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Crystallisation of Pharmaceutical Polymorphs Literature Review

1. Introduction:

Umang Shah

May 2010

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

genes 1 . 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 determination 2 . 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

crystallization 3 .

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 conditions 4 ).

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 nucleation 4 .

Conventional crystallization mechanism of the protein leads to the

Because growth in the metastable zone affords


Thus a search for nucleant, which could enhance the chances of any single trial producing crystalline material, is ongoing since last two and half decade 4-6, 8-10 . In 1988, Mc Pherson and Shlichta 5 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 substrates 4, 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 molecules 10 . 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 crystallization 10,11 .

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 science 25 . 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 solids 26 and the effects of the interfacial chemistry on the crystallisation of model protein (Hen Egg White Lysozyme) and pharmaceutical

solids 27 .

At nanometer length scale the morphology and wetting behaviour of the


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.

properties will lead to the ability to engineer crystal habits and optimise

crystallisation process. The key objectives of this study are:

Detailed understanding of the interfacial

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 unsuccessful 9,10 . 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

substrates 11 .

more uniform mesoporous pore size and functionalised surface for the purpose of

protein crystallisation.

This research aims to prepare the bioactive glass substrate with

Anodised Alumina Oxide (AAO) is widely used in the electronic industry for the various applications 32 . It can be synthesised with ease in the laboratory, and the


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 etc 94-114 . The AAO template is rarely used for the protein crystallisation, as it

is postulated to be a difficult to crystallisation on the aluminium oxide surface 31 .

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.

the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram
the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram
the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram
the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram
the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram
the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram
the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram
the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram
the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram
the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram
the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram
the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram
the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram
the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram
the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram
the detailed flow chart showing approach/ methodology of the project. Figure 1.1 Project concept flow diagram

Figure 1.1 Project concept flow diagram


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 applications 36, 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) arrays 37 . 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 junctions 38 . 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.

through melting of related oxides, at temperatures in the range of 1100–1300 o C 39 . The glass compositions were mainly based on silica, calcium, phosphate and


Originally, bioactive glasses were prepared

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 material 40,41 . Studies have shown that sol-gel glasses prepared with in three (SiO 2 - CaO-P 2 O 5 ) and two (SiO 2 -CaO) component systems, and even pure silica, can quickly develop an apatite layer in contact with simulated body fluids 42-44 . Sol-gel has the advantages of low processing temperatures and ease of control of textural properties.


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 networks 45, 46 .

A well studied alkoxide is silicon Tetra Ethyl Ortho Silicate (TEOS). The chemical formula for TEOS is given by: Si(OC 2 H 5 ) 4 , or Si(OR) 4 where the alkyl group R = C 2 H 5 . Alkoxides are ideal chemical precursors for sol-gel synthesis because they react readily with water 47 . The reaction is called hydrolysis, because a hydroxyl ion becomes attached to the silicon atom as follows:

Si(OR) 4 + H 2 O → 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 H 2 O → 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) 3 Si–O–Si(OR) 3 ] + H-O-H or (OR) 3 –Si-OR + HO–Si-(OR) 3 → [(OR) 3 Si–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.


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 technology 48-51 .

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 literature 45, 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 method 45 .

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 (HNO 3 ) 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.


reported the modified fast process to produce sol–gel monoliths using TMOS and TEOS without alcohol and with high water to alkoxide ratios 55 .

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 temperature 55 .

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 left 54, 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 pressures 45 . 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 distributions 55 . 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.


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 applications 56-58 . 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 (C 16 EO 10 ), Pluronic P123 (EO 20 -PO 70 -EO 20 ), Pluronic F127 (EO 106 PO 70 EO 106 ), 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 mesostructures 70 .

Direct templating by liquid crystal phases (copolymer surfactants) has been recognized as a method for controlling morphology and preparing large monolithic mesophases 63,64 , but the method used so far is in general restricted to only a few surfactant/water binary systems 63 . While the pore size can be varied in some cases, the long-range pore ordering is usually lost 64,65 .


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 morphology 64 .

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 processing 66,67 . However, all these strategies failed to obtain ordered mesoporous materials with pore sizes larger than 12 nm 66 . 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 enlarged 67 .

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 sucrose 72 , gelatine 73 , PEG micro/ nano spheres 78 or PMMA microbeads 74 , 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 o C and the ceramic foams sintered at 1350 o C, producing a scaffold with interconnected pore diameters of up to 300µm 75 .

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


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 reaction 76 . 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 method 77 .

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.

Monomers are added to the ceramic suspension, in situ

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 drying 45 , 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 produced 55 . 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 process 78 . 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


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 crystallisations 80-86 . 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 nanostructures 87-89 .

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 aluminum 90 . Masuda and co-workers advocated a two-step oxidation technology and process to prepare highly ordered pore arrays 91, 92 . Some special procedures for creating particular shapes and structures such as hexagonally ordered nanopore arrays with high aspect ratios 93, 94 tubular anodic aluminium oxide 95,96 , and Porous Anodised Alumina films having bone-shaped nanochannels 97 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 al 87 . Recent effort to achieve highly ordered porous structures reported synthesis of

Alumina Oxides membranes on Si and Ti substrates 102 .

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

A. Yin et al. show that


template-based anodisation process 99 .

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

force 98 .

membrane forms due to a weak mechanical driving force 98 . Figure: 2.1 Schematic diagram of

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 growth 98

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.

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

Then, a thinning process of the barrier


pore initiation, nanopores grow dierently, 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) + 3H 2 O 2Al 3 + + 6e +3O 2 Al 2 O 3 (oxide) + 6H + (aq) + 6e

alumina dissolution at oxide/electrolyte interface:

Al 2 O 3 (oxide) + 6H + (aq) 2Al 3 + (aq) + 3H 2 O

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 (10 8 – 10 11 pores/cm 2 ) 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 controlled 88-90 .

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 membrane 121 .


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

decades 112-115 . Porous anodised alumina oxide are synthesised under different

anodization voltages in different electrolyte solutions.

optimum anodisation volage is different for different electrolyte solutions, at which

the ordered pore arrangement is observed for constant current densities 119 . 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 structure 113 .

It can be observed that

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 changed 94,114 .

concentration of different electrolyte is changed 94,114 . Anodisation time: Figure 2.2 Interpore distance d in

Anodisation time:

Figure 2.2 Interpore distance d in self-organized porous alumina vs anodic voltage U a for sulphuric, oxalic, and phosphoric acid solutions 94 .

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

proportionality constant is different for the different electrolyte solution 118 . 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


ions) and dissolution (relatively constant due to its field-assistant nature) causes a

characteristic limiting thickness 113, 115 .

causes a characteristic limiting thickness 113, 115 . Ionic strength of electrolyte solution: Figure 2.3

Ionic strength of electrolyte solution:

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 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 mechanism 113, 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


conditions of the samples 94, 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 process 120 . 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 molecules 122 . 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 there 123 . 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 applications 122 .

the same for the targeted drug delivery applications 122 . Figure 2.4 test tubes using AAO

Figure 2.4

test tubes using AAO templates 124 .

Schematic illustration of template synthesis of silica nano

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


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 ratio 122-125 .

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

decades 126-135 .

different functional groups for tissue growth engineering 130 . 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 deposition 128-134 . 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

Bio active glasses are functionalised using different protocols and


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 bonds 128 .

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.

in solution prior to condensation with the solid substrate. 19 Figure 3.1 Silanization of Glass with


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 surface 134 .

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 delivery 160 .

workers reported the protocol for direct silanisation of the AAO templates 162 . In order to silanise the anodised alumina oxides the templates are coated with the fine

layer of silica, which is silanised with relative ease.

Lee and co-

Mechanism of surface silanisation:

The mechanism of grafting the silanes on the surface is explained by Leger et al 161 . 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 clear 167-168 .

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


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 characterization 136 . Mikhail et al. compared various methods of the porosity

measurements considering the scope and limitation of the methods 137 .

considering the scope and limitation of the methods 137 . Figure 4.1 applicable range. 136 .

Figure 4.1

applicable range. 136 .

Different methods for measurement of porosity and


Table 4.1 Comparison of the porosity measurements considering the scope and limitation of the methods 137




Range of





pore size


1 N 2 Adsorption

d < 50 nm


Correlation with BET surface area




program, non-

applicability of

theory in




2 Mercury

4 nm < d

Much in use, comparable data, extended range of pore sizes

Danger of breaking pore walls, large pores are filled at atmospheric pressure


60000 nm




3 Air/ He


A few

Very short time method, good reproducibility, no

influence on sample

No pore size distribution





upon gas



4 Xylene/ water

d > 100 nm

Simple method,

No pore size distribution, specimens with small diameters


short time


Small angle X

0.5 nm ≤ d


Time required for measurement is very less

Special facilities

ray scattering

100 nm

are required in XRD machine




Small angle

0.5 nm ≤ d

Time required for measurement is very less

Special facilities



100 nm

are required,

limited to



nuclear centres




≥ 20 nm


Large pores can be measured, optical checking is possible, also microporosity can be measured by analysis TEM

Limitation to large pores, significant errors in coarse grain materials, area information, for spatial information large mathematical effort

image analysis




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 m 2 g -1 ),

but this technique cannot be employed for the study of porosity 138 .

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 K 139-142 . 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 .

Six classes of isotherms, are usually distinguished 143 . 23 Figure 4.2 Types of Physorption Isotherms


Figure 4.2 Types of Physorption Isotherms 143 .

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/p 0 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/p 0 . 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 surface 142-144 .

Determination of surface area:

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

the surface area from physisorption isotherm data 145 .

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 value 143 .

A concern that must be


Barett et al. reported the method for calualtion of the pore volume and pore area distribution directly from the desorption isotherm of porous substances 146 . 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


pore type. Adsorption isotherms of pure gases on microporous sorbents can be described by means of Polanyi's potential theory 148 . 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/p 0 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.

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 carbons 150 .

The empirical formula for calculation of

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 function 151 . 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


mechanism. Various techniques such as atomic force microscopy (AFM), scanning

electron microscopy (SEM), have proven useful.

useful method in understanding the pore formation mechanism 118 .

Atomic force microscopy is a very

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 material 157-158 . 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.

gravimetric and spectroscopic methods in the literature to calculate the surface

coverage by the functional groups 159 .

Different methodologies are also refereed based on the

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 solids 26 . T.Delmas et al. reported the influence of the surface chemistry and surface topography on the crystallisation of the Hen Egg White Lysozyme 27 . 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.



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

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.

synthesis of bio-active glass templates with wide range of porosity (i.e. 5 – 150 nm)

The effect

of aging and the drying processes in the sol-gel method of synthesis on the porosity

is also the underdeveloped area.

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.

with the same pore formation approach is not reported in the literature.

porosity in the narrow pore size distribution.

It is identified that the

The crystallisation of the bio active glasses are

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.


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 silica 9 , bioglass 11 , and carbon nanotubes 163 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 proteins 11 .

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 crystallisation 12-14 , 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.

show any efforts towards the use of the role of surface properties for the crystal

engineering of polymorphs.


Literature cited also does not

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.

in order to understand the various surface characterisation methods, i.e.

spectroscopic analysis (XPS, AES, etc.) and thermal analysis (TGA, DSC).

The literature will also be referred



1. Chayen, N. E. and Saridakis, E. (2008) Protein crystallization: from purified protein to diffraction-quality crystal, Nature Methods , 5, 147–


2. Chayen, N. E. and Saridakis, E. (2009) Towards a ‘universal’ nucleant for protein crystallization, Trends in Biotechnology, 27, 2, 99–106

3. Chayen, N. E. (2004) Turning protein crystallization from an art into a science, Current Opinion in Structural Biology, 14, 577–583.

4. Mc Pherson, A.(1999) Crystallization of Biological Macromolecules, Cold Spring Harbor Lab. Press, Woodbury, NY, USA.

5. McPherson, A. & Shlichta, P. (1988) Heterogeneous and Epitaxial Nucleation of Protein Crystals on Mineral Surfaces Science, 239, 385–387.

6. Falini, G., Fermani, S., Conforti, G. & Ripamonti, A. (2002) Protein crystallisation on chemically modified mica surfaces, Acta Crystallographica. D, 58, 1649–1652.

7. Punzi, J. S., Luft, J. & Cody. V. (1991) Protein crystal growth in the presence of poly(vinylidene difluoride) membrane, J. Applied Crystallography, 24, 406–408.

8. Edwards, A. M., Darst, S. A., Hemming, S. A., Li, Y.& Kornberg, R. D.(1994) Molecular dynamics studies of proteins and nucleic acids :

Current Opinion in Structural Biology, Structural Biology, 1, 195–197.

9. Chayen, N. E. & Saridakis, E. (2001) Is lysozyme really the ideal model

protein?, J. Crystal Growth, 232, 262–264. 10.Chayen, N. E., Saridakis, E., El-Bahar, R.& Nemirovsky,Y. (2001) Porous Silicon: an Effective Nucleation-inducing Material for Protein Crystallization, J. Molecular Biology, 312, 591–595. 11.Chayen, N. E. et al. (2006) Experiment and theory for heterogeneous nucleation of protein crystals in a porous medium, Proceeding of the National Academy of Science of the U. S. A., 103, 597–601. 12. Pham. T., Lai, D.T., Ji, D., Tuntiweehapikul, W., Friedman, J.M., and Lee, T.R. (2004) Well ordered self-assembled monolayer surfaces can be used to enhance the growth of protein crystals, Colloids and Surfaces B- Bio-interfaces, 34, 3, 191 - 196. 13.Fermani, S., Falini, G., Minnucci, M., and Ripamonti, A. (2001) Protein crystallisation on polymeric surfaces, Journal of Crystal Growth, 224,


14.Hekmat, D., Hebel, D., Weuster-Bitz, D. (2008) Crystalline proteins as an alternative to standard formulations, Chemical engineering technology, 31, 6, 911–916. 15.Dressler, D.H., Mastai, Y. (2007) Controlling polymorphism by crystallization on self-assembled multilayer, Crystal Growth & Design, 7, 5, 847–850. 16.Gracia-Ruiz, J. M. (2003) Nucleation of protein crystal, Journal of structural biology, 142, 22–31. 17.Ji, D., Arnold, C. M., Grupe, M., Beadle, E., Dunn, R. V., Phan, M. N., Villazana, R., Benson, R., Colorado, R., Lee, T. R., Friedman, J. M. (2000) Improved protein crystallization by vapour diffusion from drops in contact with transparent, self-assembled monolayers on gold-coated glass cover slips, Journal of Crystal Growth, 218, 2, 4, 390–398.


18.Kam, Z., Shore, H. B., Feher, G. (1978) Crystallisation of proteins, Journal of molecular biology, 123, 4, 539–555. 19.Rong, L., Komatsu, H., Yoshizaki, I., Kadowaki, A., Yoda, S. (2004) Protein crystallization by using porous glass substrate, Journal of synchrotron radiation, 11, 1, 27 – 29. 20.Lee, A. Y., Ulamn, A., Myerson, A. S., Crystallisation of amino acids on self-assembled monolayers of rigid thiols on gold, Langmuir, 18, 15,


21.Curcio, E., Fontananova, E., Di Profio, G., Drioli, E. (2006) Influence of the structural properties of poly (vinylidene fluoride) membranes on the heterogeneous nucleation rate of protein crystals, Journal of physical Chemistry B, 110, 25, 12438–12445. 22.Liu, Y., Wang, X., Lu, J., Ching, C. (2007) Influencce of the roughness, topography, and physicochemical properties of chemically modified surface on the heterogeneous nucleation of protein crystals, Journal of Physical Chemistry B, 11, 13971–13978. 23.Plain, J., Pallandre, A. , Nysten, B., Jonas, A. M. (2006) Nanotemplated Crystallization of Organic Molecules, Small, 2, 7, 892–897 24.Desiraju, G. R. (2001) Crystal engineering: Outlook and prospects, Current Science, 81, 8, 1038–1041. 25.Bolanos-Garcia , V. M., Chayen, N. E. (2009) New directions in conventional methods of protein crystallization, Progress in Biophysics and Molecular Biology, 101, 3–12. 26.Heng J. Y. Y., Bismarck, A., Williams D. R. (2006) Anisotropic Surface Chemistry of Crystalline Pharmaceutical Solids, AAPS Pharm Sci Tech, 7, 4, 84, E1 –E9 27.Delmas T. (2008) Self assembly of closed-packed colloidal crystals:

templates for protein crystallization, Un-Published Thesis – Department of Chemical Engineering, Imperial College London, 74–94. 28.Davis, M. E., Montes, C., Hathway, P. E., Arhancet, J. P., Hasha, D. L. & Garces, J. M. (1989) Physicochemical properties of VPI-5, J. American Chemical Society, 111, 3919–3924. 29.Chen, C., Li, H. & Davis, M. E. (1993) Studies on mesoporous materials: I. Synthesis and characterization of MCM-41, Microporous Material, 2, 17–


30.W.-B. Yang et al. (2007) One-step anodization fabrication and morphology characterization of porous AAO with ideal nanopore arrays, Journal of Experimental NanoScience, 2, 3, 207-214

31.Y. Zhang et al. (2003) Oriented nano-structured hydroxyapatite from the template, Chemical Physics Letters, 376, 493 - 497 32.Li, F., Zhang, L., Metzger, R. M. (1998) On the Growth of Highly Ordered Pores in Anodized Aluminum Oxide, Chemistry of Materials, 10, 2470-


33.Losic D., Losic, D, Jr. (2009) Preparation of Porous Anodic Alumina with Periodically Perforated Pores, Langmuir, 25, 10, 5426–5431. 34.Itoh, N., Kato, K., Tsuji, T., Hongo, M. (1996) Preparation of a tubular anodic aluminum oxide membrane, Journal of Membrane Science, 117,


35.Rahimi, M.H., Saramad, S., Tabaian, S. H., Marashi, S. P., Zolfaghari, A., Mohammadalinezhad, M. (2009) Study the effect of stripping in two step anodizing process on pore arrangement of nano-porous alumina, Applied Surface Science, 256, 12–16


36.Pereira, M. M., Jones, J. R. and Hench, L. L. (2005) Bioactive glass and hybrid scaffolds prepared by sol– gel method for bone tissue engineering, Advances in Applied Ceramics , 104, 1, 36–42. 37.Shingubara, S. (2003) Fabrication of Nanomaterials Using Porous Alumina Templates, Journal of Nanoparticle Research, 5, 17 38.Zhang, F., Pan, C., Zhu, J. (2007) Nano-porous anodic aluminum oxide membranes with 6–19nm pore diameters formed by a low-potential anodizing process, Nanotechnology, 18, 1–4. 39.Hench, L. L., Wilson, J. (1993) An introduction to ceramics, Singapore:

World Scientific; 40.Pereira, M.M., Clark A.E., Hench, L.L. (1995) Effect of texture on the rate of hydroxyapatite formation on gel-silica surface. Journal of American Ceramic Society; 78, 2463–2468. 41.Filgueiras MR, LaTorre GP, Hench LL. (1993) Solution effects on the surface reactions of a bioactive glass. Journal of Biomedical Material

Research, 27, 445–453 42.Peltola T, Jokinen M, Rahiala H, Levanen E, Rosenholm JB, Kangasniemi I, Yli-Urpo A. (1999) Calcium phosphate formation on porous sol-gel- derived SiO 2 and CaO-P 2 O 5 -SiO 2 substrates in vitro. Journal of Biomedical Material Researc, 44, 12–21. 43.Pereira MM, Hench LL. (1996) Mechanisms of hydroxyapatite formation on porous gel-silica substrates, Journal of Sol-Gel Science Technology, 7,


44.Li R, Clark AE, Hench LL. (1991) An investigation of bioactive glass powders by sol-gel processing. Journal of Applied Biomaterials, 2, 231–


45.Brinker, C.J., Scherer, G.W. (1990) Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing, 1, Academic Press 46.Hench, L.L.; West, J.K. (1990). The Sol-Gel Process, Chemical Reviews , 90: 33 47.Dislich, Helmut (1971). New Routes to Multicomponent Oxide Glasses, Angewandte Chemie International Edition in English, 10, 363

48.Matijevic, E. (1986) Monodispersed colloids: art and science. Langmuir, 2,


49.Brinker, C. J.; Mukherjee, S. P. (1981). Conversion of monolithic gels to glasses in a multicomponent silicate glass system, Journal of Materials Science 16: 1980 50.Sakka, S; Kamiya, K (1980). Glasses from metal alcoholates, Journal of Non-Crystalline Solids 42: 403 51.Yoldas, B. E. (1979). Monolithic glass formation by chemical polymerization. Journal of Materials Science 14: 1843 52.Wagh P B, Begag R, Panjonk G M, Venkasteswara Rao A and Haranath D (1999) Comparison of some physical properties of silica aerogel monoliths synthesized by different precursors, Mater. Chem. Phys., 57, 214 53.BrinkerCJ, Keefer KD, Schaefer DW, Assink RA,Kay BD, and Ahley C S

(1984) Sol-gel transition in simple silicates II, J. Non-Cryst. Solid, 63, 45–


54.Bryans T R, Brawner V L and Quitevis E L (2000) Microstructure and porosity of silica xerogel monoliths prepared by the fast sol-gel method, J.

Sol-Gel Science and Technology, 17, 211–7 55.Mohammad, A., Julian, R. J., Hench L.L (2007) Fabricating sol–gel glass monoliths with controlled nano-porosity, Biomedical materials, 2, 6–10.


56.Zhao, D., Feng, J., Huo, Q., Melosh, N., Fredrickson, G. H., Chmelka, B. F., Stucky, G. D. (1998) Triblock Copolymer Syntheses of Mesoporous Silica with Periodic 50 to 300 Angstrom Pores, Science, 279, 548 – 552. 57.Xia, W., Chang, W. (2006) Well-ordered mesoporous bioactive glasses (MBG): A promising bioactive drug delivery system, Journal of Controlled Release, 110, 522–530 58.Xia, W., Chang, W. (2008) Preparation, in vitro bioactivity and drug release property of well-ordered mesoporous 58S bioactive glass, Journal of Non-Crystalline Solids, 354, 1338–1341 59.Zhao, D., Feng, J., Huo, Q., Melosh, N., Fredrickson, G. H., Chmelka, B. F., Stucky, G. D. (1998) Nonionic Triblock and Star Diblock Copolymer and Oligomeric Surfactant Syntheses of Highly Ordered, Hydrothermally Stable, Mesoporous Silica Structures, Journal of American Chemical Society, 120, 6024-6036

60.Zhao, Y. F., Loo, S. C. J., Chen, Y. Z., Boey, F. Y. C., Ma, J. (2007) In situ SAXRD study of sol–gel induced well-ordered mesoporous bio-glasses for drug delivery, Journal of biomedical research B., 85A, 4, 1032 - 1042 61.Jones, J. R., Hench, L. L. (2004) Effect of surfactant concentration and composition on the structure and properties of sol-gel-derived bioactive glass foam scaffolds for tissue engineering, Journal of Materials Science, 38,18, 3783 - 3790 62.Yan D.,* Li, X., Li, Q. (2009) Effect of Pore Size on the Growth of Hydroxyapatite from Mesoporous CaO-SiO 2 Substrate, Industrial & Engineering Chemistry Research, 48, 8829–8836 63.Chen, D. et al.(2006) Anionic surfactant induced mesophase transformation to synthesize highly ordered large-pore mesoporous silica structures, Journal of Materials Chemistry, 16, 1511–1519. 64.Feng, P., Bu, X., Pine, D. J. (2000) Control of Pore Sizes in Mesoporous Silica Templated by Liquid Crystals in Block Copolymer-Cosurfactant- Water Systems, Langmuir, 16, 5304-5310 65.Yun, H., Kim, S., Hyeon, Y. (2007) Design and p reparation of bioactive g lasses with hierarchical pore networks, Chemical Communication, 2139–


66.Matos, J. R.; Kruk, M.; Mercuri, L. P.; Jaroniec, M.; Zhao, L.; Kamiyama, T.; Terasaki, O.; Pinnavaia, T. J.; Liu, Y. (2003) Ordered Mesoporous Silica with Large Cage-Like Pores: Structural Identification and Pore Connectivity Design by Controlling the Synthesis Temperature and Time Journal of American Chemical Society, 125, 821. 67. Fan, J. et al. (2005) Low-Temperature Strategy to Synthesize Highly Ordered mesoporous silica with very large pores, Journal of American Chemical Society, 127, 10794-10795 68.Schmidt-Winkel, P.; Lukens, W. W.; Zhao, D. Y.; Yang, P. D.; Chmelka, B. F.; Stucky, G. D. (1999) Mesocellular Siliceous Foams with Uniformly Sized Cells and Windows, Journal of American Chemical Society, 121, 254. 69.Yu, C. Z.; Fan, J.; Tian, B. Z.; Stucky, G. D.; Zhao, D. Y. (2003) Synthesis of Mesoporous Silica from Commercial Poly(ethylene oxide)/Poly(butylene oxide) Copolymers: Toward the Rational Design of Ordered Mesoporous Materials, J. Phys. Chem. B, 107, 13368. 70.Anderson, M. T., Martin, J. E., Odinek, J. G., Newcomer, P. P. (1998) Effect of Methanol Concentration on CTAB Micellization and on the Formation of Surfactant-Templated Silica (STS), Chem. Mater., 10, 1490. 71.J. C. T. Andrade, J. A. Camilli, E. Y. Kawachi and C. A. Bertran, (2002) Behavior of dense and porous hydroxyapatite implants and tissue response in rat femoral defects, J. Biomed. Mater. Res., 62, 30–36.


72.V. S. Komlev and S. M. Barimov, (2002) Porous hydroxyapatite ceramics of bi-modal pore size distribution, J. Mater. Sci.: Mater. Med ,13, 295–299. 73.B. Flautre, M. Deschamps, C. Delecourt, M. C. Blary and P. Hardouin (2001) Porous HA ceramic for bone replacement: Role of the pores and interconnections – experimental study in the rabbit, J. Mater. Sci.: Mater. Med.,12, 679–682. 74.S. Callcut, J. C. Knowles (2002) Correlation between structure and compressive strength in a reticulated glass-reinforced hydroxyapatite foam, J. Mater. Sci.: Mater. Med., 13, 485–489. 75.31. P. Sepulveda and J. P. G. Binner (1999) Processing of cellular ceramics by foaming and in situ polymerisation of organic monomers, J. Eur. Ceram. Soc., 19, 2059–2066. 76.H. Oonishi, S. Kutrshitani, E. Yasukawa, H. Iwaki, L. L. Hench, J.Wilson, E.Tsuji and T.Sugihara (1997)Clin. Orthop. Relat. Res., 334, 316–325. 77.Effah Kaufmann, E.A.B., Ducheyne, P., Shapiro, I.M. (2000) Effect of varying physical properties of porous, surface modified bioactive glass 45S5 on osteoblast proliferation and maturation, Journal of biomedical materials research, 52, 4, 783-796 78.Lei, J. H., Liu, D., Guo, L. P., MinYan, X., Tong, H. (2006) Fabrication and characterization of hexagonal mesoporous silica monolith via post- synthesized hydrothermal process, J. Sol-Gel Science and Technology, 39,


79.Rizkalla, A. S., Jones, D. W., Clarke, D. B., Hall, G. C. (1996) Crystallisation of experimental bioactive glass composition, Journal of Biomedical Materials Research, 32, 119-124 80.Huang a, R., Pan, J., Boccaccini, A.R., Chen, Q.Z. (2008) A two-scale model for simultaneous sintering and crystallization of glass–ceramic scaolds for tissue engineering, Acta Biomaterialia , 4, 1095–1103 81.Cannilloa, V., Pierlia, F., Sampath, S., Siligardi, C. (2009) Thermal and physical characterisation of apatite/wollastonite bioactive glass–ceramics, Journal of the European Ceramic Society, 29, 611–619 82.Chen, Q. Z., Rezwan, K., Armitage, D., Nazhat, S. N. Boccaccini, A. R. (2006) The surface functionalization of 45S5 Bioglass®-based glass- ceramic scaffolds and its impact on bioactivity, Journal of Material Science: Mater Med, 17, 979–987 83.A. Balamurugan et al. (2006) Synthesis and characterisation of sol gel derived bioactive glass for biomedical applications, Materials Letters, 60,


84.L. Lefebvre et al. (2007) Structural transformations of bioactive glass 45S5 with thermal treatments, Acta Materialia, 55, 3305–3313 85.Xin, R., Zhang, Q., Chen, J., Leng, Y. (2008) Effects of porosity and

crystallinity of glass ceramics on the in vivo bioactive response, Biomedical Materials, 3, 1 – 5. 86.Filho, O. P., La Torre, G. P., Hench, L. L. (1996), Effect of crystallization on apetite layer formation of bioactive glasses 45S5, Journal of Biomedical Research, 30, 509 – 514. 87.C. A. Mirkin, et al. (2006) Multi-segmented One-Dimentional Nanorods Prepared by Hard-Template Synthetic Methods, Angewandete Chemie, vol.45, 2672-2692. 88.El-Sayed, H., Singh, S., Greiner, M. T., Kruse, P. (2006) Controlled growth and monitoring of tantalum oxide nanostructures, Nano Letters, 6,


89.Shijing, Lu et. al. (2009) Ionic nano-convection in anodisation of

aluminium plate, Chemical Communication, 5639-5641


90.Thompson, G. E. (1997) Porous anodic alumina: fabrication, characterization and application, Thin Solid Films, 297, 192. 91.Masuda, H.; Satoh, M. (1996) Fabrication of Gold Nanodot Array Using Anodic Porous Alumina as an Evaporation Mask, Jpn. J. Appl. Phys., 35,


92.Masuda, H.; Yamada, H.; Satoh, M.; Asoh, H.; Nakao, M.; Tamamura, T. (1997) Highly ordered nanochannel-array architecture in anodic alumina, Appl. Phys. Lett., 71, 2770 93.Li, A.-P.; Muller, F.; Birner, A.; Nielsch, K.; Gosele, U. (1999) Fabrication and Microstructuring of Hexagonally Ordered Two- Dimensional Nanopore Arrays in Anodic Alumina, Adv. Mater., 11, 483. 94.Muller, F.; Birner, A.; Schilling, J.; Li, A.-P.; Nielsch, K.; Gosele, U.; Lihmann, V. (2002) High aspect ratio microstructures based on anisotropic porous materials, Microsyst. Technol., 8,7.

95.Itoh, N.; Kato, K.; Tsuji, T.; Hongo, M. J. (1996) Preparation of a tubular anodic aluminum oxide membrane, Membr. Sci., 117, 189. 96. Itoh, N.; Tomura, N.; Tsuji, T.; Hongo, M. (1998) Strengthened porous alumina membrane tube prepared by means of internal anodic oxidation, Microporous Mesoporous Mater., 20, 333. 97.Xu, T. T.; Fisher, F. T.; Brinson, L. C.; Ruoff, R. S. (2003) Bone-Shaped Nanomaterials for Nanocomposite Applications, Nano Lett., 3, 1135. 98.Nevin Tasaltın, Sadullah Öztürk, Necmettin Kılınç, Hayrettin Yüzer·Zafer, Ziya Öztürk (2009) Simple fabrication of hexagonally well-ordered AAO template on silicon substrate in two dimensions, Applied Physics A, 95,


99.A. Yin, M. Tzolov, D. Cardimona, L. Guo and J. Xu (2007) Fabrication of highly ordered anodic aluminium oxide templates on silicon substrates, IET Circuits Devices Systems,1, 3, 205–209

100. J.W. Diggle, T.C. Downie and C.W. Goulding, (1969) Processes

involved in retainment of steady-state conditions for the anodizing of

aluminum following formation voltage changes, J. Electrochem. Soc.:

Electrochem. Sci., pp. 737–740.

101. S.J. Garcia-Vergara, P. Skeldon, G.E. Thompson and H. Habazaki,

(2006) A flow model of porous anodic film growth aluminum, Electrochim. Acta 52, pp. 681–687.

102. N. Itoh, K. Kato, T. Tsuji and M. Hongo, (1996) Preparation of tubular anodic aluminum oxide membrane, J. Membr. Sci. 117, pp. 189–196.

103. T.P. Hoar and N.F. Mott, (1959) A mechanism for the formation of

porous anodic oxide films on aluminium, J. Phys. Chem. Solids 9, p. 97.

104. J. Siejka and C. Ortega, (1977) An O-18 study of field-assisted pore

formation in compact anodic oxide films on Al, J. Electrochem. Soc. 124, p.


105. J.P. O'Sullivan and G.C. Wood, (1970) The morphology and

mechanism of formation of porous anodic films on aluminum, Proc. R. Soc. Lond. B Biol. Sci. Math. Phys. Sci. 317, pp. 511–543.

106. K. Nielsch, J. Choi, K. Schwirn, R. Wehrspohn and U. Gosele, (2002)

Self ordering regimes of porous alumina: 10% porosity rule, Nano Lett. 2

(7), 677–680.

107. F. Keller, H. S. Hunter and D. L. Robinson (1953) Structural Features of Oxide Coatings on Aluminum, J. Electrochem. Soc. 100, 411.

108. J. P. W. O’Sullivan and G. C. Wood, (1970) The Morphology and

Mechanism of Formation of Porous Anodic Films on Aluminium, Proc. Roy. Soc. Lond. Ser. A317, 511.


109. V. P. Parkhutik and V. I. Shershulsky (1992), Theoretical modelling of porous oxide growth on aluminium, J. Applied Physics D:, 25, 1258

110. O.Jessensky, F.Muller and U.Gosele, Self-organized formation of

hexagonal pore arrays in anodic alumina, Appl. Phys. Lett. (1998) 72,


111. G. E. Thompson, R. C. Rurneaux, G. C. Wood, J. A. Richardson and J.

S. Goode, (1978) Nucleation and growth of porous anodic films on aluminium, Nature, 272, 433

112. Yuan, J. H., He, F. Y., Sun, D. C., Xia, X. H. (2004) A Simple Method for Preparation of Through-Hole Porous Anodic Alumina Membrane, Chemistry of Material, 16, 1841-1844

113. Choi, Y., Hyeon, J. Y., Bu, S. D. (2009) Effects of Anodizing Voltages and Corresponding Current Densities on Self-ordering Process of Nanopores in Porous Anodic Alumina Anodized in Oxalic and Sulfuric Acids, Journal of the Korean Physical Society, 55, 2, 835-840

114. Belwalkar, A. et. al., (2008) Effect of processing parameters on pore structure and thickness of anodic aluminum oxide (AAO) tubular membranes, Journal of Membrane Science, 319, 1 – 2, 192 - 198

115. Wangon, X., Han, G. R., (2003) Fabrication and characterization of

anodic aluminum oxide template, Microelectronic Engineering, 66, 166–


116. C. Cherki and J. Siejka, (1973) Study by nuclear microanalysis and

O18 tracer techniques of the oxygen transport processes and the growth

laws for porous anodic oxide layers on aluminum, J. Electrochemical Society, 120, 784

117. Shingubara S,Okino O,Sayama Y,Sakayue Hand Takahagi T (1997)

Japanese Journal Applied Physics, 36, 7791

118. M. Ghorbani et al. (2006) On the growth sequence of highly ordered

nanoporous anodic aluminium oxide, Materials and Design, 27, 983–988

119. Smirnov, A. I. et al. (2003) Substrate-Supported Lipid Nanotube Arrays, J. American Chemical Society, 125, 8434-8435.

120. Kashi, M.A., Ramazani , A. (2005) The effect of temperature and

Concentration on theself-organized pore formation in anodic alumina, Journal of Physics D: Applied Physics, 38, 2396 – 2399.

121. Rahimi M. H. et al. (2009) Study the effect of stripping in two step

anodisation process on pore arrangement of nanoporous alumina, Applied surface science, 256, 12-16.

122. Gasparac, R., Kohli, P., Mota, M. O., Trofin, L., Martin, C. R. (2004) Template Synthesis of Nano Test Tubes, Nano letters, 4, 3, 513 - 516

123. Wang, F., Huang, H., Yang, S. (2009) Synthesis of ceramic nanotubes using AAO templates, Journal of the European Ceramic Society, 29, 1387 – 1391.

124. Son, S.J. et al, (2006) Template synthesis of multifunctional

nanotubes for controlled release, Journal of Controlled Release, 114, 143 – 152.

125. Buyukserin, F., et al. (2008) Antibody-functionalized nano test tubes target breast cancer cells, Nanomedicine, 3, 3, 283 -292.

126. Rajesh, R., (2008) Surface modification of silica nanoparticles,

Unpublished Doctoral Thesis submitted to University of Akron, 8 – 9.

127. X. Feng et al. (1997) Functionalized monolayers on Ordered

mesoporous supports, Science, 276, 923 – 926.

128. H. Azour et al. (2000), Fourier transform infrared spectroscopic

characterization of grafting of 3-aminopropyl silanol onto


aluminum/alumina substrate , Spectrochimica Acta - Part A, 56, 1627-


129. Chandran S. P. et al. (2008) Tunable surface modification of silica

nanoparticles through ‘click’ chemistry, Current Science, 95, 9, 1327 -


130. Françon, V., Chen, Q. Z., Charter, R., Boccaccini A. (2007) Surface

microstructure and chemistry of functionalised Bioglass-derived glass- ceramics, European Journal of Glass Science & Technology –A, 48, 3,


131. P. Silberzan et al. (1991), Silanisation of surfaces: A new method of

constructing pure or mixed monolayers, Langmuir, 7, 1647-1651.

132. B. J. Jeon et al. (2002) Surface modification of silica particles with

organoalkoxysilanes through two-step (acid-base) process in aqueous solution, Journal of Ceramic Processing Research, 3, 3, 216-221

133. S. A. Kulkarni et al. (2008) Tuning the hydrophobic properties of silica

particles by surface silanisation using mixed self- assembled monolayers, Journal of Colloid and Interface Science, 318, 372–379

134. C. M. Halliwell et al. (2001) A Factorial Analysis of Silanization

Conditions for the Immobilization of Oligonucleotides on Glass Surfaces, Analytical Chemistry, 73, 2476-2483

135. K. M. R. Kallury et al. (1988) X-ray photoelectron spectroscopy of

silica treated with polyfunctional silanes, Analytical Chemistry, 60, 169 -


136. K. Meyer et al. (1994) Porous solids and their characterization - Methods of investigation and application, Crystal Research and

Technology, 29, 903-930.

137. R. Mikhail et al. (1983) Microstructure and Thermal Analysis of Solid Surfaces, Wiley, Chichester.

138. K. Meyer et al. (1997) Certification of Reference Material with Special Emphasis on Porous Solids, Crystal Research and Technology, 32, 173-


139. P. Klobes et al. (2006) Porosity and specific surface area measurements

for solid materials, National Institute of Standards and Technology –

special publication, Washington, USA

140. S. Lowell et al. (2004) Characterization of porous solids and powders :

surface area, pore size, and density, Kluwer Academic Publishers,

141. M. Barkzewski et al. (2006) Solid state characterization of

pharmaceuticals, Advanced solid state analysis inc., Poland

142. S. Lowell (1984) Powder surface area and porosity, Powder Technology

series, Chapman and Hall Publication, 2.

143. K. S. W. Sing et al. (1985) Reporting physisorption data for Gas/Solid

Systems with Special Reference to the Determination of Surface Area and Porosity (Recommendations 1984), Pure and Applied Chemistry, 57, 603-


144. F., Rouquerol et al. (1999) Adsorption by Powder and Porous Solids, Academic Press, London, UK

145. S. Brunauer et al. (1938) Adsorption of Gases in multimolecular

layers, Journal of American Chemical Society, 60, 309 - 319

146. P. Barrett et al. (1950) Pore Volume and Area Distributions in Porous

Substances. I. Computation from Nitrogen Isotherms, Journal of American Chemical Society, 73, 373 – 380.

147. Dubinin, M. M., & Radushkevich, L. V., (1947) On the Characteristic

Curve Equation for Active Charcoals, Proc. Acad. Sci. USSR, 55, 327-329.


148. M. Polanyi (1963) The potential theory of adsorption, Science, 141,

1010 - 1013

149. Dubinin, M. M. (1969) The potential theory of adsorption of gases and

vapours for adsorbents with energetically nonuniform surfaces, Chemical Reviews, 60, 235-241

150. Horvath, G, & Kawazoe, K. J., (1983) Method for the Calculation of

Effective Pore Size Distribution in Molecular Sieve Carbon, Journal of Chemical Engineering of Japan, 16, 470 -475.1983

151. Evans, R. (1992) In Fundamentals of Inhomogeneous Fluids, edited by D. Henderson, Marcel Dekker Inc., New York, 1992, 85 – 177.

152. N. A. Seaton et al. (1989) "A New Analysis Method for the

Determination of the Pore Size Distribution of Porous Carbons from

Nitrogen Adsorption Measurements, Carbon, 27, 853-861.

153. C. M. Lastoskie et al. (1993) Pore Size Distribution Analysis of

microporous carbons: a Density Functional Theory Approach, Physical Chemistry, 97, 4786-4796.

154. J.P. Olivier et al. (1995) Modeling Physical Adsorption on Porous and Nonporous Solids using Density Functional Theory, Journal of Porous

Material, 2, 9-17.

155. P. Ravikovitch et al. (1997) Evaluation of Pore Structure Parameters of MCM-41 Catalyst Supports and Catalysts by Means of Nitrogen and Argon Adsorption, Journal of Physical Chemistry B, 101, 3671-3679.

156. P. Ravikovitch et al. (2001) "Density Functional Theories and

Molecular Simulations of Adsorption and Phase Transitions in Nanopores,

Physical Review E, 64, Article No. 011602, 1-20.

157. J. A. McCauley et al. (1995)Physical characterization of pharmaceutical

solids edited by H. G. Brittain, Marcel Dekker Inc., New York, USA, 223 -


158. D. Giron (1999) Thermal Analysis, Microcolorimetry and the combine

techniques for the study of pharmaceuticals, Journal of Thermal Analysis

and Calorimetry, 56, 1285 – 1304.

159. A. Ulman (1991) An introduction to ultrathin organic films from

Langmuir – Blodgett to self – assembly, Academic Press Inc., London, UK,

79 – 83.

160. A. Frey et al. (1997) Peptomer Aluminum Oxide Nanoparticle

Conjugates as Systemic and Mucosal Vaccine Candidates: Synthesis and

Characterization of a Conjugate Derived from the C 4 Domain of HIV-1 MN Gp120, Bioconjugate Chemistry, 8, 424 – 433.

161. C. Leger et al. (1996) Preparation and properties of surface modified ceramic membranes. Part III. Gas permeation of 5 nm alumina membranes modified by trichloro-octadecylsilane, Journal of Membrane Science, 120,187-195.

162. J. Qiao et al. (2005) Morphology-controllable preparation of 1D

poly(vinyl pyrrolidone) nanostructured arrays, Nanotechnology, 16, 433–


163. P. Asanithi et al. (2009) Carbon-Nanotube-Based Materials for Protein Crystallization, Applied materials and interfaces, I, 6, 1203 - 1210