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CARBON NANOTUBES SYNTHESIS USING
DOUBLE STAGE CHEMICAL VAPOR
DEPOSITION (DS-CVD) FOR SKIM LATEX
PROTEIN SEPARATION
BY
MUBARAK MUJAWAR
A thesis submitted in fulfilment of the requirement
for the degree of Master of Science (Biotechnology
Engineering)
Kulliyyah of Engineering
International Islamic University Malaysia
APRIL 2010
ii
ABSTRACT
Carbon nanotubes (CNTs) are one of the most exciting discoveries in nanoscale
sciences. The interest in CNTs is increasing due to their unique properties, large
surface area and wide range of application in biomedical and bio-engineering aspects.
The present work aims to demonstrate the optimized production of CNTs using
fabricated Double Stage Chemical Vapor Deposition (DS-CVD) followed by the
evaluation of its application in protein purification. To optimize the process
parameters of CNTs production with respect to achieving high purity and yield, a
statistical approach using Design Expert software was adopted. The operating
parameters namely reaction time, reaction temperature and flow rates of the precursor
gases, C2H2 and H2 were varied for production optimization. The CNTs produced
were analyzed for purity and morphology using Field Emission Scanning Electron
Microscope (FESEM), Transmission Electron Microscope (TEM) and Thermo
Gravimetric Analysis (TGA). Before they were applied for protein purification, CNTs
were submitted for acid purification and functionalization. In order to evaluate the
capacity of CNTs in protein purification, skim latex serum was used as model protein.
Skim latex serum is recovered from skim latex, a by-product of natural latex
concentrate industries, which are usually considered as a waste, thus lavishly thrown
away. CNTs were used as the column chromatographic media and its nanosized
structure will lead to separation of protein from skim latex. As the column
chromatographic media, CNTs were used after covalent and non-covalent
functionalization and the ability was compared with the non-functionalized ones.
Guided by the functional groups available on the surface, CNTs were used differently;
functionalized CNTs as in Ion Exchange Chromatography (IEC) media and non-
functionalized CNTs as in Hydrophobic Interaction Chromatography (HIC) media.
For optimization of the purification process, the pH as well as the salt concentration of
running buffer were varied. CNTs have been successfully produced by DS-CVD and
the statistical analysis reveals that the optimized conditions for the best yield of CNTs
production is 850°C reaction temperature, 60 mins reaction time with gases flow rates
of 40 and 150 ml/min for C2H2 and H2 respectively. The TGA analysis shows that the
purity of CNTs produced as about 95% purity. FESEM and TEM analyses reveal that
the uniformly dispersed CNTs have diameters ranging from 35 to 45nm. This work
further demonstrated that CNTs can perform as IEC and HIC column
chromatographic media. Chromatographic separation of our skim latex protein shows
that CNTs can be used as HIC media as compared to IEC. Results show that as usual
the efficiency of the protein purification is dependent upon pH and ionic strength of
the running buffer. In HIC, bound protein was observed most when chromatography
was carried out with 50mM Tris-HCl, pH 7, and using 2M ammonium sulphate as the
neutral salt. This study concludes that CNTs produced can be replace the high cost
commercialized HIC media in up-scale process. The nano -sized structured CNTs
leads to functioning as a better chromatographic media than the commercialized
product. CNTs have many uses and this work adds to another dimension in the
numerous applications of CNTs.
iii
CNTs
CNTs
CNTs
(DS-CVD)
Design ExpertCNTs
C2H2H2CNTs
FESEMTEM
TGACNTs
CNTs
CNTsCNTs
(IEC)CNTs
HICCNTs
CVD-DSCNTs°85060
40150C2H2H2TGA
CNTs95FESEMTEMCNTs
3545CNTsIEC
HICCNTs
HICIEC
HIC50mM Tris-HCl72
CNTs
HICCNTs
CNTs
iv
APPROVAL PAGE
I certify that I have supervised and read this study and that in my opinion, it conforms
to acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a thesis for the degree of Master of Science (Biotechnology Engineering).
_________________
Faridah Yusof
Supervisor
_________________
Maan Alkhatib
Co-supervisor
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
thesis for the degree of Master of Biotechnology Engineering.
___________________
Ibrahim Ali Noorbatcha
Internal Examiner
This dissertation was submitted to the Department of Biotechnology Engineering and
is accepted as a partial fulfilment of the requirements for the degree of Master of
Biotechnology Engineering.
_____________________
Md Zahangir Alam
Head, Department of
Biotechnology Engineering
This dissertation was submitted to the Kulliyyah of Engineering and is accepted as a
partial fulfillment of the requirements for the degree of Master of Biotechnology
Engineering.
______________________
Amir Akramin Shafie
Dean, Kulliyyah of
Engineering
v
DECLARATION
I hereby declare that this dissertation is the result of my own investigations, except
where otherwise stated. I also declare that it has not been previously or concurrently
submitted as a whole for any other degrees at IIUM or other institutions.
Mubarak Mujawar
Signature ____________________ Date _____________________
vi
INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA
DECLARATION OF COPYRIGHT AND AFFIRMATION
OF FAIR USE OF UNPUBLISHED RESEARCH
Copyright © 2010 by Mubarak Mujawar. All rights reserved.
SYNTHESIS OF CARBON NANOTUBES AND ITS APPLICATION IN
PROTEIN PURIFICATION
No part of this unpublished research may be reproduced, stored in a retrieval system,
or transmitted, in any form or by any means, electronic, mechanical, photocopying,
recording or otherwise without prior written permission of the copyright holder except
as provided below.
1. Any material contained in or derived from this unpublished research may
only be used by others in their writing with due acknowledgement.
2. IIUM or its library will have the right to make and transmit copies (print or
electronic) for institutional and academic purposes.
3. The IIUM library will have the right to make, store in a retrieval system
and supply copies of this unpublished research if requested by other
universities and research libraries.
Affirmed by Mubarak Mujawar.
__________________ __________________
Signature Date
vii
ACKNOWLEDGEMENTS
First of all I would like to raise my hand of syukr (appreciation) to Allah (s.w.t) for
the guidance, wisdom and barakah for all these years til now, where I have reached to
this important destination of my journey in life to accomplish my goal.
I also would like to dedicate my deepest gratitude to my supervisor, Assoc.
Prof. Dr. Faridah Yusof and my co-supervisor, Assist. Prof. Dr. Ma’an Fahmi Al
khatib for their excellent supervision, good guidance and moral support throughout the
duration of this research study. My sincere thanks also go to all the lab technicians of
Biotechnology Engineering Department and Material Engineering Department for
their good cooperation and assistance provided throughout this research study. I also
want to thank my mother and my brothers for their blessings, support and
encouragement.
Last but not the least, my special thanks goes to postgraduate colleague, Br
Emad, Khalid, Mohammad Al Saadi, Salwudin for their kind assistance provided
throughout the period of study during the experimental work as well as dissertation
writing.
viii
TABLE OF CONTENTS
Abstract in English………………………………………………………..............
Abstract in Arabic………………………………………………………………...
Approval Page…………………………………………………………………….
Declaration Page………………………………………………………………….
Copy right Page…………………………………………………………………...
Acknowledgements …………………………………………………………..…..
Table of Content……………………………………………………………….....
List of Tables ……………………………………………………………….........
List of Figures ………………………………………………………………........
List of Abbreviations ……………………………………………………………...
ii
iii
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v
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xii
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xix
CHAPTER ONE: INTRODUCTION …………………………………………..
1.1 Overview...……………………….………………………….................
1.2 Problem Statement …..………………………………….......................
1.3 Research Objectives…………………………….........….......................
1.4 Research Methodology …………………………………………...........
1.5 Research philosophy…………………………………………………..
1.5 Significance of Study…………………………………………………..
1.6 Scope of Study…………………………………………………………
1.7 Thesis Organization……………………………………………………
CHAPTER TWO: LITERATURE REVIEW ...………………………………...
2.1 Histrory of Carbon nanotubes (CNTs)…………………………………
2.2 Structure of Carbon Nanotubes (CNTs) …………………………….....
2.3 Properties of CNTs………………………….………………………....
2.3.1 Electronic Properties…….…………..………………..................
2.3.2 Mechanical Properties…...……………………….......................
2.3.3 Thermal Conductivity ………...………………………………..
2.3.4 Field Emission ………..………………………………………..
2.4 Productions Methods of CNTs………………………………………….
2.4.1Arc Discharge …….………………………………….................
2.4.2 Laser Ablation……………………………………………….....
2.4.3 Chemical Vapour Deposition……………………………………
2.4.3.1 Advantages and Limitations of CVD……………………
2.4.3.2 Vapor Phase Growth……………………………………
2.4.3.3 CVD Method using Substrate Catalyst………………….
2.4.3.4 Plasma Enhanced Chemical Vapour Deposition
(PE- CVD)............................................................................
2.4.3.5 Fluidized-Bed CVD Method.............................................
2.4.3.6 Double Stage Chemical Vapor Deposition (DS-CVD….
2.4.3.7 Statistical design for production of CNTs…………...
2.5 Comparision of Nanotube Synthesis Methods………………………….
2.6 Factors Influencing the Growth Mechanism of CNTs…………………
2.7 Functionalization of CNTs……………………………………………
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2.7.1 Covalent Functionalization …………………………………….
2.7.2 Non-covalent Functionalization of CNTs………………………
2.8 Applications of CNTs......………………………………………………
2.8.1 Biological Applications of CNTs……………………………….
2.8.1.1 Drug and Gene Delivery…………………………………
2.8.1.2 CNTs for cancer therapy…………………………………
2.8.1.3 CNTs for HIV/AIDS therapy……………………...…...
2.8.2 Nanotube Sensors………………………………………………
2.8.5.1Carbon Nanotubes as Gas sensor .....................................
2.8.5.2 Carbon Nanotubes Array –Based Biosensor……………
2.8.3 Hydrogen Storage ……………………..………………………
2.8.4 Causes and Effects on Application of CNTs………………….
2.9 Skim Latex………………………………………………......................
2.9.1 Natural Rubber …………….…………………………………...
2.9.2 Types of Protein in Skim Latex………………………………...
2.9.3 CNTs as the Filter for Protein Separation ……………………...
2.9.4 Advantage of CNTs Filter……………………………………....
2.10 Protein Purification……………………………………………………
2.11 Column Chromatography …………………………….........................
2.11.1 Ion Exchange Chromatography ………………………………
2.11.2 Hydrophobic Interaction Chromatography……………………
2.12 Summary………………………………………………………………
CHAPTER THREE: MATERIALS & METHODS ……………………….…..
3.1 Materials………………………….…………………………………….
3.2 Characterization………………..…….....................................................
3.3 Methods………………………………………………………………..
3.3.1 Design of Experiment for CNTs Production……………………
3.3.2 Design of Experiment for protein purification …………..……..
3.3.3 The Modified Double Stage Chemical Vapor Deposition
(DS-CVD)…………………………………………………………
3.3.4 Production of MWCNTs………………………………….……
3.3.5 Purification of CNTs…………………………………….........
3.3.6 Functionalization of CNTs…………………..........................
3.3.6.1 Covalent Functionalization by using two step
Carbodiimide Activated Amidation……………........
3.3.6.2 Non Covalent Functionalization of CNTs………………
3.3.7 Purity Measurement of CNTs…………………………………..
3.3.8 Preparation of Skim Latex Serum Protein…………………….
3.3.8.1 Acidification of Skim Latex ……………………………
3.3.8.2 Centrifugation of Skim Latex……………………………
3.3.8.3 Freeze Drying of Skim Latex Serum Protein…………. …
3.3.8.4 Dialyses of Skim Latex Serum Protein …………………
3.3.9 Purification of Skim Latex Proteins…………………………….
3.3.9.1 Protein Purification using Non Functionalized CNTs….
3.3.9.2 Protein Purification using Covalently Functionalized
CNTs……………………………………………………..
3.3.9.3 Protein Purification using Non Covalently
Functionalized CNTs……………………………………
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x
3.3.9.4 Protein Purification using phenyl sephrose ……………..
3.3.10 Analysis of Proteins……………………………………………
3.3.10.1 Chromatographic Protein Profile of Skim Latex
Serum………………………………………………...
3.3.10.2 Sodium Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis (SDS-PAGE) ……………………..
3.4 Summary………………………………………………………………..
CHAPTER FOUR: RESULTS AND DISCUSSION …………………………..
4.1 Introduction…………………………………………………………….
4.2 Production of CNTs ………………………………………………........
4.2.1 Statistical Analysis of CNTs………………………………......
4.2.2 Effect of Process Parameters on CNTs yield……………..…...
4.3 Purification of CNTs………………………………………………….
4.3.1 Characterization of CNTs using FESEM……….......................
4.3.2 Characterization of CNTs using TEM…………………….......
4.3.3 Characterization of CNTs using FTIR………….......................
4.3.4 Characterization of CNTs using TGA………………………...
4.4 Purification of Skim Latex Serum Protein…….……………………..
4.4.1 Protein Purification using Non Functionalized CNTs………..
4.4.2 Protein Purification using Functionalized CNTs……………...
4.4.2.1 Protein Purification using Covalently Functionalized
CNTs…………………………………………………...
4.4.2.2 Protein Purification using Non Covalently
Functionalized CNTs…………………………………..
4.4.2.3 The behavior of Covalent and Noncovalent on
Chromatography…………………….…………………
4.4.3 The Effect of pH on Purification Method……………..............
4.4. 4 The Effect of Concentration of Buffer Salts and Neutral salt
on purification method………………………………………..
4.5 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
(SDS-PAGE) Analysis of protein …………………………… ……
4.6 Comparison Between the capacity of Non Functionalized CNTs
and Phenyl Sepharose as HIC media……………………………….
4.6.1 Effect of pH on HIC media…………………………………...
4.6.2 Concentration of Buffer CNTs vs phenyl Sephrose………......
4.6.3 Effect of Neutral Salts on HIC media……………………... .
4.7 Summary……………………………………………………………..
CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS ...……
5.1 Conclusions…………………………………………………………...
5.2 Recommendations…………………………………………………….
BIBLIOGRAPHY………….....................................................................………..
PUBLICATIONS………………………………………………………………..
APPENDIX A…………………………………………………………………....
APPENDIX B………………………………………………………………........
APPENDIX C…………………………………………………………………....
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xi
LIST OF TABLES
Table No. Page No.
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3.1
3.2
3.3
3.4
3.5
3.6
3.7
Summary of synthesis of CNTs using CVD techniques
Comparison of Arc-Discharge, Laser Ablation and Chemical Vapor
Deposition
Functionalization of CNTs with wide range of molecules
Summary for some of the Hydrogen Storage Reports up to 2001
Typical Fresh Latex Composition (from Rubber Foundation
Information Centre for Natural Rubber)
Differences between proteins and their exploitation for purification
High dynamic binding capacities of ion exchange resins
Two Types of Ion Exchange Resins
Properties of phenyl sephrose Fast Flow
Four Levels Half Factorial Design with real factors values and level
ranges with four factors (A,B,C,D), three replicate, one block and
three center points per block
Two Level Full Factorial Design with real factors values and level
ranges with two factors, two replicate, one block and one center points
per block
Operating Parameters for Protein Purification by Non-Functionalized
CNTs.
Non functionalized CNTs for Protein Purification with Different
buffer, pH and Ionic Strength
Operating Parameters for Protein Purification by Covalently
Functionalized CNTs
Covalently Functionalized CNTs used for Protein Purification with
Different buffer, pH and Ionic Strength
Operating Parameters for Protein Purification by Non-Covalently
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xii
3.8
3.9
3.10
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
Functionalized CNTs
Non -Covalently Functionalized CNTs used for Protein Purification
with Different buffer, pH and Ionic Strength
Quantity (ml) of components for preparing a 12 % separating gel.
Quantity (ml) of components for preparing a 12 % stacking gel
Values of Observed Response for Production of CNTs
TGA Analysis of High Yield CNTs Samples
Analysis of Variance (ANOVA) for Selected Factorial Model for
CNTs yield
Validation Study for Selection of Best yield, high quality and high
purity of CNTs
EDX analysis of CNTs
Purification of protein from skim latex serum
Area under the elution peak for Purification of Protein by HIC media
CNTs
Comparison of area under the elution peak between HIC media CNTs
and phenyl sephrose
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151
xiii
LIST OF FIGURES
Figure No. Page No.
2.1
2.2
2.3
2.4
2.5
2.6
2.7
World of Carbon Related Materials (www.iljinnanotech.co.kr,
2002).
A graphene sheet made of C atoms placed at the corners of
hexagons forming the lattice with arrows AA and ZZ denoting
the rolling direction of the sheet to make (b) a (5,5) armchair
nanotube and (c) a (10,0)zigzag nanotube(Deepaketal., 2003)
(a) Zig-Zag Single-Walled Nanotube. Note the zig-zag pattern
around circumference and m = 0. (b) Chiral Single-Walled
Nanotube. Note twisting of hexagons around tubule body. (c)
Armchair Single-Walled Nanotube. Note the chair-like pattern
around circumference and n = m (Harris, 2001)Activated Carbon:
Surface and Pores-Scanning Electron Microscope Image
Schematic illustrations of the structures of (A) armchair, (B)
zigzag, and(C) chiral SWNTs. Projections normal to the tube axis
and perspective views along the tube axis are on the top and
bottom, respectively. (D) Tunneling electron microscope image
showing the helical structure of a 1.3- nm-diameter chiral
SWNT. (E) Transmission electron microscope (TEM) image of a
MWNT containing a concentrically nested array of nine SWNTs.
(F) TEM micrograph showing the lateral packing of 1.4-nm-
diameter SWNTs in a bundle. (G) Scanning electron microscope
(SEM) image of an array of MWNTs grown as a nanotube
forest (Ray et al., 2002)
Diagram showing rolling direction of nanotube (Dresselhaus et
al., 1998)
Structures of carbon nanotubes
Different structures of MWCNTs. Top-left: cross-section of a
MWCNT the different walls are obvious, they are separated by
0.34nm. Rotation around the symmetry axis gives us the
MWCNT. Top-right: Symmetrical or non-symmetrical cone
shaped end caps of MWCNTs. Bottom-left: A SWCNT with a
diameter of 1,2nm and a bundle of SWCNTs covered with
amorphous carbon. Bottom-right: A MWCNT with defects. In
point, P a pentagon defects and in point H a heptagon defect
(Ajayany and Ebbesenz, 1997)Structures of carbon nanotubes
(www.iljinnanotech.co.kr , 2002)
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xiv
2.8
2.9
2.10
2.11
2.12
2.13
2.14
2.14
2.15
2.16
2.17
2.18
2.19
Schematic diagram of arc-discharge apparatus
Schematic drawings of a laser ablation apparatus
TEM images of a bundle of SWCNTs catalyzed by Ni/Y (2:0.5
%) mixture, produced with a continuous laser (Thess et al.,
1996)
Schematics drawings of a CVD deposition oven
Schematic diagram of a vapour phase growth apparatus
(www.iljinnanotech.co.kr, 2002)
Schematic diagram of thermal CVD apparatus [w Schematic
diagram of spurting machine (www.angstromsciences.com,
2002 ww.iljinnanotech.co.kr, 2002)
Schematic diagram of Plasma Enhanced CVD apparatus
(www.iljinnanotech.co.kr, 2002)
Schematic diagram of the fluidized-bed reactor (Mauron et al.,
2003)
Common strategies for covalent functionalization of CNTs
via end side (Banerjee et al., 2005)
Common strategies for covalent sidewall functionalization
of CNTs (Banerjee et al, 2005)
Some specific biomedical applications of CNTs beingexplored
by various groups as novel delivery systems (Kam et al., 2006)
Schematic representation of the helical crystallization of proteins
on the outer surface of a carbon nanotube. a) Computed power
spectrum of the Fourier transform of a helical array of
streptavidin molecules. The helical repeat is 12.8 nm and the
arrow indicates 1/2.5 nmÿ1. b) Noise-free view of the helical
repeats obtained by correlation. c) Three-dimensional model of
streptavidin assemblies calculated by retroprojecting the noise-
free helical repeat shown in (b) along the Euler angles deduced
from the analysis of the diffraction pattern. The bar represents 25
nm in (b) and 12.5 nm in (c) (Balavoine et al., 1999)
Schematic diagram of the carbon nanotube array biosensor. The
enzyme immobilization allows for the direct electron transfer from
the enzyme to platinum transducer were obtained with a JEOL-
JSM 840 scanning microscope at 10 KV. Subsequently, enzyme
immobilization was achieved by incubation for 3 h of both freshly
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xv
2.20
2.21
2.22
2.23
2.24
2.26
3.1
3.2
4.1
4.2
4.3
4.4
4.5
4.6
4.7
oxidized sensors in an enzymatic solution (1500 to 2500 U/mL)
SEM images of the Pt-aligned carbon nanotube array (A) in
original state, (B) after being chemically etched with a mixture of
concentrated H2SO4 and HNO3 acids (3:1, 98% and 65% re-
spectively) for 8 h at 40 °C, washed with deionized water and
dried at 100 °C overnight, and (C) after air oxidation at 600 °C for
5 min under air flow. SEM images were obtained with a JEOL-
JSM 840 scanning microscope at 10 KV and Scale Bar is 1 µm
Temperature-dependent behavior of desorption of hydrogen
(Dillon, et al., 2001)
Segment of Natural Rubber polymer chain (smith, 1996)
Working range of anion and cation exchangers
Ion Exchange Chromatography mechanism
Structural formulae of the 8 commonly occurring amino acids that
display hydrophobic characteristics
Flow chart of the overall experiment
Schematic diagram of DS-CVD
Relationship between Predicted and Experimental values of yield
of CNTs
3D Surface Plots: Effect of process parameters on CNTs yield. (a)
interaction plot of yield of CNTs, reaction time and reaction
temperature (b)interaction plot of H2 and reaction temperature,
(c)interaction plot of C2H2 and reaction temperature, (d)
interaction plot of yield of CNTs, flow rates of C2H2 and H2
( a-d) FESEM images of CNTs produced at reaction temperature
850°C, before purification with acid HCl
( a-d) FESEM images of CNTs produced at reaction temperature
850°C, after purification with acid HCl
(a-b) FESEM images of CNTs (wall) produced at reaction
temperature 850°C
( a-b) FESEM images of CNTs produced at reaction temperature
850°C, before functionalization
( a-b) FESEM images of CNTs produced at reaction temperature
850°C, after functionalization
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xvi
4.8
4.9
4.10
4.11
4.12
4.13
4.14
4.15
4.16
4.17
4.18
4.19
4.20
4.21
(a-c)HRTEM images of CNTs produced at reaction temperature
850°C at different magnification TEM image of CNTS at 850 oC
(a-c) HRTEM images CNTs produced at reaction temperature
850°C at different magnification TEM image of CNTS at 850 oC
FTIR Absorption Spectra, a) Functionalized CNTs, b) Non-
functionalized CNTs
EDX analysis of (a) non functionalized CNTs (b) functionalized
CNTs
TGA analysis of CNTs produced at reaction temperature 850°C
TGA analysis of CNTs produced at reaction temperature 600°C
Chromatographic protein profile of skim latex serum on non
functionalized CNTs as media; U-unbound protein, B-bound
protein (run 4)
Chromatographic protein profile skim latex serum on covalently
functionalized CNTs as media; U-unbound protein, B-bound
protein (run 5)
Chromatographic protein profile skim latex serum on non
covalently functionalized CNTs as media; U-unbound protein,
B-bound protein (run 4)
SDS-PAGE for for non functionalized CNTs
Chromatographic protein profile of skim latex serum on non
functionalized CNTs as media at pH7; U-unbound protein, B-
bound protein
Chromatographic protein profile of skim latex serum on HIC using
phenyl sephrose as media at pH7; U-unbound protein, B-bound
protein
Chromatographic protein profile of skim latex serum on HIC using
CNTs as media at pH5; U-unbound protein, B-bound protein
Chromatographic protein profile of skim latex serum on HIC
using phenyl sephrose as media at pH5; U-unbound protein, B-
bound protein
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xvii
LIST OF ABBREVIATIONS
CNTs
SWCNTs
MWCNTs
DS-CVD
FESEM
TEM
FTIR
EDX
AFM
IEC
HIC
OD
OD595mm
kDa
SDS-PAGE
DMTA
NHS
EDAC
Carbon Nanotube
Single walled Carbon nanotube
Multiwalled Carbon Nanotubes
Double Stage chemical vapour deposition
Field Emission Scanning Electron Microscope (SEM)
Transmission Electron Microscope
Fourier Infrared spectroscopy
Electron Dispersive Array
Atomic Force Microscopy
Ion Exchange Chromatography
Hydrophobic Interaction Chromatography
Optical density
Optical Density at 595nm wavelength
kilo Dalton
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
Dynamic mechanical thermal analyzer
N-hydroxysuccinimide
N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide
hydrochloride
1
CHAPTER ONE
INTRODUCTION
1.1 INTRODUCTION
Research on new materials technology is attracting the attention of researchers all over
the world. Developments are being made to improve the properties of the materials
and to find alternative precursors that can give desirable properties on the materials.
Nanotechnology, which is one of the new technologies, refers to the development of
devices, structures, and systems whose size varies from 1 to 100 nanometers (nm).
The last decade has seen advancement in every side of nanotechnology such as:
nanoparticles and powders, nanolayers and coats, electrical optic and mechanical
nanodevices, and nanostructured biological materials. Nanotechnology is estimated to
be influential in the next 20-30 years, in all fields of science and technology (Harris et
al., 2001).
Great interest has recently been developed in the area of nanostructures carbon
materials. Carbon nanostructure materials are becoming of considerable commercial
importance with interest growing rapidly over the decade or so since the discovery of
buckminsterfullerene, carbon nanotubes, and carbon nanofibers (Dresselhaus et al.,
2001). Carbon nanotubes (CNTs) and carbon nanofibers (CNFs) are among the most
eminent materials in the first rank of revolution nanotechnology. The most eye-
catching features of these structures are their electronic, mechanical, optical and
chemical characteristics, which open a way to future applications. These properties
can even be measured on single nanotubes and nanofiber. For commercial application,
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large quantities of purified carbon nanotubes are needed (Dresselhaus et al., 1998 and
Thomas, 1997).
Fundamental and practical nanotube researches have shown possible
applications in the fields of energy storage, molecular electronics (transistors,
capacitors), nanomechanic devices, and composite materials. The main avenues of
potential applications of carbon nanotubes and carbon nanofibers are: ultimate
reinforcement fibers for composites (high strength, high aspect ratio, high thermal and
chemical stability), conducting nanowire, field emitters (individual nanotube field
emitters and large area flat panel displays); nanotools (tips for Scanning Tunneling,
Atomic Force, Magnetic Resonance Force and Scanning Near Field Optical,
Chemical/Biological Force Microscope tips, nano manipulators, nano tweezers)
(www.xintek.com, 2002 and http ://www.applied-nono-technology.com, 2001).
Skim latex or rubber serum is produced as a by-product during the preparation
of latex concentrate upon centrifugation. It contained a dry rubber content of only 3 to
7 % (Tangboriboonrat et al., 1999) with very low dirt content. Serum is the non rubber
aqueous portion of latex which can be obtained via acid coagulation or membrane
filtration. The serum contains a rich source of nitrogen, carbohydrates, proteins,
enzymes and lipids. Some of these proteins are important enzymes which have great
demand in pharmaceutical, food and detergent industries. Hence, there is a need in the
technology to improve the efficiency and yield of protein separation. Purification
steps will involve the use of column chromatographies, namely the Ion Exchange
Chromatography (IEC) and Hydrophobic Interaction Chromatography (HIC). This
process helps to purify and obtain high efficiency of protein separation from skim
latex, using non functionalized and functionalized CNTs as the chromatographic
media.
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1.2 PROBLEM STATEMENT
Recently, Chemical vapor Deposition (CVD) seems to be the most promising method
for CNTs production.CVD is very different from the other two common methods used
for CNTs production namely; arc discharge and laser ablation (Ebbesen and Ajayan,
1992, Journet et al.,1997 and Thess et al., 1996). At the moment, arc discharge
methods generally produce larger quantities of impure material and produces small
amount of CNTs yield (Journet and Bernier, 1998). Whereas for laser ablation, the
method was said to be uneconomically feasible due to high laser power required and
involves high-purity graphite rods, as well as it produces small amounts of nanotubes
(Kalpana et al., 2005). The material prepared by these techniques has to be purified
using chemical and separation methods (Strong et al., 2003). None of these techniques
are scalable (Lijima, 1991, Guo et al., 1995) to make the industrial quantities needed
for many applications (e.g., in composites), and this has been a bottleneck in nanotube
research and development (R&D).
In recent years, work has focused on developing Chemical Vapor Deposition
(CVD) (Dong et al., 2006) which is one of the most popular method to produce CNTs
using C2H2 and H2 as precursors, while Argon (Ar) acts as parching gas. CVD seems
to be the most promising method for possible industrial scale-up due to the relatively
low growth temperature, high yields and high purities that can be achieved (Thomas,
1997). Chemical Vapor Deposition (CVD) is a very promising process with respect to
large-scale production of different kinds of carbon nanostructures materials as for
example for multi, and single-walled carbon nanotubes.
Latex extraction from the rubber tree, Hevea brasiliensis, can be done by
tapping the tree, and as the latex exudes from the cut, it was further centrifuged to
produce concentrated latex and skim latex as by-products. The concentrated latex
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serves as the main raw material for the further process of product manufacturing while
the skim latex on the other hand is the waste product of rubber industry. The skim
latex is conventionally being treated using oxidation ditch or waste stabilization ponds
before discharging it into the stream. Instead of leaving this abundantly available
skim latex untouched and prolong affecting the environment, it is better to make use
of this useful material. This can maximize the usage of rubber product, minimize
pollution on the environment, and undeniably generate promising revenue to the
rubber growers as well as our country as a whole.
1.3 RESEARCH OBJECTIVES
1. To produce high purity of Carbon Nanotubes (CNTs) by using Double
Stage Chemical Vapor Deposition (DS-CVD).
2. To investigate the effect of production parameters on CNTs production
by varying gase flow rates (C2H2 and H2), reaction temperature and
reaction time by using Design Expert®
Version 6.0.8.
3. To optimize the process parameters in skim latex protein separation by
varying pH and concentration of buffer salt.
4. To compare the separation of protein from skim latex using non-
functionalized CNTs and functionalized CNTs as protein purification
media in column chromatography.
1.4 RESEARCH METHODOLOGY
In this research, carbon nanotube is produced using Double Stage Chemical Vapor
Deposition (DS-CVD). In order to achieve high purity of CNTs, the process
parameters were optimized, such as gas flow rates of H2 and C2H2, reaction time,
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reaction temperature, using Design Expert®
version 6.0.8. The CNTs produced were
purified by using acid washing. Covalent functionalization was carried out using two
step diimide activated amidation process. The process was carried to obtain functional
groups such as amine or carboxylic group on the surface of CNTs. The noncovalent
functionalization was obtained by irreversibly attaching 1-pyrenebutyric acid
succinimidyl ester onto the sidewall of the MWNTs via π-π stacking of the pyrene
group. In column chromatography, selection of chromatographic media namely
functionalized and non functionalized CNTs were essential in order to separate protein
from skim latex serum. The purification of CNTs using the functionalized CNTs
which guided by the functional group such as carboxylic or amine group that exists on
the surface of CNTs was performed by Ion Exchange Chromatography (IEC). Non
functionalized CNTs which contain only carbon on its surface undergo the
Hydrophobic Interactions Chromatography (HIC) which based on the hydrophobic
interaction between the proteins and CNTs matrix.
1.5 RESEARCH PHILOSOPHY
Proteins separation in conjunction with carbon nanotubes is expected to create a
major breakthrough in the coming future. Carbon nonotubes with its supernatural
ability to perform multidisciplinary functions can find niche applications in both the
immobilization of protein and selective removal of proteins. However, synthesis
conditions have major influences on the nature of the carbon nanotube product
formed. Reaction conditions including temperature, gas composition, and the nature
and composition of metallic catalysts leading to the nanotube formation which in turn
affects the properties of the final product have to be still explored to understand these
influences. Moreover, issues regarding the synthesis and purification as well as the
6
functionalization and solubilization of carbon nanotubes are relevant topics in this
rapidly growing field.
1.6 SIGNIFICANCE OF STUDY
CNTs are in the limelight globally as a new dream material in the 21st century
and are broadening their applications to almost all the scientific areas, such as
aerospace science, bioengineering, environmental energy, materials industry, medical
and medicine science, electronic computer, security and safety, and science education.
Now they are known to be superior to any other existing material in mechanical,
electrical, and hydrogen storage characteristics.
1. The effect of process parameters such as reaction temperature, reaction
time and gas flow rate of acetylene and hydrogen were essential in order
to produce carbon nanotube.
2. Separation of protein from skim latex serum using CNTs as a
chromatographic media.
3. Functionalize carbon nanotubes (CNTs), to be used as a filter in separating
the proteins from skim latex serum.
4. The research of CNTs functionaliztion has been intensified due to their
potential for biomedical and biotechnological application (Davis et al.,
1998).
5. Material for biomedical application such as fillers in property–enhanced
nanocomposites supports matrix in enzyme immobilization and
chromatographic media in protein purification.
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1.7 SCOPE OF RESEARCH
In this research, Double Stage Chemical Vapor Deposition (DS-CVD) was used to
produce a high purity of CNTs. DS-CVD was chosen because it can divide the
growing process of MWCNT or SWNT into a nucleation stage and a growth stage.
Nucleation stage is at the beginning of MWCNT or SWNT growth and in very short
time, in which the catalyst particles get ready to catalyze the growth of MWCNTs or
SWNTs and short nanotubes nucleated on the nanoparticles. In the following growth
stage, the nanotubes grow longer within certain period of time. These produced CNTs
were used in separation of protein from skim latex. This invention was a novel study
to use three types of CNTs namely non functionalized, covalent and non covalent
functionalized, as a column chromatographic media for skim latex protein separation.
The CNTs produced were characterized by using Field Emission Scanning Electron
Microscopy (FESEM), Transmission Electron Microscopy (TEM),
Thermogravimetric Analysis (TGA) and Fourier Transform Infrared Spectroscopy
(FTIR). The separation of protein from skim latex serum, the process conditions such
as pH and concentration of buffer salt will be invistigated
1.8 THESIS ORGANIZATION
Chapter one an introduction/background of the research works. Chapter two provides
a review of literature concerning the various methods of production of CNTs,
applications of CNTs and skim latex serum which contains different types of protein.
Chapter three covers the methodology of the research. Chapter four provides and
discusses the findings obtained from the research work. Chapter five is the conclusion
coupled with some recommendations on how to improve the research.
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