These materials are known as SBA-15 (Santa Barbara Amorphous) materials. SBA-15 ...... B. Muñoz, A. Rámila, J. Pérez-Pariente, I. DÃaz, and M. Vallet-RegÃ.
Fabrication of Inorganic Nanoporous Oxide and Study on their Biopolymer Adsorption Characteristics ******* A dissertation submitted to the Faculty of Engineering and Technology of Jadavpur University for the partial fulfillment of the requirement for the degree of Master of Technology In the Department of Food Technology and Biochemical Engineering 2010-2012 by Poonam Singha (Registration no. 113625) Under the guidance of Dr. Dipankar Halder & Dr. Atanu Mitra
© Copyright by Poonam Singha (2012)
Faculty of Engineering and Technology Department of Food Technology and Biochemical Engineering Jadavpur University Kolkata – 700032, India
Declaration of originality and compliance of academic ethics I hereby declare that this thesis contains literature survey and original research work by the undersigned candidate, as part of her Master of Technology in Food Technology and Biochemical engineering studies. All information in this document have been obtained and presented in accordance with academic rules and ethical conduct. I also declare that, as required by these rules and conduct, I have fully cited and referenced all material and results that are not original to this work.
Name:
Poonam Singha
Examination Roll Number:
M4FTB12-01
Thesis Title:
Fabrication of inorganic nanoporous oxide and study on their biopolymer adsorption characteristics.
Registration no.:
113265 of 2010-2011
Signature: Date:
ii
Faculty of Engineering and Technology Department of Food Technology and Biochemical Engineering Jadavpur University Kolkata – 700032, India
Certificate of Recommendation We hereby recommend the thesis entitled “Fabrication of inorganic nanoporous oxide and study on their bipolymer adsorption characteristics” prepared under supervision of Dr. Dipankar Halder and Dr. Atanu Mitra by Poonam Singha, student of second year has been evaluated by us and found satisfactory. It is therefore, being accepted in partial fulfillment for the requirement for awarding of degree of Master of Technology in Food Technology and Biochemical Engineering.
Dr. Dipankar Halder
Dr. Atanu Mitra
Assistant Professor,
Assistant Professor,
Department of F.T.B.E.
Department of Chemistry.
Jadavpur University.
Shree Chaitanya College.
Kolkata-700032.
Habra, West Bengal.
Dr. Lalita Gauri Ray Head, Department of F.T.B.E. Jadavpur University. Kolkata-700032. iii
Faculty of Engineering and Technology Department of Food Technology and Biochemical Engineering Jadavpur University Kolkata – 700032, India
Certificate of Approval This is to certify that Ms. Poonam Singha has carried out the research work entitled “Fabrication of inorganic nanoporous oxide and study on their bipolymer adsorption characteristics” under our supervision, at the Department of Food Technology and Biochemical Engineering, Jadavpur University, Kolkata. We are satisfied that she has carried out this work independently with proper care and confidence. We hereby recommend that this dissertation be accepted in partial fulfillment of the requirement for awarding of degree of Master of Technology in Food Technology and Biochemical Engineering. We are very much pleased to forward this thesis for evaluation.
Dr. Dipankar Halder
Dr. Atanu Mitra
Assistant Professor,
Assistant Professor,
Department of F.T.B.E.
Department of Chemistry.
Jadavpur University.
Shree Chaitanya College.
Kolkata-700032.
Habra, West Bengal.
iv
DEDICATION
This dissertation is dedicated to my parents, who worked very hard to support my education.
v
ACKNOWLEDGEMENTS
I would like to take this opportunity to express my sincerest thanks and appreciation to my supervisors, Dr. Dipankar Halder and Dr. Atanu Mitra, for their invaluable guidance, consistent encouragement, valuable advice and support during my studies and research work at Jadavpur University. I would also like to extend my gratitude to Dr. Uma Ghosh (ex- H.O.D.), Prof. (Mrs.) Lalita Gauri Ray (H.O.D.), Prof. Utpal Raychowdhuri, Dr. Runu Chakraborty and Prof. Amit Kumar Ghosh for their support. I sincerely thank Dr. Paramita Bhattacharyya for her support and encouragement during my post-graduate studies. It was my pleasure to work with wonderful colleagues and friends. I would like to express my thanks to my seniors Debosree Das, Gargi Dinda, Mousumi Basu, Vivek Kumar and Avneesh Kumar Singh for their help. I am thankful to research scholars, library staffs and laboratory staffs for their valuable help and co-operation. In the end I would like to acknowledge my friends: Abhijit Das, Dipanwita Roy, Shahid Haradwala and Tanumoy Mondol for their love and support. Finally, I want to express my heartfelt thanks to my family, to whom this thesis is dedicated.
vi
TABLE OF CONTENTS
1.
List of Tables
ix
List of Figures
x
Abbreviation
ix
INTRODUCTION AND OBJECTIVES
1
1.1 Bioadsorption
1
1.2 Mesoporous materials
3
1.2.1 Introduction and Historical Developments
3
1.2.2 Synthesis strategies of Mesoporous Materials
6
1.3 Examples of bimolecules adsorbed onto mesoporous silicates 1.3.1 Bovine serum albumin
9
1.3.2 Lysozyme
9
1.3.3 Deoxyribonucleic acid
10
1.4 Research Objectives and Significance
11
1.5 Dissertation Structure
11
References
2.
7
12
SYNTHESIS AND CHARACTERIZATION OF MCM-41 MATERIALS
21
2.1 Introduction
21
2.2 Experimental
22
2.2.1 Materials
22
2.2.2 Synthesis of MCM-41
22
2.2.3 Test for hydrothermal stability
vii
22
2.2.4 Characterization of MCM-41
23
2.3 Results and discussion
24
2.3.1 Characterization of MCM-41
24
2.4 Conclusions
26
References
3.
27
SBA-15 MATERIAL FOR ADSORPTION OF BIOMOLECULES
28
3.1 Introduction
28
3.2 Experimental
30
3.2.1 Materials
30
3.2.2 Synthesis of SBA-15
30
3.2.3 Test for hydrothermal stability
30
3.2.4 Characterization of SBA-15
30
3.2.5 Adsorption studies
31 32
3.3 Results and discussion 3.3.1 Characterization of SBA-15
32
3.3.2 Adsorption studies
35 39
3.4 Conclusions
40
References
4
SUMMARY
42
viii
LIST OF TABLES Table 1.1:
Possible pathways for the synthesis of ordered mesoporous materials…….............6
Table 1.2:
Adsorption of Biologically Interesting Compounds on Ordered Mesoporous Materials………………………………………………………………….………8-9
Table 2.1:
Synthesis and aging conditions of the as-synthesized materials………………….23
Table 3.1:
Characteristic peaks of the three sample obtained by FTIR……………………...34
Table 3.2:
Parameters of kinetic models of Lysozyme adsorption on SBA-15 at different temperatures………………………………………………………………………38
ix
LIST OF FIGURES Figure 1.1:
Classification of pore size ranges and examples of materials with typical pore-size distribution…………………………………………………………………………4
Figure 1.2:
Possible mechanistic pathways for the formation of MCM-41: (1) Liquid-crystalphase initiated and (2) silicate-anion-initiated…………………………………….5
Figure 2.1:
XRD pattern of the as-synthesized samples (a) sample 1(S1); (b) sample 2(S2); (c) sample 3(S3); (d) sample 4(S4)…………………………………………………..25
Figure 2.2:
XRD patterns of (a) calcined sample;
(b) calcined sample after hydrothermal
treatment………………………………………………………………………….26 Figure 3.1:
Schematic illustration of synthetic route for porous SBA-15……………………29
Figure 3.2:
XRD patterns of SBA-15 (a) as-synthesized material, (b) calcined for 6 h, (c) calcined for 12 h, and (d) 12 h calcined sample after hydrothermal treatment…..33
Figure 3.3:
FTIR spectra of SBA-15 (a) as-synthesized, (b) 6 h calcined, and (c) 12 h calcined………………………………………………………………….………..34
Figure 3.4:
1
Plot of Γ,2 vs C2 for adsorption of Lysozyme on SBA-15 at 25o C and pH 9.7………………………………………………………………………………...36
Figure 3.5:
Kinetic plots for adsorption of Lysozyme on SBA-15; (a) pseudo-second-order, and (b) diffusion model…………………………………………………………..37
Figure 3.6:
Adsorption amount of Lz on SBA-15 (8.3 nm) as a function of time……………39
x
ABBREVIATIONS
BSA
Bovine serum albumin
CPG
Controlled pore glass
CTAB
Cetyl trimethyl ammonium bromide
DNA
Deoxyribonucleic acid
FTIR
Fourier transform infrared
HMS
Hexagonal mesoporous silicates
Lz
Lysozyme
MCM
Mobil Corporation of Matter
MNP
Mesoporous nanoparticle
MSU
Michigan State University
P123
poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide)
RNA
Ribonucleic acid
RT
Room temperature
SBA
Santa Barbara Amorphous
TEOS
Tetra ethyl ortho silicate
UV-vis
Ultraviolet visible
XRD
X-ray diffraction
Formulae HCl
Hydrochloric acid
NaCl
Sodium chloride
NaHCO3
Sodium bicarbonate
NaOH
Sodium hydroxide
NH4OH
Ammonium hydroxide
xi
Nomenclature 1
Γ,2
Moles of enzyme adsorbed per kilogram of mesoporous material
C2
Molar concentration of the protein in a solution
t
C2
Molar concentration in the bulk solution at adsorption equilibrium
k2
Rate constant of pseudo-second-order adsorption
Kd
Intraparticle diffusion rate constant
Qe
Amount of Lz adsorbed at equilibrium
Qt
Amount of Lz adsorbed at time t
R2
Correlation coefficient
t
Time
t1/2
Square root of time t
V
Volume
Vt
Volume of solution per kg of solid powder
W
Grams of solid adsorbent
xii
Chapter 1 Introduction and Objectives 1.1.
Bioadsorption Protein adsorption onto solid surfaces has attracted much attention due to its scientific
importance and applications in different areas [1-3]. In industries, such as food and medical, it is essential to remove even a small amount of deposited protein which may lead to adverse biological consequences due to further adsorption of fibrous proteins [4-6]. The adsorption (immobilization) of proteins on inorganic materials is crucial because of the potential to improve the stability of enzymes under extreme conditions [7]. The controlled adsorption of proteins is essential in the fields of enzymatic catalysis, biosensors, and disease diagnostics [8-10]. Protein adsorption/immobilization onto silicate and other inorganic matrixes has been reviewed by Weetall [11] and numerous studies of protein adsorption onto silicate surfaces are to be found in the literature [12 – 14]. In the 1970s, Weetall et al. pioneered the use of porous inorganic materials for the immobilization of biological molecules and in particular the use of controlled pore glass (CPG) [15 – 20]. CPG materials of pore sizes ranging from 300 to 2000 Å have frequently been reported in such studies, and generally, it has been found that the pore size of the CPG needs to be significantly larger than the biomolecule of interest. The major disadvantages in using such materials are their cost and more importantly their surface area, which rapidly decreases with increasing pore size [16, 20]. Enzymes are excellent biocatalysts with high specificity, selectivity, and efficiency and are promising candidates for catalysis, sensor, and separation applications, but their use is currently limited due to their sensitivity to temperature, pH, organic solvents, detergents, instability, high cost, and the problems related to product contamination, separation and difficulties in recovering active enzyme for reuse [21]. There have been many approaches to immobilize enzyme or other biological molecules, such as sol–gel encapsulation, cross–linking crystals, protein–polymer conjugates [22] and microencapsulation [65]. Sol–gel matrices such as xero- and aerogels are known to possess highly variable pore sizes and their preparation involves 1
CHAPTER 1
INTRODUCTION
harsh conditions or reagents which usually cause denaturation of the proteins during the encapsulation process [23]. Enzymes bound to solid supports can overcome some of these limitations, but binding to solids often produces a change in its physicochemical properties which may distort the enzyme structure and decreases catalytic efficiency [24-26]. An important advantage of physical adsorption is that it is reversible. Denatured enzyme can be replaced by changing the pH or the ionic strength of the react ion medium, followed by adsorption of fresh enzyme. However, desorption can also be a major drawback if it occurs during reaction. The efficiency of the physical adsorption depends on several parameters - the size of the protein to be adsorbed, the specific surface area of the carrier, the texture (porosity, pore size), and the surface chemistry [28]. As most proteins adsorb with high affinity to hydrophobic surfaces, these proteins generally have less native structure than the same protein adsorbed on hydrophilic surfaces [29, 30]. It has also been seen that an increase in electrostatic interaction is generally accompanied by a reduction in the native structure [31 – 33]. The pH of adsorption is equally important since ionic interactions can either increase or decrease final enzyme loading and can also influence residual activity [28].
Usually, the
maximum adsorption is observed at a pH close to the isoelectric point of the enzyme [34]. The current challenge in adsorbent development is molecular specificity [66]. In the past decades, physical adsorption has been studied in combination with different types of particulate carrier materials including different polymers [35, 36], molecular sieves [37 – 40] silica and silica-alum in a composite [41 – 43], and carbonaceous materials [44 – 46]. Typically the use of a porous support is preferred since the enzyme will not be adsorbed only at the outer surface of the material but within the pores as well [28]. The research of the utilization of mesoporous nanoparticles (MNPs) has been interesting and burgeoning in the fields of the bio-separation due to the convenient and time-saving process [67]. Molecular crowding theory and modeling efforts have predicted that a protein inside a confined space would be stabilized by some folding forces not present for protein for bulk solution [47, 48]. Unfolding of protein chain, which could lead to protein denaturation, would be less likely to occur inside the confined space due to excluded volume. They exhibit their higher activity when confined in biological cells [49]. Mesoporous silica material belongs to a class of inorganic material which has a large pore surface area, a highly ordered pore structure with 2
CHAPTER 1
INTRODUCTION
uniform mesopores, and relatively good chemical and mechanical stability [50 – 52]. These advantages of mesoporous silica materials render them a good substrate for hosting catalysts, adsorbing proteins, and constructing biosensors [52]. Silica and silica based materials have been successfully used as gene or drug delivery system, bio-separation agents, diagnostic agents and sensors [53 – 60]. The synthesis of nanoscale magnetic particles has been intensively pursued because of their broad applications including protein and enzyme immobilization, RNA and DNA purification, separation of biochemical products and catalysts, and targeted drug delivery [61 – 64].
1.2.
Mesoporous Materials
1.2.1. Introduction and Historical Development Porous materials have been used extensively as solid supports in heterogeneous catalysis and separations. They have the ability to interact with atoms, ions and molecules not only at the surfaces, but throughout the bulk of the material [68]. According to the IUPAC system of classification, porous materials are characterized on the basis of pore dimension: microporous materials have pore size < 2 nm, mesoporous materials have pore sizes in the range of 2 to 50 nm, and macroporous materials have pores > 50 nm, as shown in Figure 1.1 [69]. Materials having pore diameter in the nanometer range (1 – 100 nm) are loosely termed as “nanoporous materials” [80]. Various kinds of porous materials (anodic alumina, pillared clays, and carbon) and their applications have been described in the literature [70]. Zeolites are microporous materials; they have a narrow and uniform pore size distribution. In the past, zeolites have been used as acidic, basic, and redox catalysts. Zeolite can separate molecules on the basis of their size by selectively absorbing a small molecule from a mixture containing molecules too large to enter its pores. This limits the use of zeolites for large molecules. Henceforth, research has been focused on increasing the pore size into the mesoporous range, which would allow larger reactants to enter the particle.
3
CHAPTER 1
INTRODUCTION
Figure 1.1 Classification of pore size ranges and examples of materials with typical poresize distribution The first ordered mesoporous materials were reported in a U.S Patent in 1971, but due to lack of analysis the immense potential and remarkable features of these materials were not recognized [71]. In 1992, Mobil Oil Corporation scientists developed a closely related material, and recognized the outstanding features of these silica based materials [50]. MCM-41 (Mobil Corporation of Matter) is the first family of materials synthesized by this group using surfactant templated synthesis. They proposed it as liquid crystal templating mechanism illustrated in Figure 1.2, in which the silicate material forms inorganic walls between ordered surfactant micelles. MCM-41 materials possess a hexagonal array of one-dimensional pores with a sharp pore-size distribution and large specific surface area.
4
CHAPTER 1
INTRODUCTION
Figure 1.2 Possible mechanistic pathways for the formation of MCM-41: (1) Liquid-crystalphase initiated and (2) silicate-anion-initiated. Other related materials such as MCM-48 (cubic) and MCM-50 (lamellar) were also reported in similar publications. The pore size of MCM materials significantly extended the range of chemical reactions that could occur in the ordered pores; however, these channels still remained inaccessible to larger biomolecules. In a separate effort, Yanagisawa et al. synthesized folded sheet mesoporous (FSM) materials in 1990 [72]. In 1998, Stucky and coworkers [73] synthesized a new class of materials with pore size in the upper mesoporous range (~ 30 nm); this synthesis used block copolymers as a structure directing agent. These materials are known as SBA-15 (Santa Barbara Amorphous) materials. SBA-15 materials proved to be superior to MCM-41 materials, even though the physical structures of the SBA-15 and MCM-41 materials are quite similar. The thicker pore walls of SBA-15 provided better hydrothermal stability to these materials. This remarkable improvement to MCM-41 type materials provided the opportunity to study adsorption and catalysis of large biomolecules on SBA-15 type large-pore materials. Ordered mesoporous materials possess high specific surface, high pore volume, highly ordered pore structure and tailored pore diameter. These properties enabled
the
researchers
to
use
these
materials
for
biocatalysis
and
bioseparations. Mesoporous materials have been shown to have immense potential in applications for the separation of biological macromolecules by chromatography. SBA-15 materials with large pore 5
CHAPTER 1
INTRODUCTION
size (~ 20-30 nm) can be used for size-selective separations of proteins, with high protein loading within the ordered cylindrical pores. 1.2.2. Synthesis strategies of Mesoporous Materials The first synthesis of MCM-41 materials in 1992 was a major breakthrough in materials engineering; since then there has been astounding progress in the development of various classes of mesoporous solids based on the templated synthesis mechanism used in the original MCM-41 synthesis [50]. Mesoporous materials can be classified on the basis of the template used for synthesis and the interaction between inorganic-organic species; Table 1.1 summarizes these synthesis strategies. MCM-41 materials are synthesized using cationic or anionic surfactants [74]; however, their large-pore counterparts, SBA-15, use tri-block copolymers as the templating agent [47]. Table 1.1 Possible pathways for the synthesis of ordered mesoporous materials [74]. Template
Interaction
Conditions
Materials
Ionic surfactant
Direct
Basic
MCM-41, MCM-48, FSM-16, metal
(ionic)
Neutral-Basic
oxides etc. AMS
Intermediate
SBA-1, -2 and -3, HMS
(Ionic) Non-ionic
Non-ionic
Acidic
HMS,MSU, SBA-15
-
-
CMK-n
Copolymer Nano-Casting
The first mesoporous materials were synthesized using ionic surfactants such as quaternary ammonium ions; the formation of the organic-inorganic assembly is driven by electrostatic interactions between the positively charged surfactant and negatively charged silicate species in the solution [74]. The mesopore size of these materials is primarily 6
CHAPTER 1
INTRODUCTION
controlled by the alkyl chain length of the surfactant used. However, auxiliary chemicals such as n-alkanes [75], fatty acids [76], and aromatics [77] are also used for the expansion of pores. The use of two surfactants with different chain length allows fine tuning of the pore size between those obtained for long- and short-chain surfactants [78].
Pinnavaia et al. [79]
developed strategies to use nonionic surfactants as structure directing agents for the synthesis of mesoporous materials. They used neutral surfactants such as primary amines poly (-ethylene oxides) to prepare MSU (Michigan State University) and HMS (hexagonal mesoporous silicate) materials with worm-like disordered structures. This system used hydrogen bonding as the driving force to form an assembly between the non-ionic surfactant and silica that allows the successful recovery of surfactant using solvent extraction with ethanol or acidified water. MSU and HMS have specific surface areas and pore volumes comparable to that of MCM-41. However, these materials have a somewhat broader pore-size distribution and disordered structure, which are major drawbacks. One of the most versatile group of surfactants are the tri-block co-polymers consisting of poly(ethylene oxide)x-poly(propylene oxide)ypoly(ethylene oxide)x, commercially known as pluronics.
Pluronics can form liquid
crystal templates that can be used for the synthesis of ordered mesoporous materials with large
pore
size
under highly acidic conditions [73]. SBA mesoporous materials ARE
synthesized using this pathway. These exhibit thick pore walls (3-5 nm) and larger pore sizes (520 nm) that significantly improve the hydrothermal properties of these materials as compared to its small-pore counterpart, MCM-41.
1.3.
Examples of biomolecules adsorbed onto mesoporous silicates
Since the synthesis of ordered mesoporous silicates (MPS) in 1992 [50, 51] the first encapsulation of enzyme on mesoporous silica was reported by Balkus et al, 1996 [22]. Since then there have a number of reports in the literature describing enzymes or proteins inside mesoporous materials, separation of the same using mesoporous materials and few works on the activity of the immobilized enzyme. The main driving forces in the adsorption of biomolecules onto porous materials are electrostatic, hydrogen bonding and weak Van der Waals interactions [88, 89, 90]. Deere et al [91] suggested hydrophobic interactions rather than electrostatic interactions dominate the adsorption of proteins onto some silica materials. In general it has been found that proteins will adsorb in pores that are larger than the hydraulic radius of the protein 7
CHAPTER 1
INTRODUCTION
[92]. Adsorption onto porous materials is a strong function of protein structure [108]. Table 1.2 shows examples of few biological molecules that have been adsorbed onto ordered mesoporous silica.
Table 1.2 Adsorption of Biologically Interesting Compounds on Ordered Mesoporous Materials [80] Adsorbate
Adsorbant
lysine, phenylalanine,
MCM-41
histidine, glutamic acid, asparaginin, etc chorophyll a
FSM-16
Vitamin E
CMK-1, CMK-3
Vitamin B2
MCM-41, MCM-48
Catalase
SBA-15
Conalbumin
SBA-15, thiol-functionalized SBA-15, APTS- modified MCF
Cytochrome c
MCM-41, MCM-48, SBA-15, AlMCM-41, AlSBA-15, CMK-3, thiol-functionalized SBA-15
HRP
FSM-16, MCM-41, SBA-15
β-lactoglobulin
SBA-15, thiol-functionalized SBA-15
Lysozyme
MCM-41, SBA-15, AlMCM-41, AlSBA-15, CMK-1, CMK-3
8
CHAPTER 1
INTRODUCTION
Myoglobin
SBA-15, thiol-functionalized SBA-15
Ovalbumin
SBA-15, thiol-functionalized SBA-15
Papain
MCM-41
Trypsin
SBA-15, thiol-functionalized SBA-15, MCM-41, MCM-48
Bovine serum albumin
SBA-15, thiol-functionalized SBA-15
1.3.1. Bovine Serum Albumin (BSA) The adsorption of BSA on mesoporous supports was studied by several groups [66, 94, 96 - 111]. BSA is reported to have dimensions of 40 Å × 40 Å × 140 Å and is a fairly large (MW= 66400 Da) ellipsoidal protein [80]. The protein contains 17 disulfide bridges and one free SH group, which can cause it to form covalently linked dimers [82]. Formation of covalent oligomers takes place when intermolecular disulfide bridges are formed by SH group exchanging with saturated S-S bridges of another monomer [83]. At the isoionic point (about pH 5.2) at which essentially all of the carboxylic acids are deprotonated and the amino, guanidino and imidazole groups are protonated, the total charge consists of about 100 each of positive and negative charges [81]. The isoelectric point in 0.15 M NaCl is about 4.7; bound chloride ions cause it to be lower than the isoionic point. BSA undergoes expansion below pH 4.3 and above pH 10.5 [84]. The helical content is high, 68%, and the content of β- sheet is 18% (pH 7.4) [85]. The tertiary structure comprises three very similar domains [86].
1.3.2. Lysozyme (Lz) Lysozyme (Lz) is a well-known enzyme with hydrolyzing ability to destroy the polysaccharides framework present in the bacterial cell wall [27]. Lz is an antimicrobial protein that is prevalent in ocular fluid, thus the adsorption of Lz on contact lenses is a considerable problem in ophthalmology [87]. It has many uses in food processing such as in food packaging films and in the preservation of meats such as sausages [93]. Four disulfide bridges are 9
CHAPTER 1
INTRODUCTION
responsible for the stabilization of the structure of the protein [112]. The Lz has a prolate spheroid shape with a molecular size of 3.0 nm × 3.0 nm × 4.5 nm. The isoelectric point of Lz is 11.4. [95.]. Lz is an unusually stable protein [113]: its thermal stability is high and characterized by a transition temperature of 77o C at neutral pH and dilute salt, and no significant change of conformation over the pH range 1.2-11.3 in dilute salt at moderate temperatures has been found. Lz is known to form dimers in alkaline solution [114-116]. It is an ideal model for adsorption onto mesopores, and extensive studies have been done with typical mesoporous materials (e.g. MCM-41, SBA-15, FDU) [117-120.]
1.3.3. Deoxyribonucleic acid (DNA)
Currently, only a few methods are used to deliver and protect DNA during cellular transfection. The most common and simple method of gene delivery is the viral vector system, whereby a nonpathogenic virus is used as the method of transport. More recently, synthetic nanoparticles with cationic surfaces have provided an alternative approach to DNA delivery [121]. Amorphous silica nanoparticles are particularly attractive due to their high chemical resistance to microbial attack, low toxicity, thermal stability, and ease of modification [122.]. Several attempts have been made to modify the external surface of silica nanoparticles for DNA binding by the attachment of cationic linkers that electrostatically bind DNA molecules [123125]. Acid-prepared mesoporous silica (APMS) can be synthesized in less than 2 h, and the particle size and pore diameter of APMS are easily controlled simply by altering a set of standard reaction conditions. Its internal and external surfaces are easily functionalized through reaction with organosilanes. Solberg and Landry [126] observed fluorescently labeled DNA within the pores of their mesoporous silica materials through confocal microscopy. This method tried to observe DNA in mesopores directly. Nevertheless, the resolution of optical microscopy was limited at the micro meter level, thus making it impossible to get clear photographs of nanomaterials [127].
10
CHAPTER 1
1.4.
INTRODUCTION
Research Objectives and Significance
The broad objective of the research reported in this dissertation is to develop and characterize ordered, siliceous support for adsorption of biomolecules. The specific objectives of this research project were to: Synthesis of large pore ordered mesoporous silica. Characterize the surface using X-ray diffraction and IR-spectroscopy. Investigate adsorption equilibrium and kinetics of SBA-15 with selected proteins.
1.5.
Dissertation structure
The significance, objectives and content of the four chapters of this dissertation has been briefly outlined below: Chapter 1: General Introduction This chapter provides general introduction to bioadsorption and briefly discusses the development of mesoporous materials and its synthesis strategies. This chapter touches on characteristics of few biomolecules used by researchers for adsorption onto mesoporous materials. Chapter 2: Synthesis of MCM-41 materials This chapter discusses a synthesis process of MCM-41 and characterizes the same. Chapter 3: Synthesis of SBA-15 materials for adsorption of biomolecules This chapter describes synthesis of large pore ordered SBA-15 and studies related to its adsorption capacities. Chapter 4: Summary
11
CHAPTER 1
INTRODUCTION
References
1. App. Biochem. Proteins at Interfaces II: Fundamentals and Applications; T. A. Horbett, J. L. Brash, Eds.; American Chemical Society: Washington, DC, 1995. 2. Proteins at Interfaces: Physicochemical and Biochemical Studies; J. L. Brash, T. A. Horbett, Eds.; American Chemical Society: Washington, DC, 1987. 3. J.D. Andrade, V. Hlady, AdV. Polym. Sci. 1986, 79, 1. 4. C. Sandu, R. K. Singh. Food Technol. 1991, 45, 84. 5. J. A. Hubbell. Bio/Technol. 1995, 13, 565. 6. K. Ishihara, H. Oshida, Y. Endo, T. Ueda, A. Watanabe, N. Nakabayashi. J. Biomed. Mater. Res. 1992, 26, 1543. 7. A. M. Klibanov. Science 1983, 219, 722. 8. G. A. Rechnitz. Chem. Eng. News. 1998, 66, 24. 9. B. D. Martin, B. P. Gaber, C. H. Patterson, D. C. Turner. Langmuir 1998, 14, 3971. 10. W. Inglis, G. H. Sanders, P. M. Williamsan, M. C. Davies, C. J. Roberts, S. J. B. Tendler. Langmuir 2001, 17, 7402. 11. H. H. Weetall. App. Biochem. Biotech. 1993, 41, 157. 12. A. Docoslis, W. Wu, R. F. Giese, C. Van Oss. J. Coll. and Surf. B: Biointerfaces. 1999, 13, 83. 13. M. Malmsten. J. Coll. Int. Sci. 1994, 166, 333. 14. U. Jönsson, B. Ivarsson, I. Lundström, L. Berghem. J. Coll. Inter. Sci. 1982, 90, 148. 15. H. H. Weetall. Science 1969, 166, 615. 16. H. H. Weetall. Met. Enzymol. 1976, 44, 134.
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INTRODUCTION
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Chapter 2 Synthesis and characterization of MCM-41 materials 2.1 Introduction Since the discovery of M41S family in 1992 by Mobil Corporation researchers [1, 2], much research has been conducted and reported on the synthesis of mesoporous materials due to their potential applications in areas like adsorption [3], drug delivery [4], biosensor [5], catalysis, separation and encapsulation [6 - 8]. These applications are favored due to their unique structural properties. The discovery opened new directions for the development of other novel mesoporous materials [9 - 13]. MCM-41 is a member of M41S family. MCM-41 is one of the most widely studied among the numerous mesoporous materials because of its structural simplicity and ease in preparation with negligible pore-networking and pore-blocking effects. MCM-41 has also been identified as the most suitable model mesopore adsorbent presently available for studying some of the fundamental features of adsorption such as the effects of pore size, hysteresis, etc., owing to its relatively uniform cylindrical/hexagonal pore channels. The prominent features of MCM41, and in general of most periodic mesoporous materials, are as follows: well-defined pore shapes (hexagonal/cylindrical); narrow distribution of pore sizes; negligible pore networking or pore blocking effects; very high degree of pore ordering over micrometer length scales; tailoring and fine-tuning of the pore dimensions (1.5-20 nm); large pore volumes (>0.6 cm3 g-1); exceptional sorption capacity (64 wt % of benzene at 50 Torr and 298 K) as a result of the large pore volume; very high surface area (~700-1500 m2 g-1); large amount of internal hydroxyl (silanol) groups (~40-60%); high surface reactivity; ease of modification of the surface properties; enhanced catalytic selectivity in certain reactions; and excellent thermal, hydrothermal, chemical, and mechanical stability [14]. The present work describes the attempt to synthesize MCM-41 following the method from an existing literature with the view to use the material for adsorption of biomolecules. The formation of ordered pore structure of the as-synthesized material and correspondingly 21
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SYNTHESIS AND CHARACTERIZATION OF MCM-41 MATERIALS
mesoporous material has been studied. This work reports the feasibility of synthesized MCM-41 as bioadsorbant.
2.2 Experimental 2.2.1. Materials TEOS (tetra ethyl ortho silicate) was purchased from Sigma Aldrich, CTAB (cetyl trimethyl ammonium bromide) was purchased from Merck, NH4OH (ammonium hydroxide), NaOH (sodium hydroxide) were of analytical grade and used without further purification.
2.2.2. Synthesis of MCM-41 MCM-41 was prepared according to a previously reported method [15] with little modifications. Reactions were performed at room temperature. Four set of experiments were carried for the synthesis. The starting compositions and aging conditions are presented in Table 2.1. As a typical run (sample 2; S2) with NaOH, synthetic procedures were as follows. 0.3 g of NaOH was mixed in 30 ml of distilled water. 1.01 g of CTAB (surfactant) was added into the solution with stirring (Magnetic stirrer, REMI 2 LH). 5.78 g TEOS was introduced under vigorous stirring condition, giving rise to white slurry. After stirring for 1 h, the resulting homogeneous mixture was crystallized under hydrothermal condition at 353 K for 1 day and then at 365 K for 4 days. The solid product was obtained by filtration, washed with distilled water, dried in air at 353 K and calcined in air at 823 K for 24 h to remove the surfactant.
2.2.3 Test for hydrothermal stability To examine the hydrothermal stability 0.5 g of the sample was continuously stirred with 50 g distilled water at 303 K for 24 hr. Then, the samples were filtered, washed with deionized water and air dried.
22
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SYNTHESIS AND CHARACTERIZATION OF MCM-41 MATERIALS
Table 2.1 Synthesis and aging conditions of the as-synthesized materials No
1
Sample
S1
Composition
5.19 (H2O)
Stirring
Stirring
Temperature
Time
RT
1h
RT
1h
pH
Aging
Aging
temperature
time
6
338 K
5 days
6
353 K
1 day
365 K
4 days
0.052(NaOH) 0.175(CTAB) 1(TEOS) 2
S2
5.19 (H2O) 0.052(NaOH) 0.175(CTAB) 1(TEOS)
3
S3
5.19 (H2O)
RT
1h
6
338 K
6 days
RT
1h
6
353 K
2 days
365 K
4 days
0.225(NH4OH) 0.175(CTAB) 1(TEOS) 4
S4
5.19 (H2O) 0.19(NH4OH) 0.175(CTAB) 1(TEOS)
2.2.4 Characterization of MCM-41 X-ray Diffraction (XRD) Low angle powder XRD for all the samples was measured by using Seifert 3000P X-ray diffractometer. The X-ray source was Cu Kα radiation (λ = 1.54186 Å).
23
CHAPTER 2
SYNTHESIS AND CHARACTERIZATION OF MCM-41 MATERIALS
2.3 Results and discussion 2.3.1. Characterization of MCM-41 The XRD patterns of the four as-synthesized MCM-41 materials are shown in figure 2.1. From the XRD patterns, it is found that NH4OH is not a good hydrolysis agent in the used concentration range (Figure 2.1 (c) and (d)). May be a prominent peak would have been possible in higher concentration of NH4OH. Whereas, more prominent peak is obtained by using NaOH as hydrolysis agent (Figure 2.1 (a) and (b)) with more clear sharp peak in case of sample 2 (S2). The d-spacing of S1 and S2 is calculated using Bragg’s equation and found to be ca. 4.3 and 4.2 nm respectively. The XRD patterns of the calcined sample and calcined sample after hydrothermal treatment are shown in Figure 2.2. We find from the XRD patterns that after calcination the structure of the material is completely lost as there is absence of any sharp peak.
24
CHAPTER 2
SYNTHESIS AND CHARACTERIZATION OF MCM-41 MATERIALS
100
d spacing = 4.3 nm
d spacing = 4.2 nm 400
S1 Intensity (a.u)
Intensity (a.u)
80
60
40
20
S2
300
200
100 0
0
2
4
6
8
10
2
4
2 (deg)
(a)
6
(b)
120
8
10
8
10
2 (deg)
100
100
Intensity (a.u)
Intensity (a.u)
80
S3 80
60
40
S4
60
40
20
20
0
0 2
4
(c)
6
8
10
2
4
(d)
2 (deg)
6
2 (deg)
Figure 2.1 XRD pattern of the as-synthesized samples (a) sample 1 (S1); (b) sample 2 (S2); (c) sample 3 (S3); (d) sample 4 (S4).
25
CHAPTER 2
SYNTHESIS AND CHARACTERIZATION OF MCM-41 MATERIALS
40 50 35 30
Intensity (a.u)
Intensity (a.u)
40
30
20
10
25 20 15 10 5 0
0 2
4
(a)
6
8
10
2
4
(b)
2 (deg)
6
8
2 (deg)
Figure 2.2 XRD patterns of MCM-41 (a) calcined; (b) calcined sample after hydrothermal treatment.
2.4 Conclusion Initially as-synthesized ordered mesostructure was obtained but they are not thermally stable. After both calcination and hydrothermal treatment, mesostructure has collapsed indicating that it is not suitable as an adsorbant. Further work is needed to prepare thermally stable MCM41 by changing synthesis conditions.
26
10
CHAPTER 2
SYNTHESIS AND CHARACTERIZATION OF MCM-41 MATERIALS
References 1. C. T. Kresge, M. E. Leonowicz, W. J. Roth, J, C. Vartuli, J. S. Beck. Nature 1992, 359, 710712. 2. J. S. Beck, J. C. Vartuli, W. J. Roth, M. E. Leonowicz, C. T. Kresge, K. D. Schmitt, C. T.-W. Chu, D. H. Olson, E. W. Sheppard, S. B. McCullen, J. B. Higgins and J. L. Schlenker, J. Am. Chem. Soc. 1992, 114, 10834. 3. Schuth, F. Ber. Bunsenges. Phys. Chem. 1995, 99, 1306. 4. B. Muñoz, A. Rámila, J. Pérez-Pariente, I. Díaz, and M. Vallet-Regí. Chem. Mater. 2003, 15 (2), 500–503. 5. C. L. Zhu, C. H. Lu, X. Y. Song, H. H. Yang, and X. R. Wang. Bioresponsive J. Am. Chem. Soc. 2011, 133 (5), 1278–1281. 6. R. Hoppe, A. Ortlam, J. Rathousky, G. Schulz, E.A. Zukel. Microporous Mater. 1997, 8, 267. 7. E. Chomski, O. Dag, A. Kuperman, N. Coombs, G.A. Ozin, Chem. Vap. Deposition 1996, 2 (1), 8. 8. C.G. Wu, T. Bein, Science 1994, 264, 1756. 9. Q. Huo, D. I. Margolese, U. Ciesla, D. G. Demuth, P. Y. Feng, T. E. Gier, P. Sieger, A. Firouzi, B. F. Chmelka, F. Schuth, G. D. Stucky. Chem. Mater. 1994, 6, 1176. 10. A. Sayari, I. Moudrakovski, J. S. Reddy, C. I. Ratcliffe, J. A. Ripmeester, K. F. Preston. Chem. Mater. 1996, 8, 2080. 11. D. M. Antonelli, J. Y. Ying. Chem. Mater. 1996, 8, 874. 12. G. G. Janauer, A. Dobley, J. Guo, P. Zavalij, M. S. Whittingham. Chem. Mater. 1996, 8, 2096. 13. U. Ciesla, S. Schacht, G. D. Stucky, K. K. Unger, Schuth, F. Angew. Chem. Int. Ed. Engl. 1996, 35, 541. 14. P. Selvam, S. K. Bhatia, and C. G. Sonwane. Ind. Eng. Chem. Res. 2001, 40, 3237-3261. 15. H. Chen, Y. Wang. Ceramics International. 2002, 28, 541–547.
27
Chapter 3 SBA-15 material for adsorption of biomolecules 3.1 Introduction Developments in biotechnology have led to an enormous increase in the exposure of proteins to non-biological solid surfaces in applications such as artificial implants, protein purification strategies, biosensors, and drug delivery systems [1]. Protein adsorption is a complex process in which the structural stability of a protein, the ionic strength and the pH of the solution and surface properties of sorbent are known to influence the affinity of a protein for a given interface [2]. As most proteins adsorb with high affinity to hydrophobic surfaces, these proteins generally have less native structure than the same protein adsorbed on hydrophilic surfaces [3, 4]. Discovery of the M41S family of materials in 1992 ended the long-standing pore-size constraint of zeolites [5, 6]; materials with pore diameters >20 Å and sharp “molecular sieve” like pore-size distributions could be synthesized. However, M41S materials are limited to a pore diameter of approximately 80 Å, and, furthermore, they have significant external surface areas. These characteristics limit its use in size-selective separations of large biomolecules such as proteins and enzymes [7, 8]. In 1998, Stucky and co-workers synthesized highly ordered Santa Barbara Amorphous (SBA) materials by using neutral triblock copolymer as structure direct agents in highly acidic medium [9, 10]. Among these, SBA-15 materials have drawn lot of interest owing to their narrow pore size distribution, high surface area and relatively high hydrothermal stability [11]. SBA-15 was synthesized using tri-block copolymer poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide), which is commercially available as pluronics P123 (PEO20PPO70PEO20). SBA-15 has proved to be very promising for the size selective adsorption or separation of large biomolecules, because pore diameters are in the range
28
CHAPTER 3
SBA-15 MATERIAL FOR ADSORPTION OF BIOMOLECULES
required for these processes, and the silica framework is well suited for the development of bonded, selective sorption phases [12–18]. The synthesis route is shown in Figure 3.1.
Figure 3.1 Schematic illustration of synthetic route for porous SBA-15
In the present study, SBA-15 supports with pore diameter ca. 8 nm have been synthesized and its performance as biomolecules loading is examined. Lysozyme (Lz) has been chosen as representative example. 29
CHAPTER 3
SBA-15 MATERIAL FOR ADSORPTION OF BIOMOLECULES
3.2 Experimental 3.2.1 Materials Lysozyme (chicken egg white, lot no. 31H 8205) was purchased from Sigma Chemicals Company and used without further purification. TEOS and Pluronic 123 (PEO20PPO70PEO20) were purchased from Sigma Aldrich. NaHCO3 (sodium bicarbonate), NaOH (sodium hydroxide) and HCl (hydrochloric acid) were of analytical grade and used without further purification.
3.2.2 Synthesis of SBA-15 materials SBA-15 materials were synthesized by following the procedure mentioned elsewhere with little modification [19]. In a typical synthesis, 4 g P123 dissolved in 30 g DW and 120 g 2M HCl was stirred for 5 h at room temperature. 9.5 g TEOS was added under vigorous stirring (Magnetic stirrer, REMI 2 LH) condition. The resulting gel was aged at 40oC for 24 h and then heated to 95oC for 24 h. After synthesis, the solid products were filtered and then washed with distilled water repeatedly. The solid products were dried at room temperature and calcinations were performed in two groups at 550oC for 6 h and at 550oC for 12 h respectively to decompose the triblock copolymer.
3.2.3 Test for hydrothermal stability To examine the hydrothermal stability we selected the 12 hr calcined sample. 0.5 g of the sample was continuously stirred with 50 g distilled water at 303 K for 24 hr. Then, the samples were filtered, washed with deionized water and air dried.
3.2.4 Characterization of SBA-15 X-ray Diffraction (XRD) Low angle powder XRD for all the samples was measured by using Seifert 3000P X-ray diffractometer. The X-ray source was Cu Kα radiation (λ = 1.54186 Å).
30
CHAPTER 3
SBA-15 MATERIAL FOR ADSORPTION OF BIOMOLECULES
FTIR Fourier Transform Infrared (FTIR) spectra of the samples were collected using PerkinElmar Spectrum rx1 by KBr palette method.
3.2.5 Adsorption studies A lysozyme (Lz) solution with a concentration of 14 µmol/L was prepared by dissolving Lz in a buffer solution of ionic strength 0.16 (pH = 9.7 sodium bicarbonate buffer). In each adsorption experiment, 20 mg (w, equal to 2 x 10-5 kg) of the mesoporous adsorbent was suspended in 5 ml (V) of the respective enzyme solution taken in 50 ml standard joint conical flask. The resulting mixture was shaken continuously at a speed of 100 rpm at 25o C for 24 h. The amount of enzyme was determined by UV absorption at 280 nm. The amount of immobilized enzyme was calculated by subtracting the amount in the supernatant liquid after adsorption from the amount of enzyme present before adding the adsorbent. Centrifugation (5000 rpm) prior to analysis was performed to avoid potential interference from scattering particles in UV–vis analysis (Hitachi U-2000). 1
The moles (Γ,) of enzyme adsorbed per kilogram of mesoporous material can be 2 calculated using eq 1.
(1) Here, C2 is the molar concentration of the protein in a solution of volume V, in ml, before t
addition of the solid adsorbent and C2
is its molar concentration in the bulk solution at
adsorption equilibrium after the addition of W grams of solid adsorbent. Vt equal to V/W, represents V volume of solution per kg of solid powder. The experiments of adsorption kinetics were carried out in stirred batch mode. For each experiment, 10 ml of the Lz solution at specified concentrations (0.15 mg/ml) was continuously 31
CHAPTER 3
SBA-15 MATERIAL FOR ADSORPTION OF BIOMOLECULES
stirred at 100 rpm with 20 mg of SBA-15 at 20, 30 and 40° C respectively. Samples were withdrawn at appropriate
time
intervals
and then centrifuged at 5000 rpm for 10 min
and the absorbance of the supernatant was measured at 280 nm.
3.3 Results and discussion 3.3.1 Characterization of SBA – 15 Panel a-d of Figure 3.2 shows the XRD patterns of the as-synthesized, calcined (6 and 12 h respectively) and 12 h calcined SBA-15 materials after hydrothermal treatment. Presence of low angle XRD peaks (figure 3.2a – d) confirms the formation of mesostructure. After calcination (figure 3.2b, c) the intensity of the XRD peak increases with little shift towards higher 2θ indicating the increase in ordered structure with little shrinkage of pore. Also, intensity of XRD decreases with increase in calcination time (figure 3.2b, c). From small-angle XRD the pore to pore distance of the as-synthesized SBA-15, SBA-15 calcined for 6 h, SBA-15 calcined for 12 h and hydrothermally treated SBA-15 are calculated to 9.8, 8.3, 7.5 and 7.3 nm, respectively. From the XRD patterns it is clear that the structure of the material is highly stable under both thermal and hydrothermal conditions. Parts a-c of Figure 3.3 shows the FTIR spectra of as-synthesized SBA-15, SBA-15 calcined for 6 h and SBA-15 calcined for 12 h, respectively. The FTIR spectra of the assynthesized SBA-15 in the 2860 – 3820 cm-1 region shows four main bands, at 2974 cm -1 (CH3), 2932 cm-1 (CH2), 2908 cm-1 (CH3) and 2876 cm-1 (CH2). After calcination (figure 3.3b, c), the peaks in the region 2860 – 3820 disappears.
This indicates that, calcination from 6 – 12 h,
completely removes the template. Figure 3.3 and Table 3.1 shows the characteristic peaks for SiO - Si and OH obtained from the FTIR spectra of the three samples.
32
SBA-15 MATERIAL FOR ADSORPTION OF BIOMOLECULES
900
400
800
200
Intensity (a.u)
Intensity (a.u)
300
d spacing =9.8 nm
700
d spacing =8.3 nm
CHAPTER 3
600 500 400 300 200
100
100 0
0 1
2
(a)
3
4
1
5
3
4
5
4
5
2 (deg)
d spacing =7.5 nm
600
500
400
Intensity (a.u)
500
d spacing =7.3 nm
600
700
Intensity (a.u)
2
(b)
2 (deg)
400
300
300
200
200 100
100
0
0 1
2
(c)
3
4
1
5
2
(d)
2 (deg)
3
2 (deg)
Figure 3.2 XRD patterns of SBA-15 (a) as-synthesized material, (b) calcined for 6 h, (c) calcined for 12 h, and (d) 12 h calcined sample after hydrothermal treatment
33
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SBA-15 MATERIAL FOR ADSORPTION OF BIOMOLECULES
SBA-15 calcined for 6 h SBA-15 calcined for 12 h As-synthesized SBA-15
60
OH Si - O - Si
50
Transmittance (%)
CHx 40
30
(a) (b)
OH
(c)
20
10
0 4000
3500
3000
2500
2000
1500 -1
1000
500
Wavenumber (cm )
Figure 3.3 FTIR spectra of SBA-15 (a) as-synthesized, (b) 6 h calcined, and (c) 12 h calcined. Table 3.1 Characteristic peaks of the three samples obtained by FTIR
Characteristic peaks for Sample
Si - O - Si (cm-1)
OH (cm-1)
OH (cm-1)
As-synthesized SBA-15
1090
1650
3394-3485
SBA-15 calcined for 6 h
1030-1153
1638
3477
SBA-15 calcined for 12 h
1030-1250
1645
3322-3629
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CHAPTER 3
SBA-15 MATERIAL FOR ADSORPTION OF BIOMOLECULES
3.3.2 Adsorption studies The adsorption isotherm of Lysozyme (Lz) on SBA-15 at pH 9.7 has been shown in 1
Figure 3.4. Moles of lysozyme, Γ,2 adsorbed per kg of SBA-15 have been plotted against C2 at 25oC keeping the pH at 9.7. The isotherm shows a sharp initial rise, suggesting a strong affinity between Lz and the adsorbant surface. Finally, the isotherm almost takes a plateau shape (type L (Langmuir) isotherm). The maximum adsorption of Lz amounts to 21.9 mol/kg at pH 9.7. The isoelectric point of Lz is around 11 [19], and hence the protein is positively charged at a pH below pI. The isoelectric point of the silica surface is around 3.6 [20] and, hence, the adsorbent is negatively charged at a pH above 3.6. These suggest that the electrostatic attraction between Lz and the silica surface should increase with decreasing solution pH. In contrast, the monolayer adsorption capacity decreases with decreasing solution pH. This indicates that the lateral repulsion between the protein molecules is more significant at lower solution pH than the electrostatic interaction between the negatively charged silica surface and the positively charged lysine and arginine amino acid residues on the protein surface. In addition, hydrophobic interactions are more dominant near the pI than electrostatic interactions. These hydrophobic interactions originate from (1) the interaction between the nonpolar side chains of the amino acids residues on the surface of Lz and surface siloxane bridges or (2) from the Lz-Lz interactions between the hydrophobic side chains of neighboring Lz molecules adsorbed on the surface of SBA-15 [21]. Moreover, the buffer used (bicarbonate) might also effect the lysozyme adsorption [22]. At pH 9.7 higher loading is obtained due to lower electrostatic repulsion.
35
CHAPTER 3
SBA-15 MATERIAL FOR ADSORPTION OF BIOMOLECULES
25
15
10
2
1
4
x 10 (mol Lyz/kg SBA-15)
20
5
0 -4
-2
0
2
4
6
8
10
12
7
C2 x 10 (M) 1
Figure 3.4 Plot of Γ,2 vs C2 for adsorption of Lysozyme on SBA-15 at 25o C and pH 9.7. In order to investigate the mechanism of adsorption various kinetic models have been suggested. In recent years, adsorption mechanisms involving kinetic-based models have been reported. In this study, some of these models were investigated to find the best fitted model for the experimental data obtained.
Figure 3.5 shows the curve-fitting plot of the pseudo second-
order equation and diffusion model and the parameters obtained for these models are shown in Table 3.2. The second-order-rate reaction can be written in the following form:
(2)
where Qe and Qt are the amount of Lz adsorbed (mg/g) at equilibrium and at time t, respectively. k2 is the rate constant of pseudo-second-order adsorption (gm/mg min). Intraparticle diffusion 36
CHAPTER 3
SBA-15 MATERIAL FOR ADSORPTION OF BIOMOLECULES
model is based on the theory proposed by Weber and Moris [27]. According to the theory, if intraparticle diffusion is the rate-controlling factor, the uptake of adsorbate is proportional to the square root of contact time in the course of adsorption. Boundary layer diffusion involves mass diffusion to the external surface of the particle through a hypothetical boundary layer surrounding the particle. When the boundary layer diffusion gives an effect, the plot of Qt versus t1/2 does not pass through the origin, and the intraparticle diffusion equation can be described as eq 3, where Kd is the intraparticle diffusion rate constant. Qt = Kdt1/2 + C
(3)
The curve-fitting plots of t/Qt versus t give a straight line, confirming the applicability of the pseudo second-order equation.
4.5
55 o
20 C o 30 C o 40 C o 20 C Model o 30 C Model o 40 C Model
3.5
t/Q t
3.0 2.5
50 45 40 Amount absorbed (mg/g)
4.0
2.0 1.5 1.0
35 30 25 o
20 C o 30 C o 40 C o 20 C Model o 30 C Model o 40 C Model
20 15 10 5
0.5
0 20
40
60
(a)
80
100
120
140
0
Time (min)
2
4
6
(b)
8
t
10
12
1/2
Figure 3.5 Kinetic plots for adsorption of Lysozyme on SBA-15; (a) pseudo-second-order, and (b) diffusion model
37
CHAPTER 3
SBA-15 MATERIAL FOR ADSORPTION OF BIOMOLECULES
Table 3.2 Parameters of kinetic models of Lysozyme adsorption on SBA-15 at different temperatures. Temperature (oC)
Pseudo-second-order kinetic model
Diffusion Model
20
K2 (g/ mg min) 2.6 x 10 -3
Qe (mg/ g) 32.57
R2 0.99849
Kd 1.79
R2 0.77844
30
1.5 x 10 -3
54.88
0.99157
2.06
0.95963
40
1.2 x 10 -3
48.15
0.98156
1.95
0.89489
The correlation coefficients for the linear plots of t/Qt against t for the secondorder equation were observed
to
be close
to
1
for
the contact time of 140 min.
If the intra-particle diffusion is involved in the adsorption processes, then the plot of the square root of time versus the uptake (Qt) would result in a linear relationship and the intraparticle diffusion would be controlling step if this line passed through the origin [23]. When the plots do not pass through the origin, this is indicative of some degree of boundary layer control and this further shows that the intraparticle diffusion is not the only rate controlling step, but also other processes may control the rate of adsorption [24]. Such plots may present a multi-linearity, indicating that two or more steps take place. The first, sharper portion is attributed to the diffusion of adsorbate through the solution to the external surface of adsorbent or the boundary layer diffusion of solute molecules. The second portion describes the gradual adsorption stage, where intraparticle diffusion is rate limiting. The third portion is attributed to the final equilibrium stage where intraparticle diffusion starts to slow down due to extremely low adsorbate concentrations in the solution [25, 26]. The rapid uptake of protein by the adsorbant when the pore size is adequate is an important characteristic for practical applications. Figure 3.6 shows the adsorption amount of Lz on SBA-15 support as a function of time. On SBA-15 with pore size of 8.3 nm, the maximum amount of Lz (pH 9.7) adsorbed is 53 mg/g at 30o C. Dimension of Lz is 30×30×45 Å [9]. The 38
CHAPTER 3
SBA-15 MATERIAL FOR ADSORPTION OF BIOMOLECULES
largest dimension of Lz is almost half of the pore diameter and hence the molecules adsorbs with great affinity.
55 50 45
35
Qt (mg g
-1
)
40
30 25
o
20 C o 30 C o 40 C
20 15 20
40
60
80
100
120
140
160
Time (min)
Figure 3.6 Adsorption amount of Lz on SBA-15 (8.3 nm) as a function of time
3.4 Conclusions SBA-15 materials with pore size as large as 8.3 nm (calculated from small angle X-ray diffraction) have been prepared. They showed stability towards thermal and hydrothermal treatment. There is insignificant change in pore to pore distance due to calcination and hydrothermal treatment. These materials are likely to find applications in downstream operation and adsorption or separation of biomolecules.
39
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References 1. M. Alkan, Ö. Demirbaş, M. Doŭan, O. Arslan. Microporous and Mesoporous Materials 2006, 96, 331–340. 2. C.A. Haynes, W. Norde, Colloids Surf. B: Biointerf. 1994, 2, 517–566. 3. A. Kondo, F. Murakami, K. Higashitani, Biotechnol. Bioeng. 1992, 40, 889. 4. A. Kondo, S. Oku, F. Murakami, K. Higashitani, Colloids Surf. B: Biointerf. 1993, 1, 197. 5. C. T. Kresge, M. E. Leonowicz, W. J. Roth, J, C. Vartuli, J. S. Beck. Nature 1992, 359, 710-712. 6. J. S. Beck, J. C. Vartuli, W. J. Roth, M. E. Leonowicz, C. T. Kresge, K. D. Schmitt, C. T.-W. Chu, D. H. Olson, E. W. Sheppard, S. B. McCullen, J. B. Higgins, J. L. Schlenker, J. Am. Chem. Soc. 1992, 114, 10834. 7. A. Katiyar, L. Ji, P. Smirniotis, N. Pinto, Microporous Mesoporous Mater. 2005, 80, 311. 8. L. Ji, A. Katiyar, N. Pinto, P. Smirniotis, Microporous Mesoporous Mater. 2004, 75, 221. 9. D. Zhao, J. Feng, Q. Huo, N. Melosh, G.H. Fredrickson, B.F. Chmelka, G.D. Stucky, Science 1998, 279, 548. 10. D. Zhao, Q. Huo, J. Feng, B.F. Chmelka, G.D. Stucky, J. Am. Chem. Soc. 1998, 120, 6024. 11. I.Rodriguez, S. Iborra, A. Corma, F. Rey, J. L. Jorda. Chem, Comman. 1999, 593. 12. A. Katiyar, L. Ji, P. Smirniotis, N. Pinto, J. Chromatogr. A 2005, 1069, 119. 13. J. Zhao, F. Gao, Y. Fu, W. Jin, P. Yang, D. Zhao, Chem. Commun. 2002, 2002, 752. 14. A. Vinu, V. Murugesan, M. Hartmann, J. Phys. Chem. B., 2004, 108, 7323. 15. H.H.P. Yiu, C.H. Botting, N.P. Botting, P.A. Wright, Phys. Chem. Chem. Phys. 2001, 3, 2983. 16. L. Washmon-Kriel, V.L. Jimenez, K.J. Balkus Jr., J. Mol. Catal. B: Enzym. 2000, 10, 453. 17. J. Lei, J. Fan, C. Yu, L. Zhang, S. Jiang, B. Tu, D. Zhao, Microporous Mesoporous Mater. 2004, 73, 121. 18. Y.L. Han, J.T. Watson, G.D. Stucky, A. Butler, J. Mol. Catal. B: Enzym. 2002, 17, 1. 19. K. P. Wilson, B. A. Malcolm, B. W. Matthews. J. Biol. Chem. 1992, 267, 10842. 40
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20. T. J. Su, J. R. Lu, R. K. Thomas, Z. F. Cui, J. J. Penfold. Colloid Interface Sci. 1998, 203, 419. 21. A. Vinu, V. Murugesan, and M. Hartmann. J. Phys. Chem. B. 2004, 108, 7323-7330. 22. V. Ball, J. J. Ramsden. J. Phys. Chem. B. 1997, 101, 5465. 23. A. S. ALzaydien. Am. J. of Env. Sc. 2009, 5 (3), 197-208. 24. G. Crini, H.N. Peindy, F. Gimbert and C. Robert. Separat. Purificat. Technol. 2007, 53, 97-110. 25. M. Ozacar, Mater. 2006, 137, 218-225. 26. K. G. Bhattacharyya, A. Sharma. J. Environ. Manage. 2004, 7, 217-229. 27. W. J. Weber, J. C. Morris. J. Sanit. Eng. Div. Am. Soc. Civ. Eng. 1963, 89, 31–60.
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Chapter 4 Summary This research work is an experimental and theoretical investigation on the synthesis procedure of mesoporous silica and their feasibility as bioadsorbant. In this context, we attempted to synthesize two classes of mesoporous materials, MCM-41 and SBA-15. SBA-15 was tested to determine the optimum performance for protein adsorption. Generally,
the
important features of materials used for bioapplications are high surface area, large pore volume, large pore diameter, and structural stability. The MCM family of materials discovered by Mobil Oil Corporation in 1992 was the first generation of ordered mesoporous materials. These materials possess pore diameters of 2-6 nm with high surface areas (1000 m2/g). The second chapter of this thesis describes the synthesis of MCM-41 materials and its characterization. The pore size of MCM-41 proved to be a major limitation for protein adsorption as the pore size range of these materials is not sufficient to capture large protein molecules. To overcome the challenges presented by the first generation of ordered mesoporous silicates, many investigators searched for possible routes to synthesize large pore ordered silicates using a templated synthesis approach. The discovery of SBA-15 materials, which have large pore diameters (4-30 nm) and characteristics similar to the MCM family of materials was a major breakthrough in this research. The third chapter of this dissertation summarizes efforts toward the development of SBA-15 materials for adsorption of protein molecules. The kinetics of adsorption and equilibrium capacity was studied with lysozyme. Ordered mesoporous silicas are important class of materials due to their remarkable features. These materials can be synthesized in different forms, such as thin films, membranes, particles and monoliths, for applications in adsorption, separation and catalysis. This work has been primarily focused on synthesis of ordered mesoporous silica for bioadsorption. Successful synthesis of large pore mesoporous silica has been achieved following an existing literature.
42