peaks of calcite were observed in Figure 5(b). The ion
concentration quickly dropped, associated with the growth of crystalline
calcite, after 3 h of soaking. Between 3 and 6 h of soaking, vaterite was totally
dissolved. It is noteworthy that 90 wt% of siloxane was also confirmed to be
released during this period. Thus, vaterite in SiV was transiently stabilized
until most of the siloxane was released.
Since the soluble silica from SiV particles was generated by hydrolysis,
their structures are likely to vary. The Si2.6V particles were soaked into de-
ionized water for 48 h and the leached products in the media were identified
by [29] Si NMR spectrometry. The spectra showed silicon species with T0, T1,
T2, and T3 species with molar fractions of 10%, 20%, 45%, and 20%,
respectively. The T0 and T1 species indicate the existence of monomeric-
silanetriols (R-Si(OH)3) and their dimers (O-[Si(OH)2-R]2), respectively. The
T2 and T3 species suggests the existence of comparably larger sized structures,
such as oligomers. The oligomeric trialkoxysilanes are known to form
polyhedral silica networks; each vertex is occupied by silicon atoms [37-39].
Detailed analysis of their size is difficult without size-exclusion
chromatography and mass spectrometry. The T2 and T3 species, however
presumably form the incomplete polyhedral structures with silanol groups.
4. TUNING IONS-RELEASING BEHAVIOR OF

SIV PARTICLES
Based on the dissolution route, the following two strategies are suggested
to extend the releasing span of Ca2+ ion. That is, the enhancement of the
siloxane/vaterite-interphase coordination and the chemical stability of
siloxane. In contrast, the loading of calcium compounds is expected to simply
increase the amount of Ca2+ ions released. These attempts are described below.
4.1. Improvement of Chemical Durability in Siloxane
Two types of SiV particles were prepared using absolute

aminopropyltriethoxysilane (APTES) or its mixture with tetraethoxysilane
(TEOS) as the source of silicon during the carbonation process. Hereafter,
these particles are referred to as SiV(100-x)ApxTe, where x represents volume%
(nearly equal to mol%) of TEOS in the silicon source (x = 0 or 30). Both
SiV100Ap and SiV70Ap30Te particles showed spherical superstructures with mean
diameters of 1.4 and 1.1 μm, respectively. These particles contained identical
amounts of siloxane, namely 2.5 wt% in a silicon mass [29]. Si MAS-NMR
spectrometry showed that the siloxane in SiV100Ap consisted of Tn species (R-
Si(OSi)n(OH)3-n, n = 1~3), while that in SiV70Ap30Te consisted of the Tn and Qn
(Si(OSi)n(OH)4-n, n = 1~4) species at a molar ratio of 76:24. This ratio nearly
follows the APTES:TEOS ratio for raw materials, where both particles showed
only vaterite patterns in the XRD pattern. Moreover, the full-width at half-
maximum values of the strongest diffraction peak, (112) were identical, that is,
the sizes of vaterite crystallites were equivalent in the SiV particle.
Figure 6. (a) Silicate and (b) Ca2+ ion concentrations in Tris buffer solution after
soaking SiV100Ap and SiV70Ap30Te particles.
Figure 6(a) and (b) shows the silicate and Ca2+ ion concentrations in the
media after soaking the SiV particles in TBS. Within the initial 2 h of soaking,
70 and 39 wt% of siloxane were released from SiV100Ap and SiV70Ap30Te,
respectively. Partial substitution of trivalent silicon atoms in the siloxane with
tetravalent silicon atoms resulted in chemical stabilization against hydrolysis.
In both particles, vaterite was observed until 6 h of soaking. Within the initial
30 min of soaking, a rapid increase of Ca2+ ion concentration, associated with
the partial dissolution of vaterite, was observed in the media supplemented
with SiV100Ap particles. This was followed by a quick drop in the
concentration, originating from the induction of crystalline calcite formation,
after 1 h of soaking. With regard to SiV70Ap30Te, the initial increase of Ca2+ ion
concentration gradually proceeded for 2 h and then decreased up to 4 h. The
formation of calcite was also observed at this point. These results illustrate that
the chemical durability of siloxane-shell strongly influences the dissolution
rate of vaterite.
4.2. Enhancement of Siloxane/Vaterite-Interphase Coordination

and Incorporation of Calcium Compounds
During the preparation of calcium carbonates, the degree of

supersaturation influences the kinetics of formation, such as the rate of crystal
growth and the dimension of the crystallites. Acetone is a conventional aprotic
solvent that has lower electrophilicity compared with methanol [40, 41]. When
these solvents are mixed in certain volume fractions, their electrophilicity
decreases in a controlled manner. This decrease subsequently raises the degree
of supersaturation for carbonate ion and accelerates the crystallization rate of
vaterite. In fact, when vaterite was prepared using the carbonation process in
absolute methanol or an equivolume mixture of methanol and acetone, its
crystallization in the latter condition completed within approximately half that
in the former [42] .
Figure 7 shows the morphologies of SiV particles, which was prepared
using absolute methanol or methanol-acetone mixture as a preparation solvent.
Hereafter, these particles are referred to as SiV-xM(100-x)A, where x is the
volume% of methanol in the solvent (x = 100, 70, 50). SiV-100M particles
show a monodispersed spherical superstructure with a mean diameter of
1.4 μm. SiV-70M30A show slightly stretched spherical morphologies with a
mean diameter of 1.7 μm and a thickness of 1.1 μm. The largest deformation
was observed with SiV-50M50A.
Figure 7. SEM image of (a) SiV-100M, (b) SiV-70M30A and (c) SiV-50M50A
particles.
Figure 8. Prospected mechanism of primary vaterite particle formation with preferred

(00l) plane orientation in SiV. During the precipitation of the primary particles,
carbamate groups of siloxane coordinate to their (00l) plane (Ca2+ ion plane), which
then regulates crystallization toward the ab-axes. Acetone in the mixed solvent
increases the supersaturation of carbonate to accelerate vaterite growth towards the
axis.
These particles showed discoid morphologies, with a mean diameter of 2.0

μm and thickness of 0.7 μm. This particle was subsequently sliced using a
focused ion beam (FIB) at a plane almost parallel to its flat face and then
characterized by electron-beam diffractometry. The diffraction projected arc-
like patterns of polycrystalline vaterite from the [301] zone axes, therefore the
vaterite primary particles in SiV-50M50A were suggested to be orientated
with their (00l) plane towards the flat face. According to the XRD of these
particles, the peak integrated area ratios of I(004)/I(110) were estimated to be
0.17, 0.20, and 0.32 for SiV-100M, SiV-70M30A, and SiV50M50A,
respectively. The degree of (00l)-plane ratio was higher in SiV with a more
flattened superstructure. The siloxane in these SiV particles contains
carbamate groups. During the carbonation process, these groups assumed to
induce the anisotropic growth of vaterite toward its ab-axes by coordination
onto their (00l) plane. The use of acetone caused acceleration of this
anisotropic growth, which induced the formation of SiV particles with
preferred crystalline orientation (Figure 8).
When the SiV particles were soaked in TBS, approximately 60 wt% of
siloxane was dissolved within the initial 2 h [Figure 9(a)], where rapid
increase in Ca2+ ion concentration was observed in all samples within the
initial 30 min. The concentrations were, however, significantly decreased with
increase in the I(004)/I(110) ratio [Figure 9(b)]. The concentration of released
Ca2+ ions at this point, were 3.1, 2.2, and 1.6 mmol･L-1 for SiV-100M, SiV-
70M30A, and SiV50M50A, respectively. All SiV particles contained siloxane
with 3.0 wt% silicon mass. Moreover, the siloxane was assumed to possess
equivalent chemical stability, since similar fractions of Tn species were
observed from [29] Si MAS-NMR spectrometry. Therefore, differences in
Ca2+ ion releasing-behavior were solely influenced by the degree of (00l)
plane-orientation. As described in the previous section, vaterite in SiV is
enclosed in siloxane and solubility requires most of the siloxane to be leached
out. These results suggest that the Ca2+ ion released from SiV particles can be
tuned by controlling the degree of coordination between siloxane and vaterite
in the SiV particle.
With regard to the SiV-50M50A, nitrogen adsorption-desorption analysis
provided type IV isotherms, indicating the presence of mesoporous structures.
Moreover, the adsorption-desorption hysteresis showed an intermediate shape
of types H2 and H3 as per IUPAC classification [43]. In particular, type H3
hysteresis suggests the existence of slit- or wedge-shaped pores, which are
formed in the aggregated planar particles. In fact, TEM observation of a SiV-
50M50A particle, which was FIB-sliced perpendicular to its flat face, revealed
the stacking of planer vaterite unit structures, each 10 to 30 nm thick at the
rim. The hysteresis may correspond to the vacancy between the unit structures.
These pores were successfully loaded with the calcium salt of lactic acid
oligomer by simply including the salt into the preparation solvent during the
carbonation process [44]. When the SiV particles were soaked in TBS,
calcium salts were released at the onset of dissolution, which contributed to
the increase in amount of Ca2+ ions.
5. PREPARATION OF NOVEL MAGNESIUM-INCORPORATED

VATERITE AND IN VITRO CELLULAR TEST
The influence of magnesium in the crystallization kinetics of calcium
carbonate has been explored widely in a geochemistry area with the aim of
accounting for the mineralization of carbonate salts in supersaturated natural
waters, such as surface seawater [45]. The Mg2+ ion strongly induces the
crystallization of aragonite from supersaturated calcium bicarbonate solution
by inhibiting the formation of vaterite and calcite [27]. Interestingly, the co-
addition of Mg2+ with Ba2+ ion, a strong inducer of calcite formation, into the
solution leads to the formation of MgCO3-CaCO3 solid solution with a calcite-
type crystalline configuration [46]. Physiologically, Mg2+ ion stimulates
osteoblast cells and enhances their differentiation, calcification, and adhesion
onto scaffolds [18, 47]. Magnesium- and siloxane-containing vaterite (MgSiV)
might be beneficial in the design of novel biomaterials to be used in bone
reconstruction remedies.
MgSiV particle has been successfully prepared with the simultaneous
addition of magnesium hydroxide and aminopropylalkoxysilane during the
carbonation process, despite the induction effect by Mg2+ ions [48]. This
particle showed a flat-spherical morphology as depicted in Figure 10. The
magnesium and silicon content in the MgSiV particle was estimated to be 2.0
and 2.8 wt% by inductively-coupled plasma atomic emission spectrometry.
XRD of this particle revealed the predominant crystalline phase to be vaterite.
Of note, the diffraction peaks that correspond to the c-axis-dependent plane
such as the (112) and (114) planes, shifted to a higher 2θ degree in comparison
with the SiV particles. On the other hand, no shift was observed at peaks
originating from the (110) plane. The lattice spacing for (004) plane was
calculated to be 4.21 Å for vaterite in the MgSiV particles and 4.26 Å in the
SiV particles. It is possible that the Mg2+ ions substituted certain sites for Ca2+
ions in vaterite. FTIR spectra of the MgSiV particles showed a slight increase
in absorption at 460 cm-1, associated with Si-O-Mg bonding [49]. A part of the
Mg2+ ions, therefore, was suggested to react with the non-bridging oxygen of
siloxane.
When MgSiV particles were soaked in TBS, about 60 wt% of the Mg2+
ions were released within the initial 12 h of soaking (Figure 11). XRD of the
particle during this period also showed the presence of vaterite, however the
shifted diffraction peaks reverted to the positions in SiV. The Mg2+ ion
concentration in the soaking media gently gradually increased in a controlled
manner for 7 d. Drastic increases in both, silicate and Ca2+ ion concentration
were observed in the soaking media within the initial 12 h, while the Ca2+ ion
concentration decreased during the next 12 h. The ion-releasing behaviors
were similar to that of SiV particles.
Figure 9. (a) Silicate and (b) Ca2+ ion concentrations in Tris buffer solution after
soaking SiV-100M, SiV-70M30A and SiV-50M50A particles for the indicated periods.
Figure 10. SEM image of MgSiV particles.

Figure 11. Silicate, Mg2+, and Ca2+ ion concentrations in Tris buffer solution after
soaking MgSiV particles for the indicated periods.
The cytocompatibility of these ions was evaluated in vitro by culturing

murine osteoblast-like MC3T3-E1 cells in a culture medium preconditioned
with MgSiV particles. In this test, a pristine culturing medium and a
preconditioned medium with SiV were also used as references. Cells were
cultured by seeding the cells into 24-well culture plates. After the initial 3 h of
culture, cells cultured in the MgSiV-preconditioned medium showed
significantly more growth in comparison with the pristine or the SiV-
preconditioned medium, which contained no Mg2+ ions. Presence of the three
ions from MgSiV resulted in the fastest proliferation rate of the cells within 1
to 3 d of culture and the earliest expression of alkaline phosphatase, a
differentiation marker of cells in earlier stages. Moreover, cells cultured in the
MgSiV-preconditioned medium showed a good degree of mineralization by
Alizarion red S staining after 35 d of culture.
CONCLUSION
In this review, the structural control of vaterite in combination with
siloxane and Mg2+ ions were discussed, aimed at achieving desirable ion-
releasing properties for biomedical applications. The carbonation process in
the presence of aminosilane led to the formation of SiV particles, which

consisted of nano-lamelae of vaterite coordinated with siloxane. Enhancement
of chemical stability in siloxane as well as the improvement of coordination at
the vaterite/siloxane-interphase is revealed as a key in the tuning of ion-
releasing properties. Besides, novel vaterite particles doped with siloxane and
Mg2+ ion (MgSiV particles) were successfully prepared using this process.
Soluble silicate, Ca2+, and Mg2+ ions from this particle enhanced cellular
activities such as initial adhesion, proliferation, differentiation, and
mineralization of murine osteoblast-like cells.
ACKNOWLEDGMENT
This work was supported in part by the Japan Society for the Promotion of
Science (JSPS) and a Grant-in-Aid for Scientific Research (KAKENHI).
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[49] Madejová, J; Bujdak, J; Janek, M; Komadel, P. Comparative FT-IR
study of structural modifications during acid treatment of dioctahedral
smectites and hectorite. Spectrochim. Acta [A], (1998), 54, 1397-1406.
Chapter 4
POROUS CALCIUM CARBONATE CORES

AS TEMPLATES FOR PREPARATION OF
PERORAL PROTEINS DELIVERY SYSTEMS:
THE INFLUENCE OF COMPOSITION OF
SIMULATED GASTROINTESTINAL FLUIDS ON
THE STRUCTURE AND MORPHOLOGY
OF CARBONATE CORES
N. N. Sudareva1*, N. N. Saprykina1, E. V. Popova1,2

and A. D. Vilesov1
1
Institute of Macromolecular Compounds of the Russian Academy of
Sciences, St. Petersburg, Russian Federation
2
St.Petersburg State Polytechnical University,
St. Petersburg, Russian Federation
ABSTRACT
One of metastable polymorphic modifications of calcium carbonate
(vaterite) has been successfully used for more than ten years in the
*
Address for correspondence: Natalia N. Sudareva, Institute of Macromolecular Compounds of
the Russian Academy of Sciences, Bolshoi av. 31, 199004, St. Petersburg, Russian Federation.
Tel: +78123286896. Fax: +78123286896. E-mail: nnsas@mail.ru.
74 N. N. Sudareva, N. N. Saprykina, E. V. Popova et al.
formation of drug delivery systems (DDS). Porous calcium carbonate

systems containing biologically active compounds serve as templates for
layer-by-layer polyelectrolyte assembly and formation of a multilayer bi-
polymer shell. After dissolving carbonate cores with ethylenediamine-
tetraacetic acid (EDTA), a biologically active compound remains in the
polymeric microcapsule; the main disadvantage of this capsule is its low
mechanical strength.
If DDS are used for peroral administration, it is necessary to protect
biologically active load from the action of acidic gastric juice and provide
its gradual release in weakly alkaline intestinal medium. Sodium alginate
polyanion is used in the formation of polymeric shells, among other
polymers, and this compound meets all the necessary requirements. It
does not dissolve in acidic medium and swells in weakly alkaline liquids;
besides, it is biocompatible, biodegradable and does not cause any side
effects. In the studies of DDS behavior in vitro in simulated
gastrointestinal fluids, 0.05 – 0.10 M HCl solutions are used (acidic
gastric medium), as well as various weakly alkaline buffers. Phosphate
buffer or Tris-HCl buffer (with pH varying from 7.4 tо 8.2) are most
frequently used as models for intestinal medium.
Carbonate cores are used as “half-finished product” for preparation
of different variants DDS. The templates without protective coating
dissolve in acidic gastric medium. We have studied the influence of ionic
composition of intestinal medium on the structure and morphology of
carbonate cores and release profiles of model and therapeutic proteins.
When phosphate buffer is used, ionic exchange between phosphate and
calcium carbonate takes place; this process results in considerable
changes in core structure and leads to fast protein release. Prolonged
release was observed in the experiments with other buffer systems (e.g.,
duodenal juice which reproduces natural intestinal fluid as accurately as
possible). Scanning electron microscopy allows visualizing morpho-
logical changes in carbonate cores which correlate with release profiles of
proteins. The EDS data allow determining atomic composition of the
structures formed from carbonate vaterites during prolonged exposure to
various ionic media.
The obtained results give the ability to make an expert choice of the
medium for controlling quality of DDS in vitro. Besides, the protocol of
DDS formation by polyelectrolyte assembly on carbonate cores can be
simplified. Elimination of the stage involving dissolution of carbonate
cores with EDTA will decrease losses of the encapsulated object during
DDS formation and strengthen the structure of delivery systems.
Keywords: CaCO3 templates, simulated gastrointestinal fluids, proteins

release, SEM
Porous Calcium Carbonate Cores As Templates for Preparation ... 75
INTRODUCTION
Porous vaterite (a polymorph of calcium carbonate (СаСО3)) can be
applied in medicine and diagnostics as a carrier for drugs, dyes or magnetic
particles. There are several ways of using vaterites as templates for the
formation of polymer–based drug delivery systems (DDS). One method has
been proposed more than a decade ago; this so-called polyelectrolyte
adsorption method (PEA) includes the formation of a polymeric shell around
carbonate templates containing a target object. Oppositely charged
polyelectrolytes are deposited onto carbonate templates layer-by-layer. The
target object (e.g., a protein) is loaded into carbonate cores during co-
precipitation of solutions containing a protein, Na2CO3 and CaCl2. After the
formation of polyelectrolyte shell, templates are removed by treatment with
ethylenediaminetetraacetic acid (EDTA) solution [1].
The systems obtained by this method were used for encapsulation of
various model [2] and therapeutic proteins (insulin [3], fibroblast growth
factor [4], and HIV-1 p24 antigen [5]). The enzymes immobilized inside
carbonate-polymer carriers retain their biological activity, as was
demonstrated with lactate dehydrogenase and urease [6]. The influence of
СаСО3 synthesis conditions on core morphology and regularities of protein
loading were studied in [7]. Controlled release of fibroblast growth factor from
polyelectrolyte microcapsules based on carbonate templates regulates cell
proliferation in vitro [4]. In the majority of cases, CaCО3 cores serve as
sacrificed templates; after the formation of polyelectrolyte (PE) shell, the cores
are dissolved by treatment with acidic solution or EDTA [1]. It should be
noted that after removing СаСО3 with EDTA, the shell loses mechanical
strength, and after centrifugation and washing, significant losses of
encapsulated object were detected [8].
Delivery systems containing non-dissolved CaCО3 templates and their use
were studied in [9]. The authors have demonstrated that CaCО3 particles
without shells can be successfully used for intranasal administration of
loperamide (analgesic drug) to the brain via the olfactory route, which allows
bypassing the blood-brain barrier. The efficient use of CaCО3 vaterites in vivo
as carriers of photosensibilizers (compounds which possess targeted
cytotoxicity and should be introduced into tumor cells) was demonstrated in
[10].
In the majority of the above-mentioned studies, authors suggested
possibility of using these carriers in peroral drug delivery. Peroral DDS should
provide integrity of the encapsulated object in acidic gastric juice and its
gradual release in weakly alkaline intestinal medium. This quality of DDS is

tested by studying release of the target object in vitro. In these studies, 0.05 –
0.10 N HCl solutions are used for simulating gastric environment, and
sometimes a proteolytic enzyme (pepsin) is added. Intestine medium is
simulated by weakly alkaline solutions of different ionic composition with pH
varying from 7.5 to 8.0. Various buffer systems are used as simulated
intestinal fluids, e.g., phosphate buffer [11, 12], citrate-phosphate buffer [12]
or Tris buffer [11-13]; sometimes, intestinal proteases are added [13]. In [14],
gastrointestinal fluid was simulated by sodium borate-phosphate buffer (with
pH varying from 2.0 to 8.0). In another study [15], simulation of digestion was
based on the knowledge of human physiology. The duodenal juice used in
these in vitro experiments included seven components.
In the above-mentioned works, release of proteins from peroral DDS of
various configurations (alginate and pectinate beads, polyelectrolyte capsules,
protein microparticles, liposomes etc.) was studied. It was demonstrated [12]
that the rutin release from calcium pectinate beads depends on the composition
of dissolution media. Release of rutin into Tris buffer medium proceeds slower
than that in the experiments with phosphate-containing media (H2РO4- or
HPO4 2-).
Carbonate templates serve as a “half-finished product” for preparation of
DDS. The studies of the influence of composition of simulating media on
protein behavior in carbonate templates give the ability to make an expert
choice of the medium for controlling quality of DDS in vitro.
In this Chapter, we give the results of our studies of ionic composition of
simulating media on the structure of carbonate templates and release profiles
of proteins from these templates.
MATERIALS AND METHODS

Materials
The following reagents were used in CaCO3 preparation (puriss.,

purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further
purification): sodium carbonate (Na2CO3), calcium chloride (CaCl2•2H2O) and
sodium chloride (NaCl). Chemical reagents used for preparation of different
simulated intestinal fluids were the following (p.a., purchased from Sigma-
Aldrich): calcium bicarbonate (Са(НСО3)2), sodium dihydrogen phosphate
(NaH2PO4), potassium dihydrogen phosphate (KH2PO4), disodium hydrogen
phosphate (Na2HPO4), potassium chloride (KCl), magnesium chloride

(MgCl2) and Tris (tris(hydroxymethyl)aminomethane, ((HOCH2)3CNH2).
Hydrochloric acid (HCl, puriss.) was purchased from “Component-Reactiv
LLC” (Moscow, Russian Federation). Solutions for obtaining CaCO3 cores
and different simulated fluids were prepared using deionized water with a
conductivity of 0.056 mS/cm (Vital Diagnostics, St. Petersburg, Russia). The
proteins used in the experiments (α-lactalbumin, bovine serum albumin) were
purchased from Sigma-Aldrich. Superoxide dismutase was purchased from the
State Research Institute of Highly Pure Biopreparations (St. Petersburg,
Russian Federation). Characteristics of these proteins are given in Table.
The following three solutions were used as simulated intestinal fluids
(SIF): 0.07 М Na phosphate buffer, pH = 7.9 (PB); Tris•HCl buffer, pH = 8.0
(Tris) and multicomponent solution approximating the composition of
duodenal juice (with the exception of urea) and prepared according to the
method described elsewhere [15], (0.18M pH = 7.75 (DJ)). Composition of DJ
was the following: 40 mL of NaCl — 175.3 g/L; 40 mL of NaHCO3 – 84,7
g/L; 10 mL of KH2PO4 - 8 g/L; 6.3 mL of KCl – 89,6 g/L; 10 mL of MgCl2 - 5
g/L; 180μl HCl - 37% g/g.
In this Chapter, we used the following polymers: high-viscosity sodium
alginate (Sigma-Aldrich) with a molecular mass of about 1300 kDa and acid-
treated gelatin A (Lysychansk Gelatin Plant, Lysychansk, Ukraine) with a
molecular mass of 355 kDa (determined by chromatographic analysis).
Methods
Preparation of Carbonate Cores

The CaCO3 cores were formed during precipitation in the course of the
reaction between calcium chloride and sodium carbonate. We used the
procedure described by authors [1] as a basic method, but the techniques of
core washing and drying were modified according to [7]. Briefly, a certain
volume of 0.33 mol/L water solution of CaCl2 (in most cases, it was 3 mL)
was rapidly added to water solution of Na2CO3 (of the same volume and
concentration) during mixing with a magnetic stirrer (400 rpm). The mixture
was stirred for 30 s, and then the obtained suspension was left for 15 min.
Washing and drying of cores were performed using Schott filter glasses (# 16);
cores were washed with acetone/water mixtures of increasing acetone
concentration (33%, 50% and 100%). Final drying was carried out in
thermostat at 30-50°С, since vaterites are formed during drying СаСО3
precipitate in this temperature region. At lower temperatures, calcites are

mainly formed, and at higher temperatures, aragonites predominate in the final
product.
The dried cores were stored in a sealed container at room temperature.
Loading Proteins into Cores

The loading of proteins into carbonate cores was performed by co-
precipitation in the following manner: protein solution with an initial
concentration of 2.0–2.5 mg/mL was added to the solution of CaCl2•2H2O. In
subsequent experiments, this procedure was carried out as described above
(see the section “Preparation of carbonate cores”). The amount of protein
loaded into cores (I) was calculated according to the following equation:
(C  V ) s  ( (Ci  Vi ))
I (%)  i
100% , (1)
(C  V ) s
where Vs is the volume of the initial protein solution; Cs is the concentration

of the initial solution; Σi is the total amount of products present in filtrate and
the liquid used for washing filters; Vi and Ci are the volume and concentration
of filtrate and the liquid used for washing filters, respectively.
The efficiency of protein introduction into cores was defined as protein
load (L), i.e., the amount of the included protein per unit weight of cores:
L = I × (Ppr/ Ptempl), (2)
where Ppr is the initial weight of protein, and Ptempl is the weight of cores. Each
measurement was carried out three times. Protein concentration was
determined using calibration curves obtained from optical density
measurements in the corresponding solvents at a wavelength of 280 nm. In the
used concentration ranges, calibration curve plots were linear for all proteins.
Optical measurements were carried out using a Specord M40
spectrophotometer (Carl Zeiss, Oberkochen, Germany).
Formation of Polyelectrolyte Shells around CaCO3 Templates

The formation of polyelectrolyte shell was performed by layer-by-layer
coating of the SOD-containing templates using solutions of a polyanion
(sodium alginate) and a polycation (gelatin A, isoelectric point рI = 8.0),
according to the technique described elsewhere [2]. Briefly, aqueous solution

of sodium alginate (C = 2 mg/mL) was poured onto protein-containing
templates (the ratio was 2.5 mL of the solution per 20 mg of the templates).
The suspension was subjected to ultrasound treatment for 10 s, then it was
stirred using a Multi Bio RS-24 rotator (Biosan, Riga, Latvia) for 10 min. The
rotation was programmed as follows: the orbital range of rotation was 7 rpm,
the reciprocal range was 90 and the vibration range was 5. Then, the obtained
suspension was centrifuged for 3 min at 3 000 rpm. Supernatant was removed,
water was added (in order to wash templates and remove free polymer); this
suspension was intensively stirred manually and centrifuged in the same
conditions. Washing was carried out twice. Another polyelectrolyte (gelatin A)
was added to the precipitated templates, and the same manipulations were
performed. Thus, the shell consisting of one polyelectrolyte pair was formed.
The final product (SOD delivery system) included CaCO3 templates
surrounded by three pairs of polyelectrolyte layers. Protein loss occurring
during the coating procedure was calculated as a total amount of protein in
supernatant liquids (about 15%).
Release Experiments
The experiments were carried out at the temperature approximating the
human body temperature (37°С). The ratio between weight of templates and
volume of a simulated intestinal fluid (SIF) was constant in all experiments
(20 mg/2.5 mL). Continuous stirring was performed using the programmable
rotator (Multi Bio RS-24, Biosan). Rotation was programmed in the similar
way to that used during the formation of polyelectrolyte shells
(see “Formation of polyelectrolyte shells around CaCO3 templates”).
Rotation intensity was continuous and constant throughout the
experiment. The amount of released protein was determined at certain time
intervals after the start of experiment. Suspensions of templates were
centrifuged for 3 min at 3 000 rpm. 0.5 mL of a supernatant was taken to
determine protein concentration, 0.5 mL of the corresponding SIF was added
to templates, and release experiment was resumed. Protein concentration was
determined spectrophotometrically (λ = 280 nm) using calibration curves
obtained for the corresponding media. Supernatant obtained after incubation of
protein-free CaCO3 cores in the same conditions was used as a reference
solution. Release was expressed as a percentage of protein included in the
initial templates. The mean error of determination of the amount of released
protein (in three experiments performed under the same conditions) was 8%.
Characterization of CaCO3 Cores

The SEM images of CaCO3 cores were obtained using a Supra 55 VP
scanning electron microscope (Carl Zeiss). The samples were attached to the
adhesive tapes and then coated with gold.
Elemental composition of the samples was determined by energy-
dispersive X-ray spectroscopy (EDX) using an X-Max 80 detector (Oxford
Instruments, Abingdon, UK).
The pore volume and pore size distribution of CaCO3 cores were
determined by the Brunauer-Emmet-Teller (BET) method involving nitrogen
adsorption/desorption at 77.3˚ K. The data were collected using a Nova 1200e
surface area and pore size analyzer (Quantachrome Instruments, Boynton
Beach, Fl, USA). Quantachrome NovaWin software package developed for
Nova instruments and intended for data acquisition and reduction was used.
RESULTS AND DISCUSSION

The main characteristics of a peroral DDS are the load value and release
profile of an encapsulated drug into simulated gastric fluids (SGF) and
simulated intestinal fluids (SIF). These parameters determine dosage and
frequency of drug administration. The conditions of preliminary in vitro
experiments should be as close as possible to real in vivo conditions.
In this Chapter, we have studied CaCО3 cores which are used as “half-
finished product” for preparation of various DDS. The carbonate cores without
protective coating dissolve in acidic gastric medium. In weakly alkaline media
(simulated intestinal fluids), carbonate cores undergo certain morphological
and chemical transformations. Our main goal was to compare the influence of
three simulated intestinal fluids with different ionic composition on the
behavior of CaCО3 templates. Two of these media are frequently used in
similar studies (0.07 M sodium phosphate buffer with pH = 7.9 (PB) and
0.2 М Тris·HCl buffer with рН = 8.0 (Tris)). The third system was a
multicomponent medium with the composition reproducing that of real
duodenal juice as accurately as possible (with the exception of urea),
according to [15], 0.18 М, рН = 7.75 (DJ).
Two model proteins (α-lactalbumin and bovine serum albumin) and one
antioxidant therapeutic protein (superoxide dismutase) were used as objects
for encapsulation. Characteristics (molecular mass - MM, isoelectric point – pI
and hydrodynamic radius - Rg) of the used proteins and its average load values
are given in Table. The loading of proteins into carbonate cores was performed
according to the technique described above.
Table. Characteristics of the used proteins and the load values obtained in
the experiments with CaCО3 cores
N Protein MM pI Rg (nm) Load

(kDa) (μg/mg)
1 α-Lactalbumin (Lact) 14 4.5 2.1 16.1±1.4
2 Superoxide dismutase (SOD) 32 5.4 2.6 32.2±1.9
3 Bovine serum albumin (BSA) 65 4.9 3.5 55.4±2.3
Release of Proteins from CaCO3 Templates

The conditions of experiments aimed at determination of amount of
protein released from carbonate templates have a significant influence on the
results.
After irregular discontinuous stirring of templates in the studied medium
at room temperature, the error of determination of the amount of released
protein sometimes reached 50%. Therefore, competent analysis of the results
obtained in vitro requires carrying out experiments in identical conditions, i.e.,
in thermostat at 37°C, at continuous stirring (with constant intensity). These
observations add further credence to the opinion that one drug may have
various effects on different individuals. Specific features of physiology
(particularly, peristalsis) and diet of patients necessitate individual approach to
prescription of a medicine.
Figures 1-3 demonstrate time profiles of protein release into various
simulated intestinal fluids (SIF). The duration of experiments (24 h) was
selected with account of physiological needs of an organism assuming that a
medicine is taken once every day. As can be seen from comparison between
curves presented in Figures 1 and 2, common simulating media (phosphate or
Tris buffers) give widely different profiles of protein release from CaCО3
templates.
In phosphate buffers, Lact and SOD are virtually completely released in 2
hrs. In Tris buffer, in all three cases, less than one third of a protein is released
in 24 hrs.
Figure 1. Release profiles of Lact (1), SOD (2), and BSA (3) proteins into 0.07 M
phosphate buffer (рН= 7.9).
Figure 2. Release profiles of Lact (1), SOD (2), and BSA (3) proteins into 0.2 M Tris
buffer (рН= 8.0).
In PB and DJ, bovine serum albumin is released from CaCО3 templates

much slower than SOD and Lact (Figures 1 and 3). The estimated
hydrodynamic radii (Rg) of the used globular proteins (see Table) differ only
slightly. The average pore radius of CaCО3 core samples prepared according
to the technique given in the “Materials and Methods” section was determined
using nitrogen adsorption (the BET method) and was found to be 6.7 nm.
Higher values (not less than 10 nm) are given in the literature [1]. Apparently,
differences in release profiles of these proteins cannot be explained solely by
steric hindrances. According to the authors of [16], albumin serves as a
transport protein for transferring ions of several metals (including Ca++) into
blood circulatory system. ВSA binds Ca++ in a relatively nonspecific manner.
This interaction may also be realized in the course of formation of carbonate
template containing the protein. During co-precipitation of products from
solutions containing BSA, CaCl2 and Na2CO3, BSA molecules are surrounded
by dissociating salts (including Ca++ ions). It can be assumed that these
interactions hold BSA molecules in carbonate template during the release
procedure.
Figure 3. Release profiles of Lact (1), SOD (2), and BSA proteins (3) into duodenal
juice (pH=7.75).
To explain these significant differences in release profiles of proteins into

simulating media of various compositions, we should consider changes in the
structure of CaCО3 templates occurring with changing ionic composition of
the medium.
Morphological Modification. Scanning Electron Microscopy of CaCO3

Templates
Carbonate cores (templates) included in the systems used for peroral
protein delivery undergo certain morphological and chemical transformations
in simulated intestinal fluids. The samples (both templates containing proteins
and protein-free cores) prepared according to the technique described in the
“Materials and Methods” section; initially represent porous СаСО3 polymorph
(vaterite). Scanning electron microscopy (SEM) allows visualizing

morphological changes in carbonate cores which correlate with release profiles
of proteins. Figure 4 illustrates the influence of ionic composition of
simulating fluid on СаСО3 morphology. SEM images of BSA-containing
templates after exposure for 24 hrs in phosphate buffer (B), Tris buffer (C) and
duodenal juice (D) as well as these for intact templates (A) are given.
Comparison of SEM images of carbonate templates taken under corresponding
conditions can explain considerable differences in release profiles obtained for
three proteins in various media.
The most noticeable changes in СаСО3 morphology occur in 0.07 М
sodium phosphate buffer (pH = 7.9). Dynamics of this process is presented in
Figure 5; here, SEM images of intact empty СаСО3 cores (A) and СаСО3
cores exposed to PB for 1 (B), 2 (C) and 24 (D) hrs are given. In seven days,
СаСО3 cores did not undergo any further changes.
Figure 4. (Continued).
Figure 4. SEM images of CaCO3 templates containing BSA. A – The intact template
(A); the template after 24 h contact with phosphate buffer (B), with Tris buffer (C) and
with duodenal juice (D).
Figure 5. (Continued).
Figure 5. SEM images of empty СаСО3 cores. The intact cores (A); cores exposed to
phosphate buffer for 1 (B), 2 (C) and 24 (D) hrs.
Calcium carbonate structure depends only on the duration of its contact

with PB. The presence of protein inside carbonate templates does not have an
effect on their morphology. SEM images of carbonate particles taken after
24 hrs exposure to PB are similar in the following cases: for protein-free
СаСО3 (Figure 5 D), BSA-containing СаСО3 (with 54% of the protein
released) (Figure 4B), and SOD-containing СаСО3 (100% of the protein was
released), the image is not given. Considerable changes in the СаСО3 porous
structure occurring in PB medium lead to fast release of loaded proteins (as
can be seen in Figure 1A). In the case of Tris buffer medium, certain
compactization of СаСО3 templates can be noticed at 50 000 times
magnification (Figure 4С). Possibly, this compactization causes considerable
decrease in the amount of released protein. This problem needs further
investigation.
In the duodenal juice medium, the structure of templates does not change
significantly (Figure 4D) and resembles that of the intact templates (Figure
4A). After prolonged exposure (24 hrs), the same regularities as in the case of
PB are observed, i.e., template structure does not depend on its contents. SEM
images of BSA-containing templates after release of 70% of the protein
(Figure 4D) are similar to the SEM images of the templates which are
completely free of released SOD (the image is not presented).
Thus, CaCO3 vaterites turn into more porous structures in РВ medium
containing Na2HPO4 and NaH2PO4 salts. Pore sizes of cores increased in
alkaline media as a result of the ion exchange reaction (CaCO3 → CaHPO4);
the possible reaction paths are given below.
CaCO3 + Na2HPO4 + 4 H2O → Na2CO3 • 2H2O + CaHPO4 • 2H2O (3)
Na2CO3 • 2H2O → NaOH + H2CO3 (in aqueous medium) (4)
H2CO3 → H2O +CO2↑ (5)
There is a qualitative confirmation of this reaction proceeding after

contact of CaCO3 templates with one of PB components (рН = 7.9); this is
liberation of carbon dioxide gas bubbles and increase in pH of the medium at
the expense of the appeared NaOH. The value of pH of the phosphate buffer
containing submerged CaCO3 templates (50 mg in 10 mL of the solution)
increased from 7.9 to 8.9 in 2 hrs.
Modification of Atomic Composition of the Templates. Energy Dispersive

X-ray Spectroscopy Data
Chemical modification of CaCO3 cores in PB medium is confirmed by the
EDX data. The structures with changed morphology (exposed to phosphate
buffers for 24 hrs) contain not only Ca, C and O atoms, but also P atoms
(approximately 16%); this result can be explained by the formation of СаНРО4
molecules according to Equation 3.
According to [15], gastric juice contains Na+, К+, Ca++ and Мg++ cations
as well as Cl-, HPO4-- and СО3-- anions. Phosphates are present in small
proportion; thus, exposure to duodenal juice does not lead to significant
modification of CaCO3 structure. The studies of atomic composition of CaCO3
cores after exposure to this medium for 24 hrs reveal only slight increase in
Mg and Cl contents (less than 1%). After prolonged contact between CaCO3
cores and Tris buffer, atomic composition of the cores also remains virtually
unaltered.
The authors of [12] have demonstrated that release of rutin from calcium
and zinc pectinate beads into Tris buffer medium proceeds slower than that in
the case of phosphate-containing media. These data correlate with the results
of experiments involving protein release from CaCO3 templates performed in
this Chapter (Figures 1 and 2). X-ray diffraction data confirmed that during
swelling of cаlcium pectinate beads in phosphate buffer media, CaHPО4•
2Н2О crystals are formed. The use of phosphate buffers in cаlcium-containing
systems leads to destabilization of these systems and fast release of
encapsulated substances; similar process occurs in the system described in this
Chapter.
CaCO3 Templates and DDS Based on these Templates in Simulated

Intestinal Fluid (SIF)
Now, another important question arises: how accurately do the protein
release data obtained for non-coated CaCO3 templates represent behavior of a
complete DDS? This point was illustrated by the DDS based on CaCO3
templates containing SOD and coated with polyelectrolyte shell. The shell was
formed layer-by-layer and included three pairs of layers (alginate and gelatin
A). Isoelectric point of polyampholyte gelatin A (pI) is 8.0. In the conditions
of polyelectrolyte assembly (рН = 6.5), gelatin molecules carry excess positive
charge and thus interact with polyanion alginate molecules. Figure 6 presents
SEM images of initial SOD-containing carbonate templates coated with three
pairs of alginate-gelatin layers (A) and the templates exposed to PB (SIF) for
24 hrs (B). The coating defect in Figure 6A allows estimating its thickness.
During dissolution of polymeric shell in PB, СаСО3 templates undergo the
same modification as the free cores, but this process takes longer and has an
influence on the rate of release of protein from the carrier.
Figure 7 presents profiles of SOD release from two types of structures into
PB (simulated intestinal fluid), namely: from the structures based on non-
coated СаСО3 templates (1) and from СаСО3 templates covered with three
pairs of alginate-gelatin A polyelectrolyte layers (2).
Since the non-coated particles dissolve in acidic medium, SOD release

from these carriers was studied only in SIF. СаСО3 templates with
polyelectrolyte shells were first placed for 2 hrs into simulated gastric fluid
(SGF) (0.1N HCl), then into SIF (0.07M PB, pH=7.9). Polyelectrolyte layer
containing alginate (which does not dissolve in acidic medium) protects
СаСО3 templates and the encapsulated protein against SGF. In SIF, SOD
release from DDS is delayed as compared to the case of non-coated СаСО3
templates; the additional time is required for swelling and dissolving
polyelectrolyte coating.
Figure 6. SEM images of CaCO3 templates containing SOD and coated with three
pairs of alginate-gelatin layers (layer-by-layer method). The initial structure (A), the
structure after 24 h exposure to phosphate buffer (B).
Figure 7. Figure 7. Release profiles of SOD into phosphate buffer (рН = 7.9) from non-
coated СаСО3 templates (1) and СаСО3 templates covered with three pairs of alginate-
gelatin A polyelectrolyte layers (2).
Curve 2 given in Figure 7 demonstrates efficiency of the SOD release

process when using DDS with remaining СаСО3 templates.
Let us now turn our attention to pharmacokinetics of peroral DDS
containing non-dissolved CaCO3 templates. As pointed out above, carbonate
templates are coated with polymeric shells for the purpose of protection in
acidic gastric juice. In weakly alkaline media of different parts of intestine,
polymeric shells dissolve, and the target object (TO) included in the templates
is released gradually. Protective shell is commonly formed from alginate,
chitosan and several other polymers. Due to mucoadhesive properties of these
macromolecules, DDS are localized in the vicinity of mucosal surface. The
exact mechanism of mucoadhesion is not fully understood; possible types of
interaction are listed in [17]. Mucoadhesion increases the time of residence of
the released TO in immediate proximity to mucosal surface and thus increases
probability of its entry into bloodstream. Empty СаСО3 cores are either
removed from intestine or find way into circulatory system. Here we shall not
be concerned with possible mechanisms of a very complicated and virtually
unstudied process of penetrating various substances from gastrointestinal tract
to the bloodstream. It will be recalled that the average size of CaCO3 cores is
4-6 μm, and, therefore, one possible way of their penetration through cell wall
is endocytosis [18].
Now consider transformations of carbonate cores in the intestinal tract.

The duration of stay of CaCO3 cores in intestines depends first of all on
physiological features of an organism. As shown above (Figure 4D), neither
empty cores, nor protein-containing templates undergo significant
morphological modification after exposure to simulated DJ for 24 hrs (and
even after 7 days). However, the studies of vaterite stability in the presence of
various polymeric and ionic additives have demonstrated that depending on
the nature of an additive, morphological transformation of vaterites is possible
[19]. Subsequent behavior depended on the polymer or other additive present.
The influences of ionic composition and the presence of macromolecules on
the process of СаСО3 crystallization are reviewed in [20].
Thus, calcium carbonate introduced into intestinal tract and real medium
of an organism may undergo morphological transformations which depend on
the composition of intestine fluid. All three СаСО3 polymorphs (vaterite,
calcite and aragonite) were found in human organism.
Calcium is a necessary element for human organism and plays important
regulatory and structural roles. Calcium makes up approximately 2% of human
body weight.
Now we give an example of efficient use of СаСО3 as a prophylactic
treatment. Calcium deficiency leads to bone fragility in elderly people,
particularly in postmenopausal women. In this case, calcium preparations are
administered; the most popular ones are Calcium-D3 Nycomed and Calcimine
advance (with one pill containing 500 mg of СаСО3). The authors of [21] have
compared efficiency of prophylactic administration of Calcium-D3 Nycomed
and pure СаСО3 aimed at prevention of bone tissue losses in postmenopausal
women for 2 years. Drug tolerance is similar in both cases. No negative trends
in bone density parameters were observed (during administration of Calcium-
D3 Nycomed, these parameters increased by 1.3% due to the presence of
vitamin D3 in the preparation), in contrast to reference group of women who
received recommendations concerning diet and physical activity.
The data given above confirm safety of using DDS containing carbonate
cores, their compatibility with human organism and, in a number of cases,
their positive influence on calcium exchange in organism.
CONCLUSION
SEM and EDX were used in the studies of morphological and chemical
transformations of carbonate cores (templates for the preparation of DDS for
peroral protein administration) in solutions of various ionic compositions

which simulate intestinal medium.
Comparison between types of behavior of СаСО3 templates in simulating
media of various compositions indicates that it is very important to make
expert choice of an ionic simulating medium for in vitro studies. Three
dissolution media were used in order to imitate intestinal fluid.
It was demonstrated that phosphate and Tris buffers (which are commonly
used in the studies of release of encapsulated substances in weakly alkaline
SIF) are not ideally suited for these purposes. First, phosphate-containing
media may destabilize calcium-containing carriers (this effect was
demonstrated in experiments with СаСО3 templates in this Chapter and with
calcium-pectinate beads in [12]). Second, the rate of protein release in Tris
buffer is much lower than that in PB. Composition of duodenal juice provides
the medium rate of protein release. The ionic composition of duodenal juice
was based on human physiology (in vitro digestion model [15]). This fluid
should provide the most appropriate behavior of peroral DDS in SIF. This
solution can be recommended as a simulated intestinal fluid.
When peroral dosage form is prescribed, the burst of encapsulated drug is
highly undesirable. Drug formulations with durable action allow regulating
dosage and making drug intake more convenient for patients. Proteins are
released completely and fast enough from СаСО3 templates into duodenal
juice medium. Polyelectrolyte coating formed around СаСО3 templates
prolongs the process of protein release from these DDS in intestines for
several hours. The duration of action of this drug formulation increases.
Calcium carbonate is present in human organism, its safety was
confirmed, and prophylactic administration of this substance efficiently
normalizes calcium exchange.
The obtained results suggest changing protocol of the formation of DDS
by polyelectrolyte adsorption on CaCO3 cores [1]. Exception of the stage
involving dissolution of carbonate templates with EDTA will decrease losses
of encapsulated substances and increase mechanical strength of DDS.
ACKNOWLEDGMENTS
The authors thank G.A. Pankova for determination pore structure of
CaCO3 cores by BET method and S.G. Petunov and A.S. Radilov for fruitful
discussion.
Declaration of Interest
The authors report no conflicts of interest. The authors alone are

responsible for the content and writing of the article.
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Reviewed by Professor Gleb Sukhorukov, Queen Mary University of London,

UK School of Engineering and Materials Science.
INDEX
ammonium, 2, 3, 14, 39, 53

A ammonium salts, 2
analgesic, 75
access, 32
antigen, 75
accessibility, 40
antioxidant, 80
accounting, 64
aqueous solutions, 43, 46
acetone, 61, 63, 77
aragonite, viii, 30, 31, 32, 35, 37, 38, 39, 40,
acid, ix, 2, 9, 32, 36, 45, 46, 57, 71, 74, 75,
45, 47, 50, 52, 53, 64, 69, 91
77, 93
aspartic acid, 32, 35
acidic, ix, 10, 35, 74, 75, 80, 89, 90
atmosphere, 58
acrylic acid, 42, 43
atomic emission spectrometry, 64
active compound, ix, 74
atoms, 59, 61, 87
additives, viii, 13, 19, 29, 30, 32, 33, 43, 45,
attachment, 69
52, 53, 91
avian, 30, 44
adhesion, ix, 50, 51, 64, 67, 71
adsorption, vii, 1, 2, 3, 8, 11, 12, 13, 14, 15,
16, 18, 21, 22, 31, 37, 40, 41, 43, 63, 75, B
80, 82, 92, 93
adsorption isotherm(s), vii, 1, 3, 8, 11, 12, bacteria, 30
21, 22 barium, 71
AFM, 36 base, 9
aggregation, 12, 38, 46, 57 behaviors, 65
aging society, 51 bicarbonate, 64, 71, 76
albumin, 82, 94 biological activity, 75
alkaline media, 80, 87, 90 biological systems, 30
alkaline phosphatase, 51, 66 biologically active compounds, ix, 74
alkane, 9 biomaterial(s), viii, 30, 42, 49, 57, 64
allylamine, 39, 44 biomechanics, 67
amine(s), 2, 3, 12, 13, 14, 15, 36, 40 biomedical applications, vii, ix, 50, 66, 69
amine group, 40 biopolymer(s), 30, 31, 32, 33, 36
amino, 56 birds, 34
98 Index
blood, 71, 75, 83 chicken, 34

blood-brain barrier, 75 chitin, 35
bloodstream, 90 chitosan, 33, 90
body fluid, viii, 49 chromatography, 4, 59
bonding, 13, 51, 55, 58, 64 classes, 33
bone, viii, 49, 50, 51, 52, 64, 67, 68, 70, 71, classification, 63
91 CMC, 3, 11, 21
bone cells, 68 CO2, viii, 13, 14, 21, 36, 49, 56, 70, 87
bone form, viii, 49, 51, 52, 68 collagen, 31, 44, 45, 46, 51, 93
bone resorption, 50 commercial, 7
bovine serum albumin, 32, 47, 77, 80, 82 compatibility, 91
brain, 75, 94 composites, 2, 35
composition, vii, ix, 30, 34, 53, 55, 71, 74,
76, 77, 80, 83, 84, 88, 91, 92
C compounds, viii, 49, 59, 75, 93, 94
condensation, 3, 55, 70
Ca2+, viii, 13, 33, 37, 38, 39, 42, 49, 51, 52,
conductivity, 18, 77
56, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
configuration, 64
68
constant rate, 11
calcification, 64
contact angle, vii, 1, 3, 4, 5, 8, 9, 11, 15, 16,
calibration, 78, 79
17, 18, 19, 22, 23
capillary, vii, 1, 4, 8, 9, 10, 11, 19, 21, 22,
COOH, 42
23
coordination, viii, 50, 56, 59, 63, 67
capillary rise method, viii, 1, 8, 19, 23
copolymer(s), 30, 36, 41, 42, 45, 46
capsule, ix, 74
correlation, 13, 21
carbamate, viii, 13, 50, 56, 62, 63
critical value, 3, 18, 22
carbon, 13, 38, 87
crystal growth, 31, 34, 37, 40, 43, 53, 57, 61
carbon dioxide, 13, 38, 87
crystal structure, 37, 41
carbonate cores, vii, ix, x, 74, 75, 78, 80, 81,
crystalline, vii, viii, 3, 30, 32, 35, 46, 49, 52,
84, 91, 93
59, 61, 63, 64, 70
carbonization, 38
crystallinity, 30
carboxyl, 37
crystallites, 46, 53, 60, 61
carboxylic acid, 43
crystallization, viii, 31, 32, 33, 34, 35, 37,
carboxylic groups, 42
38, 39, 41, 42, 44, 45, 46, 47, 50, 52, 53,
Carrageenan, 33
55, 56, 61, 62, 64, 91
cation, 37, 42
crystallization kinetics, 64
cationic surfactant, vii, 1, 5, 7, 8, 21, 22
crystals, 6, 15, 22, 31, 32, 33, 34, 35, 36, 37,
cell culture, 51
38, 39, 40, 41, 42, 43, 45, 46, 69, 88
cell line, 68
CTAB, 53
cellulose, 52
culture, ix, 50, 51, 66
chemical, viii, 40, 50, 52, 59, 61, 63, 67, 80,
culture medium, ix, 50, 51, 66
83, 91
cuticle, 34
chemical stability, viii, 50, 52, 59, 63, 67
cytocompatibility, ix, 50, 66
chemical structures, 40
cytotoxicity, 75
chemisorption, 56
chemotaxis, 51, 68
Index 99
ethanol, 38
D ethylene, 40, 41, 42, 43, 45
ethylene glycol, 40, 41, 42, 43, 45
DDS, ix, x, 74, 75, 76, 80, 88, 89, 90, 91, 92
ethylenediamine-tetraacetic acid, ix, 74
decomposition, 53, 57
examinations, 35
deficiency, 91
exclusion, 59
deformation, 61
experimental condition, 42
deposition, 46
exposure, x, 74, 84, 86, 87, 88, 89, 91
deposits, 30
derivatives, 33
desorption, 63, 80 F
diet, 81, 91
diffraction, 7, 56, 59, 60, 62, 64 fabrication, 94
diffusion, 36, 39, 53 fatty acids, 2
digestion, 76, 92 fibers, 35, 36, 39
discs, 39 fibroblast growth factor, 75, 93
dispersion, 3, 5, 22 fillers, 52, 70
distilled water, 53 filling materials, 51
distribution, 5, 14, 33, 80 films, 36, 44
dodecylammonium hydrochloride, vii, 1, 5, filters, 78
7, 11, 12, 14, 15, 18, 19, 21, 22 fish, 30
doping, viii, 50 fixation, 51
dosage, 8, 80, 92 flexibility, 33
drug delivery, ix, 74, 75, 94 flocculation, 41
drugs, 75 flotation, vii, 1, 2, 5, 11, 13, 19, 20, 21, 22,
drying, 77 25, 26
durability, 61 flotation recovery, vii, 1, 11, 13, 20
dyes, 75 fluid, ix, 4, 51, 74, 76, 79, 84, 88, 89, 91, 92
force, 4, 47
formamide, 8, 15, 16, 17, 18
E formation, viii, ix, x, 3, 14, 17, 21, 22, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
EDTA, ix, x, 74, 75, 92
42, 44, 45, 50, 52, 53, 56, 57, 61, 62, 63,
egg, 34, 35, 46
64, 67, 68, 69, 70, 74, 75, 78, 79, 83, 87,
Eggshell, 34
92
electrolyte, 13
fractures, 50, 67
electron, ix, 5, 10, 17, 22, 31, 62, 74, 80, 84
fragility, 91
electron microscopy, ix, 31, 74, 84
free energy, vii, 1, 3, 5, 9, 17, 19, 22
e-mail, 29
FTIR, 7, 54, 55, 56, 58, 64
encapsulation, 75, 80, 93
energy, 9, 18, 22, 31, 55, 80
England, 23 G
environment, 76
enzymes, 75 gastrointestinal tract, 90
epidemiology, 67 gel, viii, 37, 50, 53, 54, 55, 56, 57, 94
equilibrium, 4, 14 gene expression, 51, 68
100 Index
Germany, 78 incidence, 67
glasses, 69, 77 individuals, 81
glycol, 46 inducer, 64
granules, 36, 45 induction, 33, 61, 64
gravitation, 4 induction time, 33
grounding, 17 industry, 5
growth, 30, 31, 32, 34, 36, 38, 40, 43, 44, inflammation, 52
46, 47, 51, 53, 59, 62, 63, 66, 68, 69, 75 infrared spectroscopy, 7
growth factor, 51, 68, 75 inhibition, 37
growth mechanism, 43 inhibitor, 36
insulin, 51, 68, 75
integrins, 67
H integrity, 75
interface, 3, 4, 32, 40
Hallimond tube, vii, 1, 10, 11, 19, 22
interface energy, 40
height, 8, 11, 19
interphase, 56, 59, 67
helium, 5
intestinal tract, 91
heptane, 8, 9, 10, 15, 16
intestine, 90, 91
HIV, 75, 93
ionic polymers, 36, 40
HIV-1, 75, 93
ions, viii, 8, 12, 14, 29, 30, 31, 32, 33, 37,
human, 68, 76, 79, 91, 92
38, 39, 42, 44, 45, 49, 50, 51, 52, 53, 56,
human body, 79, 91
59, 63, 64, 66, 71, 83
hybrid, 70
isotherms, 63
hydrogen, 13, 76
hydrolysis, 14, 59, 61, 70
hydrophilic materials, 18 J
hydrophilicity, 4
hydrophobicity, 2, 3, 4, 13, 18, 19, 21, 22, Japan, 49, 67
23
hydroxide, 38, 53, 64
hydroxyapatite, 51 K
hydroxyl, 36, 42
KBr, 7
hysteresis, 63
kinetics, 11, 19, 20, 61, 71
I
L
identification, 34, 46
lactate dehydrogenase, 75
image(s), 5, 6, 16, 22, 55, 56, 62, 65, 80, 84,
lactic acid, 63, 70
85, 86, 87, 87, 88, 89
laminar, 4
immersion, 7, 14, 19
Latvia, 79
implants, 71
lead, 3, 30, 37, 39, 40, 42, 87, 88
improvements, 2
liberation, 87
in vitro, ix, x, 30, 34, 42, 44, 51, 57, 64, 66,
limestone, 17, 18
68, 69, 74, 75, 76, 80, 81, 92, 94
liposomes, 76
in vivo, 75, 80
Index 101
liquids, vii, ix, 1, 8, 9, 10, 17, 18, 22, 23, 74, modified Washburn equation, vii, 1, 8
79 moisture, 58
lysozyme, 34, 35, 46, 47 mole, 40
molecular mass, 77, 80
molecular weight, 37, 38, 40, 41, 43, 45
M molecules, 2, 3, 11, 12, 14, 21, 32, 33, 36,
42, 43, 52, 56, 57, 83, 87, 88
macromolecules, vii, viii, 29, 30, 34, 36, 40,
mollusc shell, 32, 35
46, 69, 90, 91
monolayer, 3, 32, 33, 47
magnesium, viii, 31, 32, 44, 45, 50, 51, 53,
monomers, 53
64, 71, 77
morbidity, 50
magnetic particles, 75
morphogenesis, 44
majority, 75
morphology, vii, viii, ix, 29, 30, 31, 32, 33,
mapping, 56
34, 37, 38, 39, 40, 41, 42, 44, 45, 46, 47,
MAS, 56, 60, 63
55, 64, 69, 74, 75, 84, 86, 87
mass, vii, 1, 4, 9, 11, 19, 35, 59, 60, 63, 77
mortality, 50
mass spectrometry, 59
Moscow, 77
material surface, 26
motivation, 30
materials, vii, viii, 2, 18, 29, 30, 51
mRNA, 68
materials science, 30
matrix, 2, 44, 46
measurement(s), 3, 4, 5, 8, 13, 14, 15, 17, N
18, 19, 21, 22, 78
media, x, 52, 53, 58, 59, 61, 64, 74, 76, 79, Na+, 88
80, 81, 83, 84, 88, 92 NaCl, 76, 77
median, 5 nanocomposites, 69
medical, 51, 52 nanocrystals, 33, 38
medicine, 75, 81 nanofibers, 44
medium composition, 94 nanoparticles, 33, 37, 38, 44, 47
melting, 52 nanorods, 39, 45, 47
metabolism, 51 neutral, 3, 13, 14
metals, 83 NH2, 13
methacrylic acid, 41 nitrogen, 5, 63, 80, 82
methanol, 53, 61, 69 NMR, 56, 59, 60, 63
Mg2+, 32, 51, 53, 64, 66 novel materials, 51
microorganisms, 44 nuclear magnetic resonance, 56
microparticles, 52, 76, 93 nucleation, 18, 32, 33, 34, 38, 40, 41, 43
microscope, 5, 80 nuclei, 56, 57
microscopy, 47
microspheres, 38
microstructure(s), 5, 30, 34 O
mineralization, viii, 30, 43, 45, 47, 50, 64,
occlusion, 34
66, 67, 69
oligomers, 43, 59
mixing, ix, 14, 50, 52, 77
optical density, 78
models, ix, 74
optimization, 93
modifications, ix, 73
102 Index
ores, 93 polymer(s), ix, 2, 32, 33, 36, 38, 40, 41, 42,
organic polymers, 32 43, 44, 51, 74, 75, 77, 79, 90, 91
organic solvents, 51 polymer matrix, 2
organism, 30, 81, 91, 92 polymer molecule, 32, 41
organs, 50 polymorphism, 69
Osteopontin, 34 polypeptide, 31
osteoporosis, 67 polysaccharide(s), 30, 31, 33, 36, 44, 45
oviduct, 34 porosity, 9, 15, 30, 71
oxalate, 46 potassium, 76
oxygen, 37, 52, 64 precipitated calcium carbonate, vii, 1, 2, 5,
6, 7, 11, 12, 14, 15, 16, 17, 20, 21, 22, 45
precipitation, 3, 15, 31, 35, 36, 37, 38, 39,
P 40, 41, 42, 44, 46, 53, 62, 75, 77, 78, 83
preparation, vii, viii, ix, 17, 32, 49, 53, 61,
PAA, 36, 37, 38, 42, 47
63, 74, 76, 80, 91
parallel, 62
prevention, 91
pathology, 50
principles, 23
pathway, ix, 50
probability, 90
pepsin, 76
probiotics, 94
peristalsis, 81
proliferation, 51, 66, 67, 68, 75, 93
peroral administration, ix, 74
prophylactic, 91, 92
peroral proteins, vii
protection, 50, 90
Peroral Proteins Delivery Systems, v, 73
protective coating, ix, 74, 80
pH, viii, ix, 8, 12, 13, 14, 15, 21, 37, 38, 39,
protein constituent, 34
41, 42, 50, 52, 57, 70, 74, 76, 77, 80, 83,
protein synthesis, 68
84, 87, 89
proteinase, 94
pharmacokinetics, 90
proteins, vii, ix, 30, 31, 34, 35, 36, 46, 51,
phase transformation, 38, 46
74, 75, 76, 77, 78, 80, 81, 82, 83, 87
phosphate(s), ix, 2, 36, 51, 68, 74, 76, 77,
proteolytic enzyme, 76
80, 81, 82, 84, 85, 86, 87, 88, 89, 90, 92
protons, 12
photosensitizers, 94
pumps, 30
physical activity, 91
purification, 76
physicochemical properties, 4
purity, 13
physiology, 76, 81, 92
plants, 30
platelets, 40 R
PM, 69
Poland, 1, 5, 23, 27, 29, 47 radiation, 7
polar, 9, 13, 22, 31, 43 radius, 4, 9, 15, 80, 82
polarization, 56 Raman spectroscopy, 70
poly(allylamine hydrochloride), 39, 44 raw materials, 60
Poly(ethylene glycol), 40, 41 reactant, 40
polyacrylamide, 38, 39, 45, 47 reaction temperature, 38
polyacrylic acid, 36, 45 reaction time, 32, 34, 39, 40
polyamines, 40, 46 reagents, 26, 76
polyanion, ix, 74, 78, 88 receptor, 68
Index 103
recommendations, 71, 91 skeleton, 67

reconstruction, viii, 49, 51, 52, 64 sodium, 38, 45, 52, 69, 76, 77, 78, 80, 84
recovery, vii, 1, 11, 13, 19, 20 software, 7, 8, 80
regeneration, 70 sol-gel, 69
regenerative medicine, 70, 71 solubility, vii, viii, ix, 3, 13, 19, 21, 30, 50,
repulsion, 3, 38 52, 53, 57, 63
requirements, ix, 74 solution, viii, 2, 8, 11, 13, 14, 15, 16, 17, 18,
researchers, 3, 30 19, 21, 22, 32, 33, 34, 36, 37, 38, 39, 40,
resolution, 7, 36 41, 42, 50, 53, 57, 58, 59, 60, 64, 65, 66,
response, 69, 93 69, 71, 75, 77, 78, 79, 87, 92
restoration, 50 solvents, 61, 70, 78
restrictions, 4 species, 13, 14, 21, 32, 38, 59, 60, 63
riboflavin, 34 specific adsorption, 43
risk, 50, 67 Specord M, 78
risk factors, 67 spectroscopy, 80
room temperature, 13, 18, 38, 53, 78, 81 spindle, 30
roughness, 37 sponge, 44
routes, 53 St. Petersburg, 73, 77
Royal Society, 25 stability, vii, viii, 49, 91, 93
Russia, 77 stabilization, 61
stabilizers, 52
state, 3, 4
S structural modifications, viii, 49, 71
structure, vii, viii, ix, x, 7, 29, 31, 33, 34,
safety, 91, 92
36, 37, 39, 41, 44, 45, 49, 52, 55, 56, 57,
salt concentration, 44
69, 70, 74, 76, 83, 86, 87, 88, 89, 92
salts, 2, 12, 19, 21, 63, 64, 83, 87
styrene, 38, 42, 43, 45
saturation, 15
subsidy, 43
science, vii, ix, 25, 50
substitutes, 68
scientific papers, 4
substitution, 61
seeding, 66
substrate, 38, 39
self-assembly, 70
Sun, 26
sensing, 68
surface area, 5, 30, 71, 80
serum, 32, 47, 77, 80, 81, 82
surface energy, viii, 2, 18, 23, 52, 53
serum albumin, 32, 47, 77, 80, 81, 82
surface free energy, vii, 1, 3, 5, 9, 17, 18,
shape, 32, 33, 36, 37, 40, 63
19, 22
side effects, ix, 74
surface modification, viii, 2, 8, 23
signaling pathway, 68
surface properties, vii, 2, 23
silane, viii, 50, 53, 55, 56, 57, 70
surface tension, 4, 9, 18, 32
silanol groups, 59
surfactant(s), vii, 1, 2, 3, 5, 7, 8, 11, 12, 13,
silica, 51, 53, 56, 59, 70
15, 16, 17, 21, 22, 41
silicon, 36, 53, 56, 58, 59, 60, 61, 63, 64, 70
suspensions, 14
siloxane, viii, 49, 50, 52, 53, 56, 57, 59, 60,
swelling, 88, 89
61, 62, 63, 64, 66, 71
synthesis, 30, 32, 33, 36, 39, 40, 42, 43, 44,
simulation, 70, 76
52, 71, 75
SiO2, 51, 69
104 Index
uterus, 34
T
target, 75, 76, 90 V

TBS, 57, 61, 63, 64
techniques, vii, 1, 4, 77 van Oss equation, vii, 1, 9
TEM, 52, 55, 63 vapor, 4, 9, 39, 53
temperature, 18, 37, 44, 47, 69, 78, 79 vascular endothelial growth factor (VEGF),
TEOS, 60 51
terminals, 56 vaterite, v, vii, viii, ix, 30, 31, 33, 36, 37,
testing, 17, 23, 57 38, 39, 40, 41, 42, 43, 45, 46, 47, 49, 50,
tetraethoxysilane, 60 52, 53, 55, 56, 57, 59, 60, 61, 62, 63, 64,
TGF, 51 66, 69, 70, 71, 73, 75, 84, 91
thermodynamic stability, vii, viii, 49 velocity, 4
thin films, 39, 44, 46 vibration, 55, 79
tissue, 34, 50, 67, 68, 91 viscosity, 4, 9, 77
tissue engineering, 67, 68 vitamin D, 91
titanium, 94
transactions, 51
transformation(s), 32, 37, 44, 58, 70, 80, 83, W
91
transforming growth factor, 51 Washburn method, vii, 1, 4
transmission, 52 water, 2, 3, 8, 11, 12, 14, 15, 16, 17, 18, 19,
transmission electron microscopy, 52 21, 22, 32, 37, 38, 42, 46, 59, 77, 79
transport, 83 wettability, 3
treatment, 3, 37, 68, 71, 75, 79, 91 wetting, vii, 1, 3, 4, 15, 22
tumor, 75 workers, 32, 39, 40, 41
tumor cells, 75
tunable solubility, vii, ix, 50 X
turnover, 68
X-ray diffraction (XRD), 6, 7, 31, 32, 33,
39, 41, 42, 56, 58, 60, 62, 64, 88
U
X-ray diffraction data, 88
Ukraine, 77
ultrasound, 22, 79 Z
uniform, 23, 39
urea, 77, 80 zeta potential, vii, 1, 8, 11, 13, 15, 19, 21
USA, 76, 80, 95 zinc, 88, 94

Calcium Carbonate

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Calcium Carbonate

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BIOCHEMISTRY RESEARCH TRENDS

Additional books in this series can be found on Nova’s website

Additional e-books in this series can be found on Nova’s website

NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

Library of Congress Control Number: 2015946956

Published by Nova Science Publishers, Inc. † New York

the conventional crystallization pathway that includes a solution mixing

microscopy allows visualizing morpho-logical changes in carbonate cores

Izabela Polowczyk*, Anna Bastrzyk and Tomasz Koźlecki

and surface energy of non-modified calcium carbonate as well as after its

In another proposed adsorption model, a condensation theory (CT) [23],

and hydrophilicity, when characterized by the contact angle, should be

where:  - equilibrium contact angle,

There are many methods of contact angle measurements, but three

where h is the length wetted by the liquid (wetting perimeter), r capillary

measurements of contact angle of modified particles [17, 29]. However, there

MATERIALS AND METHODS

Figure 1. Particle size distribution of precipitated calcium carbonate.

A surface area of precipitated calcium carbonate was measured by the

calcium carbonates formed aggregated rhombohedral calcite crystals with

Figure 2. SEM image of precipitated calcium carbonate particles.

Figure 3. XRD pattern of precipitated calcium carbonate.

The crystallographic structure of calcium carbonates was determined by

Figure. 4. FTIR spectrum of precipitated calcium carbonate.

The XRD pattern (Figure 3) and FTIR spectrum (Figure 4) of precipitated

where; γl – surface tension of liquid,

The value of C must be determined from a slope of m2= f(t). It is possible

 l 1  cos    2  sLW  lLW  2  s l  2  s l (5)

 sAB  2  s s (6)

 s   sAB   sLW (7)

In Equation 7 the surface free energy is regarded as a sum of Lifshitz-van

Table 1. Properties of investigated liquids [46]

Liquid ρ η γl γlLW γl- γl+

Figure 5. Extended Hallimond tube.

The flotation tests of modified and unmodified calcium carbonate

RESULTS AND DISCUSSION

The adsorption isotherm of dodecylammonium hydrochloride onto

(CMC) and is called the critical surface aggregation concentration (CSAC)

Figure 6. Adsorption isotherm of dodecylammonium hydrochloride onto precipitated

Dissolution of Calcium Carbonate

Dissolution of calcium carbonate sustained in deionized water was

pH and collector concentration ranges [5]. Comprehension of the mechanism

The zeta potential values of non-modified calcium carbonate particles as

Figure 7. Zeta potential of CaCO3 modified with dodecylammonium hydrochloride in

When the calcium carbonate particles were dispersed in deionized water,

The electrokinetic measurements [19, 62] proved that the increasing

The use of the Washburn equation requires the reference liquid

Table 2. Calculated values of contact angle of precipitated calcium

Amount of C [m5] Contact angle

Surface Free Energy

Table 3. Surface free energy components of non-modified and surfactant

Based on the results shown in Table 3, the decrease in the value of

To confirm the obtained data, the flotation experiments of modified and

Figure 9. The kinetics of flotation of the precipitated calcium carbonate particles.

By comparing the data in Figure 9 and 10 it can be seen that higher

Figure 10. The flotation recovery of calcium carbonate particles.

amount of dodecylammonium hydrochloride, the zeta potential of calcite

On the other hand, the production of non-porous, uniform compressed

[32] Nguyen, A. V.; Schulze, H. J. Colloidal science of flotation; Marcel

Reviewed by Przemyslaw B. Kowalczuk, Ph.D.; Mineral Processing Division,

Zygmunt Sadowski, Anna Bastrzyk

THE ROLE OF ORGANIC BIOPOLYMERS IN CALCIUM

Depending on the concentration of Mg2+ irregular lumpish, discoid and