Crystallisation of Pharmaceutical Polymorphs

Literature Review

Umang Shah
May 2010

1. Introduction:

The success of the Human Genome Project has opened up exciting opportunities
for the treatment of disease. However, it is often not the genes themselves that are
the targets of potential drugs but the thousands of proteins encoded by these
genes1. To understand how proteins perform their various tasks, we need to know
the structure of these proteins. The current projects worldwide have set out to
determine the structures of all proteins having potential pharmaceutical
application, yet, in spite of investing considerable funds and effort, they have had
limited success so far. The most effective technique for determining protein
structure is X-ray crystallography, which requires high quality crystals of the
proteins. The main problem of crystallization lies in either getting no crystals at all
or, even more frustrating, getting crystals that are not of high enough quality to
enable structure determination2. Obtaining such crystals has always been the
bottleneck to structure determination and, with the advent of genomic projects,
this problem is becoming increasingly acute; there is an urgent need for new and
improved crystallization methods to enable progress 1,2.

The study of the nucleation and growth of protein crystals is one of the most
important and underdeveloped areas of structural biology. The ability to control the
nucleation phase is pivotal in the quest to overcome the bottleneck of protein
crystallization3. Conventional crystallization mechanism of the protein leads to the
growth of the protein crystals from supersaturated aqueous solutions.
Homogeneous nucleation takes place in the bulk of the solution, when the
supersaturation is high enough for the free-energy barrier to nucleus formation to
be overcome. Heterogeneous nucleation is caused by the presence in the solution
of solid material, which can be an already formed crystal of the molecule to be
crystallized (a ‘‘seed’’ crystal) or a different type of solid substance that has
nucleation-inducing properties (a ‘‘generous” nucleation, in what is called the
metastable zone of conditions4). Because growth in the metastable zone affords
kinetic advantages that often lead to larger and better-ordered crystals than those
grown at higher supersaturations, an aim of protein crystallizers is to be able to
induce heterogeneous nucleation4.

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Thus a search for nucleant, which could enhance the chances of any single trial
producing crystalline material, is ongoing since last two and half decade4-6, 8-10.
In 1988, Mc Pherson and Shlichta5 introduced the idea of controlling nucleation
using mineral substrates as epitaxial nucleants for protein crystallization. This
initiative has been pursued over the past 20 years by employing a variety of
substrates4, 6-9, but none have proved to be generally applicable as nucleants.

In 2001, Chayen et al proposed the idea of using a porous substrate containing

pore sizes that are comparable with the size of the protein molecules10. Such pores
may confine and concentrate the protein molecules and thereby encourage them to
form crystalline nuclei. Successful use of porous silicon in crystallizing several
proteins indicated that it is feasible to apply porous material to crystallization10,11.

In a multidisciplinary effort, involving biomedical sciences, material science,

engineering and even theoreticians, new materials that contained nanopores with
sizes of the order of magnitude of the sizes of protein molecules were designed and
tested 10,11. In addition to this various specifically engineered surfaces with desired
geometrical and chemical interaction are found to be promoting nucleation for
specific proteins 11-25.

Although large number of publications is reported on the crystallisation of various

proteins under different conditions, there is no one perfect method for achieving
protein crystallization and that to a great extent the field remains an empirical
science25. It is evident that the thorough understanding of crystallisation of the
molecular and macromolecular compounds needs to be developed.

This study targets to understand crystallisation behaviour of the organic molecule

in general and nucleation process in particular. It is also proposed to utilise the
understanding developed in engineering the crystals with controlled size, habit and
polymorphism.

The project is built upon the findings of anisotropic wettability for crystalline
pharmaceutical solids26 and the effects of the interfacial chemistry on the
crystallisation of model protein (Hen Egg White Lysozyme) and pharmaceutical
solids27. At nanometer length scale the morphology and wetting behaviour of the

2
surface is assumed to play a significant role in the crystallisation of the organic
molecules.

The goal of the project is to develop thorough understanding of the effect of the
surface topography and the interfacial chemistry on the crystallisation of molecular
and macromolecular compounds. Detailed understanding of the interfacial
properties will lead to the ability to engineer crystal habits and optimise
crystallisation process. The key objectives of this study are:
1. To evaluate the Classical Nucleation Theory and understand the role of the
interface in nucleation/ crystallisation processes by the coupling of
topography and chemistry;
2. To utilise this understanding in order to rationalize and ultimately control
the crystals size distribution, crystal aggregation and crystal shapes
obtained from a crystalliser; and
3. To exploit the role of surface properties, especially at the nano-scale level
which is thought to be the range of the critical nucleus size, for crystal
engineering of polymorphs.

These objectives will be achieved via preparation of nano-templates for nucleation

studies and crystal engineering of polymorphs. In order to achieve the objectives
two different substrates for preparation of nano-templates is selected, which are bio
active glasses and the anodised alumina oxide membrane.

Different porous materials, such as Sephadex beads of various sizes, carbon

powder, alumino-silicates [VPI-5]28, mesoporous molecular sieves [MCM-41]29, and
zeolites of various mesh sizes are tested for protein crystallisation and found to be
unsuccessful9,10. The bioactive glasses, which are chemically different from the
silica, are already used for the crystallisation of the various proteins, and due to
the wide variation in the pore sizes (2 – 5 nm) obtained during synthesis of the
material, 14 different proteins have been crystallised on the bioactive glass
substrates11. This research aims to prepare the bioactive glass substrate with
more uniform mesoporous pore size and functionalised surface for the purpose of
protein crystallisation.

Anodised Alumina Oxide (AAO) is widely used in the electronic industry for the
various applications32. It can be synthesised with ease in the laboratory, and the

3
pore diameter, pore structure and the aspect ratio of the pores can also be
controlled by controlling different parameters i.e. anodisation voltage, temperature,
time etc94-114. The AAO template is rarely used for the protein crystallisation, as it
is postulated to be a difficult to crystallisation on the aluminium oxide surface31.
Considering the controlled nano-porosity and the pore structure it is postulated to
be a potential candidate for the understanding role of interface in nucleation/
crystallisation processes by the coupling of topography and chemistry. Figure 1.1
represents the detailed flow chart showing approach/ methodology of the project.

Figure 1.1
Project concept flow diagram

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2. Synthesis of Nano-Templates:

The preparation of the bioactive glasses and the Anodised Alumina Oxide is
extensively studied since last three decades. The bioactive glasses have wide
variety of applications in the tissue growth engineering and in other biomedical
applications. The bioactive glasses have been widely used as fillers, spacers and
bone graft substitutes in orthopaedic and maxillofacial applications36, 40-43. Nano-
porous anodic aluminium oxide (AAO) membranes have been widely used as
templates in the synthesis of one-dimensional (1D) nanomaterials or quantum dot
(QD) arrays37. This low-cost membrane has attracted much interest for its wide
use and high efficiency. Various nanostructures have been created within the
membranes, such as solar cells, carbon nanotubes, catalysts, metal nano-wires
and hetero junctions38. Although literature refers different methods for synthesis
and controlling porosity of the bioactive glasses and AAO templates, it is still
challenging to controll the porosity in narrow range as per the requirement of the
application.

2.1 Synthesis of Bioactive glasses:

Bioactive glasses are a group of surface reactive glass-ceramics. Bioglass glasses
was developed by Professor Larry Hench in late 1960s at University of Florida,
using melt derived synthesis method and further developed by the him and his
team at the Imperial College London. Originally, bioactive glasses were prepared
through melting of related oxides, at temperatures in the range of 1100–1300 oC39.
The glass compositions were mainly based on silica, calcium, phosphate and
sodium.

The sol-gel route enabled production of glasses which are specifically designed for
the tissue growth engineering, with enhanced bioactivity, compared to melt-derived
glasses with same composition, because of the highly porous nature of this
material40,41. Studies have shown that sol-gel glasses prepared with in three (SiO2-
CaO-P2O5) and two (SiO2-CaO) component systems, and even pure silica, can
quickly develop an apatite layer in contact with simulated body fluids42-44. Sol-gel
has the advantages of low processing temperatures and ease of control of textural
properties.

5
 2.1.1 Sol-Gel Route for synthesis:

Sol-gel Method:
The sol-gel process, also known as chemical solution deposition, is a wet-chemical
technique widely used in the fields of materials science and ceramic engineering.
Such methods are used primarily for the synthesis of materials (typically a metal
oxide) starting from a chemical solution which acts as the precursor for an
integrated network (or gel) of either discrete particles or network polymers. Typical
precursors are metal alkoxides and metal chlorides, which undergo various forms
of hydrolysis and polycondensation reactions. Thus, the sol evolves towards the
formation of a gel-like diphasic system containing both a liquid phase and solid
phase whose morphologies range from discrete particles to continuous polymer
networks45, 46.

A well studied alkoxide is silicon Tetra Ethyl Ortho Silicate (TEOS). The chemical
formula for TEOS is given by: Si(OC2H5)4, or Si(OR)4 where the alkyl group R = C2H5.
Alkoxides are ideal chemical precursors for sol-gel synthesis because they react
readily with water47. The reaction is called hydrolysis, because a hydroxyl ion
becomes attached to the silicon atom as follows:
Si(OR)4 + H2O → HO-Si(OR)3 + R-OH
Depending on the amount of water and catalyst present, hydrolysis may proceed to
completion, so that all of the -OR groups are replaced by -OH groups, as follows:
Si(OR)4 + 4 H2O → Si(OH)4 + 4 R-OH
Any intermediate species [(OR)2–Si-(OH)2] or [(OR)3–Si-(OH)] would be considered
the result of partial hydrolysis. In addition, two partially hydrolyzed molecules can
link together in a condensation reaction to form a siloxane [Si–O–Si] bond:
(OR)3–Si-OH + HO–Si-(OR)3 → [(OR)3Si–O–Si(OR)3] + H-O-H
or
(OR)3–Si-OR + HO–Si-(OR)3 → [(OR)3Si–O–Si(OR)3] + R-OH
Thus, polymerization is associated with the formation of a 1, 2, or 3- dimensional
network of siloxane [Si–O–Si] bonds accompanied by the production of water and
alcohol species.

By definition, condensation liberates a small molecule, such as water or alcohol.

This type of reaction can continue to build larger and larger silicon-containing
molecules by the process of polymerization.

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Polymerization of silicon alkoxide, for instance, can lead to complex branching of
the polymer, because a fully hydrolyzed monomer Si(OH)4 is tetrafunctional (can
branch or bond in 4 different directions). The mechanisms of hydrolysis and
condensation, and the factors that bias the structure toward linear or branched
structures are the most critical issues of sol-gel science and technology48-51.

Synthesis of sol-gel glass monoliths:

Several methods have been well described in the literature for synthesis of the
monoliths of the bio active glass using the sol-gel method. The effect of reaction
conditions, i.e. reactant concentration, amount of catalyst, reaction time, aging
time, drying conditions etc, have been extensively reported in the literature45, 52-55.

Commonly, a one-step method is used to make sol– gel glasses 45, which involve the
simultaneous hydrolysis and polycondensation of alkoxide precursors. The alkoxide
precursor commonly used is Tetra Ethyl ethOxy Silane (TEOS); however,
alternatives can be used, such as Tetra MethOxy Silane (TMOS), or Tetran-PropOxy
Silane (TPOS). Water is the reactant which leads to the hydrolysis of the alkoxide,
so the water to alkoxide ratio is very important in the one step sol-gel method45.

A two-step method has been devised to produce sol–gel materials in a

comparatively shorter time 52. In the two-step method, first hydrolysis of TEOS is
carried out under either acidic or basic conditions at specific water to alkoxide ratio.
Then water and alcohol is added to initiate polycondensation reaction 53. There are
also some one-step sol–gel fabrication methods available where alcohol is not used
with an higher molar ratio of water to alkoxide 52,53. The pH of the acid (HNO3)
used is 0.5 and the gelation time for this system is ∼50h at 40 ◦C and ageing
requires 4 days. This method works well for powder production. However, when
monoliths are made, the process is very sensitive to drying conditions and
monoliths greater than 10 mm diameter and 2 mm in height often crack. The
percentage yield of small monoliths is also low and variable. Recently, a more rapid
method of sol–gel fabrication has been developed 53 and has been modified for the
fabrication of dye doped sol–gel materials 54. This was a direct hydrolysis reaction of
TEOS with water and alcohol at over 70◦C for bio active glass powder production.
Alcohol and TEOS are not used in scaffold production. Mohammad, A. et al.

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reported the modified fast process to produce sol–gel monoliths using TMOS and
TEOS without alcohol and with high water to alkoxide ratios55.

2.1.2 Pore Formation Mechanism:

Synthesis of the bio-active glass monolith with controlled and uniform pore size
distribution is the prima-facie objective of the project. In order to tune the porosity
as per the requirement of the application, it is essential to understand the
mechanism of pore formation. Sol–gel derived glasses have a textured porosity, the
size distribution of which is dependent on the processing conditions such as
precursor type, pH, reactant concentration and temperature55.

The pores are a result of the condensation (gelation) reaction, which leaves an
aqueous by product. Aqueous droplets prevent the formation of a complete silica
network and leave –OH groups as effective network modifiers. As the droplets
evaporate small pores are left54, 55.

Mohammad, A. et al. Reported that the use of very low acid concentration results in
changes in texture of the sol–gel samples produced through fast method. By
heating the sol during hydrolysis and condensation, some excess water and
polycondensation by product was evaporated before full gelation and before drying.
The early removal of these liquids meant that there was less to remove from the
samples during drying and therefore lower capillary pressures45. This not only
enabled the production of large monoliths, but the evaporation process also
reduced the pore size, i.e. it produced narrow size distribution. Therefore sol–gel
samples of the same compositions can be prepared as monoliths using the same
precursors but with different nanopore size distributions55. Such prepared
samples results in the pores, which are microporous in nature.

The natural pore formation mechanism results in the mean pore diameter from 2 –
5 nm. Higher mean pore diameter, achieved via natural pore formation is not
reported in the referred literature.

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Controlling porosity with Sacrificial Templates:

Block Co-polymer Surfactants:

Bioactive glass ceramics with ordered mesoporous pore structure have attracted
more attention considering their emerging application in the field of catalyst,
adsorptions and other biomedical applications56-58. Ordered mesoporous materials
with very large pores are in urgent demand for applications in the encapsulation
and separation of proteins where biomolecules with large molecular weights are
involved. It is challenging to have the ordered mesoporous structure with the
larger pore sizes. The concept of controlling porosity of the silica based glasses
using the sacrificial templates is widely studied.

Hexagonal mesoporous bio active ceramic monoliths, fabricated with different non-
ionic surfactants i.e. Brij 56 (C16EO10), Pluronic P123 (EO20-PO70-EO20), Pluronic
F127 (EO106PO70EO106), etc have been reported widely in the literature 59-62.

It is very important to understand the phenomenon of the orderd mesoporous pore

formation in order to synthesise the bioactive glass monoliths with tuned porosity.
Upon introduction of surfactants (or any surface active materials) into the sol they
will initially partition into the interface, reducing the system free energy by
removing the hydrophobic parts of the surfactant from contacts with water.
Subsequently, when the surface coverage by the surfactants increases and the
surface free energy (surface tension) has decreased, the surfactants start
aggregating into micelles, thus again decreasing the system free energy by
decreasing the contact area of hydrophobic parts of the surfactant with water.
Upon reaching Critical Micelle Concentration (CMC), any further addition of
surfactants will just increase the number of micelles. Pre-formation of micelles is
crucial to produce mesoporous material from a dilute sol, and the cooperative
assembly of composite micelles leads to ordered mesostructures70.

Direct templating by liquid crystal phases (copolymer surfactants) has been

recognized as a method for controlling morphology and preparing large monolithic
mesophases63,64, but the method used so far is in general restricted to only a few
surfactant/water binary systems63. While the pore size can be varied in some
cases, the long-range pore ordering is usually lost64,65.

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Method for the synthesis of periodic mesoporous silica by direct liquid crystal
templating in multi-component surfactant and cosurfactant systems is reported by
Feng P. et al.64. This method direct liquid crystal templating to more complex
ternary (surfactant/cosurfactant/water) and microemulsion-type quaternary
(surfactant/cosurfactant/water/oil) systems and thus leads to a greatly enhanced
control over surfactant phase domains, mesoporous structural diversity, and
macroscale morphology64.

Various synthesis strategies to meet the challenge of ordered larger pore diameter
often include the different choice of templates, the addition of swelling agents such
as 1,3,5-trimethylbenzene (TMB), and the utilization of high temperature
processing66,67. However, all these strategies failed to obtain ordered mesoporous
materials with pore sizes larger than 12 nm66. Fan, J. et. al. demonstrated a novel
low-temperature pathway to synthesize highly ordered face-centered cubic (fcc)
mesoporous silica structure with the largest pore sizes (up to 27 nm) and unit cells
(up to 44 nm). Another significant feature of synthesis strategy reported by Fan, J.
et. al is that the mesostructures remain highly ordered even when the pore sizes
and cell parameters have been greatly enlarged67.

Other Polymeric materials as Sacrificial Templates:

Many techniques have been used to produce porous bioactive ceramics, but the
templates discussed in this section don’t form the ordered pore structures,
although the interconnectivity of the pores is reported. Relatively simple method is
the use of polymer nano/ micro beads or other organic agents (porogens) that can
be burnt off to leave pores. Porosity can be increased by adding fillers to the
powder and the wetting solution, for example sucrose72, gelatine73, PEG micro/
nano spheres78 or PMMA microbeads74, which burn out on sintering. In polymer
foam replication, open celled polyurethane foams can be immersed in ceramic
slurries under vacuum to allow the slurry to penetrate into the pores of the foam.
The organic components are burnt off at 250 oC and the ceramic foams sintered at
1350 oC, producing a scaffold with interconnected pore diameters of up to 300µm75.

Highly porous ceramics can be produced by foaming of particulate suspensions or

colloidal sols to obtain pores in the size range 20µm up to 1–2 mm. The

10
incorporation of bubbles is achieved by injection of gases through the fluid medium,
mechanical agitation, blowing agents, and evaporation of compounds or by
evolution of gas by in situ chemical reaction76. A surfactant is generally used to
stabilise bubbles formed in the liquid phase by reducing the surface tension of the
gas/ liquid interfaces. A successful modification of the direct foaming method is the
gel-casting method77. Monomers are added to the ceramic suspension, in situ
polymerisation is carried out and cross-linking occurs, forming a 3D polymeric
network (gel) which produces strong green bodies. Porous samples are then
sintered to provide mechanical strength and to burn out the organic components.

The porous scaffolds synthesised using the different sacrificial templates do not
indicate narrow pore size distribution. The pore size achieved with the sacrificial
templates, discussed here are large, more detailed study is required to tune the
porosity in narrow pore size distribution with the sacrificial templates at the nano-
meter length scale.

2.1.3 Effect of aging and drying on pore formation:

During the process of aging the poly-condensation reaction proceeds further and
the liquid by product is formed, which is sequentially removed during the drying45,
so it is very important to understand the effect of ageing and drying in order to
control the porosity at the nanometer length scale. Two different aging methods
have been discussed by Mohammad, A. et al., utilizing a fast gelation process; the
pore size of sol–gel derived glasses can be controlled and large monoliths
produced55. Hydrothermal aging treatment discussed by Jia-Heng Lei et al. ,
promote the condensation and dehydration reaction of silanol groups, with the
result that cross-linking degree, the flaws and moisture content in gels are reduced
notably. The process is found promising for the integrity of gels in the following
drying process78. For the natural pore formation mechanism, the process of aging
and drying is postulated to have considerable effect. The effect of drying on the
formation of pore in the sol-gel process is not a well studied subject. .

2.1.4 Sintering and crystallisation of bioactive glass ceramic:

The first effort to study the kinetics of crystallisation of the bioactive glass is
reported by Rizkalla, A. S. et al.79. After this number different studies on the

11
crystallisation behaviour and the effect of sintering on different bioactive ceramics
have been presented. Studies referred to different optimal conditions for surface
and bulk crystallisation of the bioactive ceramic monoliths, and different kinetic
parameter of the crystallisations80-86. The focus of these studies is to consider the
effect of crystallisation of the bioactive glass substrate on the growth of
hydroxyapetaite layer. It appeared from the literature referred is that the effect of
crystallisation of the bioactive glass on the porosity is not a well understood area.

2.2 Synthesis of Anodised Alumina Oxide:

In recent years, the fabrication of one-dimensional (1D) nanostructures has

attracted ever-increasing interest for its applications in many field, including
magnetics, self-assembly, electronics, biology, catalysis, and optics. Among all the
synthesis techniques, the template method for 1D nanostructures synthesis has
become a very simple yet powerful process, with the advantages of low cost, high
throughput, high volume, and ease of production. Anodic Aluminium Oxide (AAO)
template is a well-established nano-technique and has become a method of choice
for scientists interested to synthesise and characterizes small quantities of multi-
segmented nanostructures87-89.

In the past 80 years, many methods have been developed to prepare or improve
porous alumina membrane. Thompson gave a detailed review on the anodic
alumina film formation from aluminum90. Masuda and co-workers advocated a
two-step oxidation technology and process to prepare highly ordered pore arrays91,
92. Some special procedures for creating particular shapes and structures such as
hexagonally ordered nanopore arrays with high aspect ratios93, 94 tubular anodic
aluminium oxide95,96, and Porous Anodised Alumina films having bone-shaped
nanochannels97 were developed for different application.

Recent advances in synthesis, properties, and applications of multi-segmented 1D

nanostructures prepared by template methods were reviewed by Mirkin et al87.
Recent effort to achieve highly ordered porous structures reported synthesis of
Alumina Oxides membranes on Si and Ti substrates 102. A. Yin et al. show that
highly ordered AAO templates can be grown on Si substrates with either thick or
thin Al films using two complementary techniques: self-organised nanopore arrays
formation by direct anodisation of aluminium on silicon and a second-order

12
template-based anodisation process99.

2.2.1 Pore formation mechanism:

The structure of self-ordered AAO membranes is formed during the transformation

of aluminium into alumina by volume expansion and mechanical driving forces.
The maximum volume expansion ratio (thickness of AAO layer formed / thickness
of original aluminium) corresponds to the largest mechanical driving force; under
the condition, pores are unable to form. Lowering the ratio below a critical value, a
non-ordered porous alumina membrane forms due to a weak mechanical driving
force98.

Figure: 2.1 Schematic diagram of

the pore-formation mechanism for
fabrication of AAO template.

Regime 1: formation of barrier oxide

on the entire area; Regime 2: local
field distributions caused by surface
fluctuations; Regime 3: creation of
pores by field-enhanced or/and
temperature- enhanced dissolution;
Regime 4: stable pore growth98

The mechanism by which pores grow is still in debate, but pore formation models
have been developed 104-108. A generalised possible formation process of the
nanopores in porous anodised alumina oxide is described herewith.
At the initial state of the anodisation process, a barrier layer is formed, which leads
to an exponential decay of the current. Then, a thinning process of the barrier
layer occurs until a stable thickness of the barrier layer is reached. This leads to an
increase in current. The stable thickness is determined by the applied voltage 111-113.

Simultaneously, the pores are initiated at random positions on the convex-curved

surface due to the enhanced electric field at the convex-curved surfaces 115. After

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pore initiation, nanopores grow differently, depending on the current densities.
During this process, nanopores grow by the equilibrium of two reactions 112:

One is the reaction of the alumina formation at the metal/oxide interface and the
other is that of the alumina dissolution at the oxide/electrolyte interface 111-113.

Two reactions can be simply expressed as alumina formation at metal/oxide

interface:
2Al(m) + 3H2O → 2Al 3+ + 6e−+3O2− → Al O (oxide) + 6H (aq) + 6e
2 3 + −

alumina dissolution at oxide/electrolyte interface:

Al2O3(oxide) + 6H+(aq) → 2Al 3+(aq) + 3H2O
At the condition of a moderate voltage that cause a moderate and stable current,
both the reactions may uniformly occur due to the stable current density. These
uniform reactions may lead to the formation of uniform pores (pore homogenization
process). During this process, the stress, such as the repulsive force, between the
pores caused by the current makes the pore arrangement become homogeneous
(pore arrangement process) 114. As a result, hexagonally ordered arrays of pores with
uniform diameters can be formed.

2.2.2 Effect of process parameters on the pore arrangement and diameter:

Controllable variation of the anodisation conditions gives rise to the possibility of

tailoring the size and density of the template pores over a wide range. AAO
templates are available in high porosity (108 – 1011 pores/cm2) and the pore size
can be controlled from 10 to 250 nm 92, 93. Effect of the process parameters, i.e.
anodic oxidation voltage, temperature, concentration of electrolyte, purity of
aluminium, and electrochemical conditions are discussed in the literature 94, 102, 112-

114.

By varying the anodisation voltage of the aluminium foil or film with a specific
electrolyte such as phosphoric acid, sulphuric acid, and oxalic acid, the density
and diameter of the nanopores can be readily controlled88-90.

It is reported that pore diameter and interpore spacing is linearly proportional to

the applied voltage 109. The self-ordering of pores requires a porosity of 10% which
is independent of the specific anodisation conditions 106,110. The pore opening of
the bottom layer or barrier layer removal is also considered an important step
towards making a through-hole porous anodic alumina membrane121.

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Anodisation Potential and Current Density:

The effect of anodizing voltages, and the corresponding current densities on the
pore morphologies have been investigated by different authors in past four
decades112-115. Porous anodised alumina oxide are synthesised under different
anodization voltages in different electrolyte solutions. It can be observed that
optimum anodisation volage is different for different electrolyte solutions, at which
the ordered pore arrangement is observed for constant current densities119. The
current density is having direct relationship with the process of pore formation, it
increases rapidly during the formation of the barrier layer and then stabilised,
which leads to the ordered pore structure113.

Interpore distance increased with respect to the applied anodisation voltage in

direct proportion. While the applied voltage was kept constant, the interpore
distance remained at constant value even when the concentration of different
electrolyte is changed94,114.

Figure 2.2 Interpore distance

d in self-organized porous
alumina vs anodic voltage Ua
for sulphuric, oxalic, and
phosphoric acid solutions94.

Anodisation time:

Thickness of the membrane is directly proportional to the anodisation time. The

proportionality constant is different for the different electrolyte solution118. This
suggests that the membrane thickness is dictated by dynamic equilibrium of
oxidation and chemical dissolution. The dynamic equilibrium of the oxide
formation (highly dependent on the acid concentration and availability of oxygen

15
ions) and dissolution (relatively constant due to its field-assistant nature) causes a
characteristic limiting thickness113, 115.

Figure 2.3
Variation of AAO
tubular membrane
thickness with respect
to anodization time
(Electrolyte used is
sulphuric acid with 2%,
10% and 20% (v/v))114.

Ionic strength of electrolyte solution:

Ionic strength and concentration of the electrolyte solution affects the pore size. If
sulphuric acid is used as the electrolyte solution the pore size obtained is smaller
than the same obtained with the oxalic acid as electrolyte. Increase in the
concentration of electrolyte solution results in the smaller pore size. The effect of
the ionic strength of the electrolyte solution on the pore diameter is not fully
understood. The pore formation depends on the oxide layer formation and
dissolution during the anodisation process. It is suggested that the dissolution and
growth of the oxide layer depends on the concentration of the electrolyte solution.
Higher electrolyte concentration increases the oxide layer formation and dissolution
and thus smaller pores for higher electrolyte concentration, although literature
fails to explain the detailed mechanism113, 114.

Temperature of electrolyte solution

Self ordering mechanism of the pores is greatly influenced by the temperature of

the electrolyte solution during the anodisation process. Although the detailed
mechanism for explaining the dependence of the ordering on the temperature of the
electrolyte is not quite simple, two different factors may be related to the optimal

16
conditions of the samples94, 114. Increasing the temperature in the first anodization
step increases the growth in the longitudinal direction, which in turn increases the
self-ordering configuration 91 and raising the temperature increases the growth rate
and reduces the interaction time for the self-organization process120. These two
factors may together create a situation in which the cells grow with the least strain
resulting into highly ordered structures.

2.2.3 Synthesis of silica nanotubes/ nano test tubes using AAO

The nanotubes are ideal vehicles for delivery of drugs, DNA, proteins, or other
bio molecules122. Tubular structures are ideally suited for such applications
because they are hollow. Increasing applications of the nanotubes attracted
attention of reaserchers in recent past. Although the carbon nanotubes, peptide
nanotubes and self assembled lipid nano tubes are in application, due to their high
cost and other constraints requirement for the less expensive and easy to tune
option is there123. Martin and co-workers have first reported the synthesis of silica
nano test tubes using the anodised alumina oxide templates. Using such
templates the diameter and the aspect ratio of the nano tubes/ test tubes can be
controlled as per the requirement, which lead the increased demand of the same for
the targeted drug delivery applications122.

Figure 2.4 Schematic illustration of template synthesis of silica nano

test tubes using AAO templates124.

Template synthesis is especially useful to make nanotubes because the cylindrical

wall of nanopores can be a starting point of chemical reactions. This is to date the

17
easiest way to form a tubular structure whereby nearly any material can be
integrated into nanotubes. First, thin layers of nano materials are formed onto the
cylindrical wall of nanopores of an alumina membrane including surfaces of
membranes through the sol-gel chemistry, and then the top layers of one are
removed by mechanical polishing. It is non expensive and easy method to
synthesise the nanostructure with tuned porosity and aspect ratio122-125.

3. Surface functionalisation of templates:

Surface functionalisation introduces chemical functional groups to a surface. This

way, materials with functional groups on their surfaces can be designed from
substrates with standard bulk material properties. Different chemical functional
groups grafted on the template substrate mainly depend on the surface reactivity.
Generally the surfaces of inorganic materials are functionalized with polymer
chains either chemically (through covalent bonding) or physically (by
physisorption)126.

3.1 Surface functionalisation of Bioactive glass surfaces:

Bio active glasses are silica based material. The surface functionalisation of the
silica based materials is comprehensively reported in the literature over past three
decades126-135. Bio active glasses are functionalised using different protocols and
different functional groups for tissue growth engineering130.
The chemical properties of the silica surface are mainly determined by the various
silanol and siloxane groups that are present on the external as well as the internal
structure. The hydroxyl groups on the surface of silica particles can be easily
tailored with organic compounds or polymers. Silanol groups can be easily
functionalised by different chemical procedures. The most convenient technique
for silica surface functionalization is the use of the reaction of silanol groups with
suitable silane reagents.
A range of different procedures have been described for the silanization of silica
materials, including elevated and room-temperature organic phase aqueous phase,
vapour phase, and chemical vapour deposition128-134.
Mechanism of surface silanisation:
In theory, the silanization of surfaces such as silica with mono-, di-, or
trialkoxysilanes is relatively simple; hydrolysis of the alkoxy groups yields hydroxyl

18
groups that can covalently interact with the silanol surface, the number of bonding
interactions with the surface being the same as the number of hydrolysable
alkoxysilanes. The silane can be cleaved and the silanol surface regenerated by the
action of strong base on the generated hydroxyl linkages between silane and
surface. However, in reality the situation is more complicated and the exact
modification method is still not fully understood. It is thought that the initial step
is indeed the rapid hydrolysis of the alkoxy groups to liberate silanols and release
alcohols (i)131. The silanol groups then condense with the surface residues to form
siloxane linkages. In the case of trialkoxysilanes the presence of three silanol
residues in the hydrolysis product can lead to the possibility of multiple surface
attachments. The work of Kallury et al. with (3-aminopropyl)triethoxysilane
(3APTES)135 has shown that providing the number of attachment sites on the
surface is not limiting, the preferred conformation of the reaction product of
monomeric 3-APTES on a glass or metal oxide has two sites of attachment and the
third silanol remains free (ii). This leads to two possible effects. First, with
3-APTES, the activity of the amine group is considerably lowered due to internal
self-cyclization and ionization produced by intramolecular hydrogen bonds128.
This lowered reactivity occurs even after thermal curing, which cross-links the
remaining silanol groups (iii). The second effect is common to all trialkoxysilanes,
namely, that polymerization can occur at the free silanol group on the surface or in
solution prior to condensation with the solid substrate.

Figure 3.1
Silanization of Glass with
3-MPTS in
(i) The reactive groups of
physisorbed silane are
hydrolyzed by the surface
water on a hydrated silanol
(glass) surface, followed by
condensation
(ii) Which leaves the silane
covalently bound to the
oxide surface, and
(iii) Thermal curing of the
film, which, in cross-
linking the free silanol
groups, reduces the effect
of hydrolysis of one or
more of the siloxane
linkages to the surface134.

19
 3.2 Surface functionalisation of AAO membrane surfaces:

For functioanlisation of any surfaces depends on the surface reactivity. The

surfaces of the Anodised Alumina Oxide are inert in nature. It is interesting to graft
different functional groups on the AAO membrane surfaces, as the AAO templates
with altered polarity has increasing number of application in the different fields
including chemical sensors, energy conversion and drug delivery160. Lee and co-
workers reported the protocol for direct silanisation of the AAO templates162. In
order to silanise the anodised alumina oxides the templates are coated with the fine
layer of silica, which is silanised with relative ease.

Mechanism of surface silanisation:

The mechanism of grafting the silanes on the surface is explained by Leger et al161.
In order to expose the hydroxyl groups on the surface the surface of the AAO is first
cleaned by heating moderately high temperature in air, boiling in 30% hydrogen
peroxide followed by soaking in boiling distilled water and drying at moderately
high temperature. Then the membranes are silanised with different silanes.

Octadecylsilanes were used in this study. For such long chain aliphatics the
intensity of the C-H absorption band is relatively high and thus the detection of
successful grafting becomes easier. When dimethyl octadecyl chlorosilane was
used as a reactant no C-H bonds was detected on the alumina surfaces and so no
reaction takes place between the chlorosilane and the hydroxyl group of the
alumina. This lack of reactivity could be due to the steric effect of the octadecyl
chain and the two methyl groups limiting the access of the Si-C1 bond to hydroxyl
groups of the alumina surface. This reaction was expected to produce a permanent
modification because the chemical grafting is by a very stable Si-O bond.

The precise structure of the bonding on the surface is still unresolved, although the
reaction mechanism is clear167-168.

4.0 Characterisation of the templates:

Characterisation of the templates is also a very important aspect of the synthesis

process. Texture analysis of the surface allows synthesising the templates with

20
tuned porosity and helping in optimisation of the process parameters and synthesis
methods. Characterisation methods are also useful in understanding the effect of
various synthesis parameters on the porosity, pore structure and surface chemistry
of the templates.

4.1 Porosity and specific surface area measurement:

Several methods have been developed for determining the surface area and the pore
size distribution in porous systems. The operations of these different methods
generally are based on different physical principles. It should be expected,
therefore, that they effectively represent probes of different sizes and, hence, that
the pore size ranges in which they are most reliable are necessarily different. Figure
4.1 compares the ranges of validity of a selection of methods commonly used for
pore characterization136. Mikhail et al. compared various methods of the porosity
measurements considering the scope and limitation of the methods137.

Figure 4.1 Different methods for measurement of porosity and

applicable range.136.

21
Table 4.1 Comparison of the porosity measurements considering the scope and
limitation of the methods137

Sr Method Range of Assumption Advantages Disadvantages

No pore size
1 N2 Adsorption d < 50 nm Correlation with Complicated
BET surface area evaluation
program, non-
applicability of
theory in
microporous
region.
2 Mercury 4 nm < d ≤ Much in use, Danger of
comparable data, breaking pore
Intrusion 60000 nm
extended range of walls, large
pore sizes pores are filled
Cylindrical at atmospheric
Pores pressure
3 Air/ He A few Very short time No pore size
method, good distribution
penetration nanometers
reproducibility, no
, depending influence on
sample
upon gas
applied
4 Xylene/ water d > 100 nm Simple method, No pore size
short time distribution,
impregnation
specimens with
small diameters
5 Small angle X 0.5 nm ≤ d Time required for Special facilities
measurement is are required in
ray scattering ≥ 100 nm
very less XRD machine
(SAXS)
6 Small angle 0.5 nm ≤ d Time required for Special facilities
measurement is are required,
neutron ≥ 100 nm
very less limited to
scattering nuclear centres
7 Quantitative d ≥ 20 nm Spherical Large pores can Limitation to
be measured, large pores,
image analysis Pores
optical checking is significant
possible, also errors in coarse
microporosity can grain materials,
be measured by area
analysis TEM information, for
spatial
information
large
mathematical
effort

22
Specific Surface Area and Pore Analysis by Gas Adsorption:
Pore analysis:
Gas adsorption measurements are widely used for the characterization of a variety
of porous solids. Particular importance is the application of physisorption (physical
adsorption) for the determination of the surface area and pore size distribution.
Nitrogen (at 77 K) is the recommended adsorptive for determining the surface area
and mesopore size distribution, but it is necessary to employ a range of probe
molecules to obtain a reliable assessment of the micropore size distribution. An
alternative technique to gas adsorption is mercury porosimetry used for macropore
size analysis. For operational reasons, krypton adsorption (at 77 K) is usually
adopted for the determination of relatively low specific surface areas ( < 2 m2g-1),
but this technique cannot be employed for the study of porosity138.

Volumetric and gravimetric methods have been devised for determining the amount
of gas adsorbed. Volumetric methods are generally employed for measuring
nitrogen or krypton isotherms at cryogenic temperatures, 77 K139-142. The isotherm
is usually constructed point-by-point by the admission and withdrawal of known
amounts of gas, with adequate time allowed for equilibration at each point. For the
evaluation of the measured isotherm, it is necessary to chose among several
evaluation procedures that differ according to the qualitative nature of the isotherm.
Six classes of isotherms, are usually distinguished 143.

Figure 4.2
Types of
Physorption
Isotherms143.

23
Type I isotherms are observed for microporous solids having relatively small
external surfaces. Type II isotherm is the normal form obtained with a non-porous
or macroporous adsorbent. Type III isotherms are convex to the p/p0 axis over its
entire range. This type is not common. Type IV isotherms are distinguished by two
characteristic features. This type of isotherm exhibits a hysteresis loop, which is
associated with capillary condensation taking place in mesopores, and has a
limiting uptake over a range of high p/p0. Type V isotherms are uncommon. They
are related to the Type III isotherm in that the adsorbent-adsorbate interaction is
weak, and they also exhibit hysteresis as in Type IV. Type VI isotherm exhibits
stepwise multilayer adsorption on a uniform nonporous surface142-144.

Determination of surface area:

It is standard practice to apply the Brunauer-Emmett-Teller (BET) method to derive

the surface area from physisorption isotherm data145. A concern that must be
recognized is that the theory underlying the BET method is based on an
oversimplified extension of the Langmuir mechanism to multilayer adsorption.
Sing et al. reported the that the constant (C) used in the theory is having a very
low value for the microporosity, which is associated with an appreciable overlap of
monolayer and multilayer adsorption; for that case, the application of the BET
analysis leads to doubtful value143.

Microporosity:
Barett et al. reported the method for calualtion of the pore volume and pore area
distribution directly from the desorption isotherm of porous substances146.
According to the IUPAC Recommendations 143, micropores are defined as pores with
internal width less than 2 nm. They are characterized by the micropore volume and
the micropore distribution. Different models are proposed for calculation or the
porosity in the microporous material, although the model applicable for all different
pore types and structure is still in search. Major model are based on the sorption
methods. The field-proven methods of gas sorption are of special interest with
respect to the use of microporous material as specific sorbents, molecular sieves,
and carriers for catalysts.

Dubinin - Radushkevich method originally developed to investigate the

microporosity of activated carbons 147 can be used for microporous material similar

24
pore type. Adsorption isotherms of pure gases on microporous sorbents can be
described by means of Polanyi's potential theory148. Each gas/sorbent system is
characterized by an adsorption potential E which is influenced in particular by the
chemical properties of the sorbent. The pore volume filled at a given relative
pressure p/p0 as a part of the total micropore volume is a function of the
adsorption potential.

According to Dubinin, the adsorption potential equals the work required to bring
an adsorbed molecule into the gas phase. The empirical formula for calculation of
isotherm is suggested by Dublin et al.149
Horvath and Kawazoe (HK) described a semi-empirical, analytical method for the
calculation of effective pore size distributions from nitrogen adsorption isotherms in
microporous materials. The original HK approach considers a fluid (nitrogen)
confined to a slit-pore, such as can be found in some carbon molecular sieves and
active carbons150.

Two important methods have been developed to describe the sorption and phase
behavior of inhomogeneous fluids confined to porous materials, computer
simulation and density functional theory (DFT). DFT is a computational method
that treats the intrinsic free energy of a system as a functional of the particle
distribution function151. Seaton et al. were the first to apply the DFT to the
calculation of the pore size distribution (PSD) in both the meso-and micropore
range. Although the local DFT provides qualitatively reasonable description of
adsorption in the pores, it is quantitatively inaccurate especially in the range of
small pores (i.e., micropores)152.

A significant improvement in accuracy was obtained with the Non-Local

Density Functional Theory (NLDFT) which was first used for the pore size analysis
of microporous carbons 153. Since then, the NLDFT has been applied frequently to
the pore size analysis of microporous and mesoporous materials 154-156.

4.2 Pore arrangement and pore size measurement for the Anodised Alumina
Oxide templates:

Microscopy has been used to determine the pore structure and pore size. It is used
widely to understand the effect of various parameters on the pore formation

25
mechanism. Various techniques such as atomic force microscopy (AFM), scanning
electron microscopy (SEM), have proven useful. Atomic force microscopy is a very
useful method in understanding the pore formation mechanism118.

4.3 Characterisation of functionalised surfaces:

Gravimetric techniques are used to determine the changes in weight before and
after modification of the surface. Thermal analysis, such as thermo-gravimetric
analysis (TGA) and differential scanning calorimetry (DSC) provide a bulk
compositional property of the hybrid material157-158. Spectroscopic techniques such
as NMR, FT-IR and X-ray photoelectron spectroscopy (XPS) are widely used to
characterize functionalised surfaces. XPS in particular is useful for monitoring the
presence of different oxidization states of atoms, relative abundance of atomic
species and the bulk of the material. Various authors investigated the elemental
and chemical surface composition of the initial templates and the functionalised
templates by XPS. Different methodologies are also refereed based on the
gravimetric and spectroscopic methods in the literature to calculate the surface
coverage by the functional groups159.

5. Conclusion, Perspective and future work:

The dream of being able to set up one crystallization experiment that will produce
protein crystals with certainty is still very distant. The crystallisation of the
macromolecular structure is underdeveloped area and the urgent requirement is
there to understand the mechanism of nucleation and/ or crystallisation of
proteins. The project is motivated on the results of the influence of colloidal
templates on the crystallisation of the molecular and macromolecular structures
and the anisotropic wettability behaviour of the crystallised pharmaceutical solids26.
T.Delmas et al. reported the influence of the surface chemistry and surface
topography on the crystallisation of the Hen Egg White Lysozyme27.
In order to understand the effect of morphology and chemistry of interface at the
nano-meter length scale in nucleation or crystallisation, it is proposed to have the
template assisted crystallisation approach. The background information on the
synthesis and surface modification of the nano-templates has been studied and the
gap identified in the literature with respect to the perspectives of the project.

26
Summary:

Template Synthesis:

The synthesis of the templates selected for the project i.e. bio active glasses and the
Anodised Alumina Oxide templates are well established techniques. Various
methods are also reported in the literature to control the porosity and the pore
structures. Considering the perception of the project, for verification of the effect of
porosity on the nucleation, it is essential to synthesise the surfaces with the
porosity in the narrow pore size distribution. It is also crucial to alter the porosity
in wide range to develop the understanding the effect of the porosity in nucleation
of the proteins with different molecular weights. All different methods reported to
control the porosity of the bioactive glass are not able to produce mono-dispersed
porosity and the control of the pore size is also limited. It is identified that the
synthesis of bio-active glass templates with wide range of porosity (i.e. 5 – 150 nm)
with the same pore formation approach is not reported in the literature. The effect
of aging and the drying processes in the sol-gel method of synthesis on the porosity
is also the underdeveloped area. The crystallisation of the bio active glasses are
discussed in detail, but the effect of the crystallisation on the porosity is not well
understood. As far as Anodised alumina oxide is concerned, the synthesis method
is well understood. The literature also reports the controlled porosity of the
membrane with different electrolyte solutions, although the effect of different
electrolyte solutions on the pore size is not fully defined. The effect of the ionic
strength and the concentration of the electrolyte solution on the porosity are also
not fully understood.

Surface modification of the bio active glasses/ silica based material using different
protocols are widely referred in the literature for the purpose of the verification of
its effect on the tissue growth engineering. Very few papers referred the surface
functionalisation of the AAO templates and the mechanism for the same is not well
studied. It is proposed here to coat the surface of the AAO with the silica sol
prepared with sol-gel method to silanise the surface with relative ease.

27
Protein Crystallisation:

Considering the project objectives, it is proposed to understand the role of interface

in nucleation and crystallisation process. In recent years there are intense efforts
to understand the effect of surface porosity on nucleation and crystallisation has
been reported. Different porous materials like silica9, bioglass11, and carbon
nanotubes163 etc. are utilised to understand the effect of porosity on the
crystallisation of proteins. The approach adopted in the past is to understand the
effect of the disordered porous media on the crystallisation of proteins11. The
substrate proposed to synthesise in the present study is the substrate with the
ordered pore size and pore structure, no efforts in the past have referred the effect
of such substrates on the crystallisation. Silanised glass, mica and polymeric
surface have been used as the templates for the protein crystallisation12-14, it is not
reported in the literature referred that the surfaces with tuned porosity are
silanised for protein crystallisation. No efforts in the literature combined the effect
of the surface chemistry and morphology to understand the mechanism of
nucleation and crystallisation of complex protein molecules. The effect of the
crystallisation of the surface on the crystallisation of the protein molecules is also
not published. Any effort to crystallise the crystals with controlled shape using the
present approach are also not cited in the literature. Literature cited also does not
show any efforts towards the use of the role of surface properties for the crystal
engineering of polymorphs.

Future Work:
The literature referred here in above is specifically focused on the synthesis of the
templates with tuned porosity and surface chemistry and surface area and porosity
characterisation. More extensive literature needs to be referred in near future for
the other conventional protein crystallisation methods i.e. vapour diffusion hanging
drop method, sitting drop method, etc. The need to understand the methods for
screening of the metastable conditions for particular proteins for crystallisation and
detailed literature will be referred on this area. The literature will also be referred
in order to understand the various surface characterisation methods, i.e.
spectroscopic analysis (XPS, AES, etc.) and thermal analysis (TGA, DSC).

28
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