Synthesis and Characterization of Polymeric Membranes for...
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Synthesis and Characterization of Polymeric Membranes for Dialysis Applications By Hizba Waheed Registration No: NUST201290041TPSCME2512F Thesis Supervisor: Dr. Arshad Hussain School of Chemical and Materials Engineering (SCME) National University of Sciences and Technology (NUST) H-12 Islamabad, Pakistan 2019
Transcript of Synthesis and Characterization of Polymeric Membranes for...
Synthesis and Characterization of Polymeric
Membranes for Dialysis Applications
By
Hizba Waheed
Registration No: NUST201290041TPSCME2512F
Thesis Supervisor: Dr. Arshad Hussain
School of Chemical and Materials Engineering (SCME)
National University of Sciences and Technology (NUST)
H-12 Islamabad, Pakistan
2019
Synthesis and Characterization of Polymeric
Membranes for Dialysis Applications
By
Hizba Waheed
Registration No: NUST201290041TPSCME2512F
Thesis submitted to the National University of Science and Technology,
Islamabad, in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in
Chemical Engineering
Thesis Supervisor: Dr. Arshad Hussain
School of Chemical and Materials Engineering (SCME)
National University of Sciences and Technology (NUST)
H-12 Islamabad, Pakistan
2019
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Dedicated to my Parents and Sons
Turab Ali Haider
Zoraiz Wali Haider
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ACKNOWLEDGMENTS
I thank ALLAH, the Most Gracious the Most Beneficent, for all the kindness and
blessings including this learning opportunity of PhD work. Hazrat MOHAMMAD (peace
be upon him) is the role model for every mankind.
I am thankful to my PhD supervisor, Prof. Dr. Arshad Hussain, for his trust, support,
encouragement and valuable instructions throughout the course of this research. I would
like to thank Prof. Dr. Abdul Qadeer Malik, as he always guided and encouraged me
throughout my PhD research work. Sincere thanks to Prof. Dr. Habib Nasirand Prof. Dr.
Muhammad Mujahid for their valuable guidance being members of the Guidance and
Examination Committee.
I want to thank my parents for their continuous support and guidance throughout my life;
especially my Abu. Ammi’s prayers are most precious thing ever happened. I appreciate
the kind support and encouragement from my loving brothers &caring sisters.
Many thanks to late Dr. Fouzia Tabassum Minhas, Dr. Sarah Farrukh and Dr.
Muhammad Ahsan for their support at every step towards this dissertation. They always
encouraged me to work hard for achieving my goals. I would like to thank Prof. Dr.
Xianshe Feng, University of Waterloo (UWaterloo), Canada for his support, guidance and
help during my stay at UWaterloo. I will never forget his lab in my life, as I have learnt a
lot from there. The experimental facilities provided by Department of Chemical
Engineering, UWaterloo played the key role in this dissertation.
It would not be out of place to thanks School of Chemical and Materials
Engineering (SCME), NUST my alma mater for providing me state of the art facilities to
accomplish this task.
I am thankful to all the Egyptian and European scholars in UWaterloo for their
contribution to my research work, guidance and living support during my stay at
UWaterloo, Canada. The technical discussion with Tabassum Fozia, M. Aftab Akram,and
Sofia Javed during this dissertation always took me out of the problem, I was facing.
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I love my kids Turab Ali Haider and Zoraiz Wali Haider whose smiles always relieved
the pressures of different phases during the PhD work. The finishing line of my
acknowledgment is reserved for my husband, Maj. Amir Mukhtar, whose presence;
support, care and love are invaluable.
I am thankful to NUST, for funding my PhD studies and supporting me throughout this
work. It would not have been possible to complete my PhD if HEC hasn’t provided the
international platform with IRSIP.
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ABSTRACT
Membrane is a thin, delicate, flat sheet which acts as a barrier for selective transport of
species under the impact of some driving force. Membrane technology has gained
important place in industrial and medicinal field because of its easy utility, efficient
performance and low cast. Hemodialysis is an extensively used medical therapy for renal
failure and dialysis membranes are essential components of a hemodialysis. The essential
properties of a dialysis membrane are high mass transfer of toxic solutes (urea and uric
acid) to reduce the dialysis time, maximum protein rejection ability and moderate water
flux. Protein adsorption or deposition on the surface or in its pores results in reduction in
flux, change of selectivity of the membrane and the low toxin elimination. Polymeric
membrane fabricated from cellulose, regenerated cellulose and synthetic polymers are
well known for dialysis.
Asymmetric Cellulose Acetate (CA) membranes were prepared through phase inversion
method and they were modified by blending various organic and inorganic additives. The
effects of these additives on membrane’s morphology were investigated using Atomic
Force Microscopy, Scanning Electron Microscopy, Fourier Transform Infra-Red
Spectroscopy and Contact Angle. Fabricated membrane’s performance was studied in
terms of pure water flux, porosity, water uptake, BSA rejection and urea clearance tests.
Biocompatibility and blood mimic tests were conducted to find the interaction of
synthesized membrane towards cell culture and blood comparable fluids.
In first part, CA was blended with poly-ethylene glycol (PEG). The membranes were
modified by blending CA/PEG casting solution with glycol. The modified membrane
showed good selectivity for urea but was not suitable for dialysis operation. Hence, the
composition was reformed using Hydroxyapatite particles (inorganic additive). The
results showed enhanced BSA rejection and urea clearance but the obtained percentages
were low to be utilized in dialysis. The biocompatibility outcomes of CA/PEG/HA
membrane make it appropriate for other biomedical applications.
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In the second part, CA was blended with organic additives including sericine, Poly
vinylpyrolidone (PVP) and polyethylene imine (PEI) to improve BSA rejection and Urea
clearance of polymeric membrane. These membranes possess moderate pure water flux
and hydrophilicity. Performance evaluation investigations confirmed that all these
membranes had good pure water flux and BSA rejection above 90%. Membranes
fabricated using blend of CA and PEI have highest urea clearance of 67.2%. So, this
membrane was selected for further adjustment.
During the last part, effect of solvent on CA/PEI dialysis membrane was investigated.
Various solvents including acetic acid, formic acid, N, N-Dimethylacetamide (DMAC)
and 1-Methyl-2-pyrolidone (NMP) were tested. The performance efficiency of
synthesized membranes verified to, when CA was blended with formic acid (F.A) have
desired dialysis characteristics. It possesses moderate hydrophilicity, desired pure water
flux value, optimum water uptake, above 98% BSA rejection and urea clearance
percentage of 69%.
The biocompatibility tests were conducted for CA/PEI/FA membrane using MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide )assay. It was found that the
materials selected for this membrane fabrication were most suitable for dialysis
application. Highest cell viability and cellular attachment found in biocompatibility
analysis was higher in comparison with commercial dialysis tubing and non-treated
control standard. CA/PEI/F.A membrane was further inspected via blood mimic solution
to find the performance of membrane commensurate with blood type feed.
The up short of the present work is that CA/PEI/F.A membrane is the best possible
solution for dialysis. Providing new insight in the dialysis domain; this membrane is not
only cost effective but also has high BSA rejection and Urea clearance. Accordingly,
biocompatibility and blood mimic results prove it to be the finest device/implant for
hemodialyser unit.
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PUBLICATIONS
Relevant with this Thesis 1. Hizba Waheed, Arshad Hussain, Sara Farrukh, Fabrication,
Characterization and permeation study of ultrafiltration membranes:
glucose absorption membranes, Desalination and water treatment 57(2016)
24799 – 24806.
2. Hizba Waheed, Arshad Hussain, Fouzia Tabassum, Cellulose
Acetate/Sericin Blended Membranes for Use in Dialysis. Journal of
Polymer Bulletin 75(2018) 3935 – 3950.
3. Hizba Waheed, Arshad Hussain, Effect of Polyvinyl Pyrolidone on
Morphology and Performance of Cellulose Acetate Based Dialysis
Membrane.Engineering technology and applied science researchVol. 9,
No. 1, 2019, 3744-3749.
4. Hizba Waheed, Arshad Hussain, Fabrication of Cellulose
acetate/Polyaziridine Blended Flat Sheet Membranes for Dialysis
Application.Bio-nano science. https://doi.org/10.1007/s12668-019-0600-5.
Conference proceedings
5. Hizba Waheed, Arshad Hussain, Modification of Cellulose acetate based
ultrafilteration membranes using Hydroxyapetite nanoparticles for dialysis
application, International Journal of Advances in Science Engineering and
Technology 5(2017) 39 – 43.
“Conference of on chemical and biochemical Engineering (ICCBE) 2017”
6. Hizba Waheed, Arshad Hussain, Preparation and solvents effect study of
asymmetric cellulose acetated/polyethyleneimine blended membranes for
dialysis application. International Journal of Health and Medicines 2(2017)
5 – 9.
“International Conference of Engineerinhg, Management, Technology and
Science (ICEMTS) 2017”
Other Publications
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7. Hizba Waheed, Arshad Hussain, Utilization of Carbon dioxide for the
production of Ethanol, Journal of Chemical Society of Pakistan 39(2017)
896 – 902.
8. Aneela Hayder, Arshad Hussain, Ahmad Nawaz Khan, Hizba
Waheed,Fabrication and characterization of cellulose
acetate/Hydroxyapetite composite membranes for the solute separations in
Hemodialysis. Journal of Polymer Bulletin 75(2018) 1197 – 1210.
9. Amir Mukhtar, Habib Nasir, Hizba Waheed, Pressure time study of slow
burning rate Ap/HTPB based composite propellant by using closed vessel
test (CVT), Key Engineering Materials 778(2018) 268 - 274.
Table of Contents
Chapter 1 : General Introduction to Membranes Vis-a-vis Biomedical
Applications ..................................................................................................................... 1
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1.1 Introduction .......................................................................................................................... 3
1.2 Fundamental Concepts ........................................................................................................ 4
1.3 Classification of Membranes ............................................................................................... 4
1.3.1 Classification of Membranes Based on Membrane Materials .......................... 5
1.3.2 Classification of Membranes Based on Morphology and Structure ................. 6
1.4 Preparation of Porous Polymeric Membranes ................................................................... 8
1.4.1 Phase Inversion Technique ............................................................................... 8
1.4.2 Precipitation by Solvent Evaporation ............................................................... 9
1.4.3 Precipitation from the Vapor Phase .................................................................. 9
1.4.4 Precipitation by Controlled Evaporation .......................................................... 9
1.4.5 Thermal Precipitation ..................................................................................... 10
1.4.6 Immersion Precipitation.................................................................................. 10
1.4.7 Coating ............................................................................................................ 10
1.5 Different MembraneModules ........................................................................................... 11
1.5.1 Tubular Membrane Modules .......................................................................... 11
1.5.2 Flat Sheet Membranes .................................................................................... 13
1.6 Membrane Based Separation Processes ........................................................................... 14
1.6.1 Microfiltration................................................................................................. 15
1.6.2 Ultrafiltration .................................................................................................. 15
1.6.3 Dead-end filtration .......................................................................................... 15
1.6.4 Cross-flow filtration........................................................................................ 16
1.6.5 Nano-filtration ................................................................................................ 16
1.6.6 Reverse Osmosis (RO) ................................................................................... 17
1.7 Transport mechanism ........................................................................................................ 18
1.8 Research Objectives ........................................................................................................... 18
Chapter 2: Background of Polymeric Membranes for Dialysis ............................... 22
2.1 Kidneys ............................................................................................................................... 22
2.1.1 Kidney failure ................................................................................................. 23
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2.1.2 Medical treatments.......................................................................................... 24
2.2 Hemodialysis, membranes and principles ....................................................................... 24
2.2.1 Commonly used hemodialysis membranes .................................................... 25
2.2.2 Basic principles of hemodialysis .................................................................... 27
2.3 Uremic toxins...................................................................................................................... 29
2.3.1 Water-soluble toxins ....................................................................................... 29
2.3.2 Protein-bound toxins ....................................................................................... 30
2.3.3 Large toxins .................................................................................................... 30
2.4 Properties of Hemodialysis Membranes .......................................................................... 30
2.5 Membrane preparation techniques .................................................................................. 31
2.6 Cellulose acetate Membranes............................................................................................ 32
2.7 Additives ............................................................................................................................. 33
2.7.1 Poly etylene glycol (PEG) ....................................................................... 33
2.7.2 Monosodium glutamate (MSG) ................................................................ 33
2.7.3 D-glucose monohydrate .................................................................................. 34
2.7.4 Chitosan .......................................................................................................... 34
2.7.5 Glycerol .......................................................................................................... 35
2.7.6 Lithium Additives ..................................................................................... 35
2.7.7 Oxides ............................................................................................................. 35
2.8 Solvents ............................................................................................................................... 36
References................................................................................................................ 37
Chapter 3: Experimental Procedures & Materials .................................................... 40
3.1 Introduction ........................................................................................................................ 40
3.1.1 Polymer- Organic Additive Blended Membranes for Dialysis ....................... 40
3.1.2 Polymer- Inorganic particles Blended Membranes for Dialysis ..................... 40
3.2 Materials ............................................................................................................................. 41
3.2.1 Selection of Polymer ...................................................................................... 41
3.2.2 Organic filler ................................................................................................... 42
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3.2.3 Inorganic Additives ........................................................................................ 46
3.2.4 Solvents .......................................................................................................... 46
3.2.5 Others .............................................................................................................. 47
3.3 Fabrication Processes ........................................................................................................ 47
3.4 Characterization Techniques ............................................................................................ 48
3.4.1 Scanning Electron Microscopy ....................................................................... 48
3.4.2 Atomic Force Microscopy .............................................................................. 49
3.4.3 Fourier Transform Infrared Spectroscopy ...................................................... 50
3.4.4 Contact Angle Measurement .......................................................................... 51
3.4.5 Porosity of Membrane .................................................................................... 52
3.4.6 Water Absorption Measurements (Degree of swelling) ................................. 52
3.5 Methods to Test Membrane dialysis efficiency ............................................................... 53
3.5.1 Pure water flux (PWP) .................................................................................... 53
3.5.2 Molecular weight cut-off measurement (Solute transport) ............................. 54
3.5.3 BSA rejection.................................................................................................. 55
3.5.4 Urea Clearance................................................................................................ 55
3.5.5 Biocompatibility test ....................................................................................... 56
3.5.6 Blood Mimic testing ....................................................................................... 58
References................................................................................................................ 60
Chapter 4: Optimization of Polymeric Material ........................................................ 63
4.1 Introduction ........................................................................................................................ 63
4.2 Results and Discussions ..................................................................................................... 64
4.3 SEM Analysis ..................................................................................................................... 65
4.4 Atomic Force Microscopy analysis ................................................................................... 67
4.5 FTIR Spectroscopic Analysis ............................................................................................ 68
4.6 Membrane Performance ................................................................................................... 69
4.7 Conclusion .......................................................................................................................... 73
References................................................................................................................ 74
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Chapter 5: Result and Discussion of membranes with In-organic Additive ........... 75
5.1 Inorganic Additives ............................................................................................................ 75
5.2 Blend of cellulose acetate/PEG with Hydroxyapatite inorganic additive) for dialysis 75
5.2.1 Introduction..................................................................................................... 75
5.1.2 Results and Discussions .................................................................................. 76
5.1.2.1 SEM Analysis .............................................................................................. 76
5.1.2.2 AFM Analysis .............................................................................................. 77
5.1.2.3 FTIR Results ................................................................................................ 78
5.1.2.4 Contact angle measurement ......................................................................... 79
5.1.3 Permeation Testing ......................................................................................... 80
5.1.3.1 Pure water flux ............................................................................................. 80
5.1.3.2 BSA rejection% ........................................................................................... 80
5.1.3.3 Urea clearance % ......................................................................................... 81
5.1.4 Biocompatibility testing.................................................................................. 82
5.1.5 Conclusion ...................................................................................................... 88
References................................................................................................................ 89
Chapter 6: Result and Discussion of Dialysis via Polymer-Organic Material Blended
Membranes .................................................................................................................... 90
Organic Additives .................................................................................................... 90
6.1 Blend of Cellulose acetate-Sericin (organic additive) ..................................................... 90
6.1.1 Introduction..................................................................................................... 90
6.1.2 Results and Discussion ................................................................................... 92
6.1.2.1 SEM Analysis .............................................................................................. 92
6.1.2.2 AFM Analysis ............................................................................................ 94
6.1.2.3 FTIR Analysis .............................................................................................. 95
6.1.2.4 Contact angle and water uptake ................................................................... 95
6.1.3 Permeation Testing ......................................................................................... 96
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6.1.3.1 Pure Water Flux and MWCO ...................................................................... 96
6.1.3.2 Porosity ........................................................................................................ 98
6.1.3.3 BSA rejection % .......................................................................................... 99
6.1.3.4 Urea clearance % ......................................................................................... 99
6.1.4 Comparison Study ........................................................................................ 100
6.2 Blend of Cellulose acetate-polyvinyl pyrolidone PVP (organic additive) ................... 101
6.2.1 Introduction................................................................................................... 101
6.2.2 Results and Discussions ................................................................................ 102
6.2.2.1 SEM Analysis ............................................................................................ 102
6.2.2.2 FTIR Analysis ............................................................................................ 103
6.2.2.3 Influence of PVP concentration on hydrophilicity and water uptake ........ 104
6.2.3 Permeation Testing ....................................................................................... 105
6.2.3.1 Effect of PVP concentration and evaporation time on pure water flux ..... 105
6.2.3.2 BSA rejection% study of CA/PVP blended membranes ........................... 106
6.2.3.3 Urea clearance study of CA/PVP blended membranes ............................. 107
6.2.4 Conclusion .................................................................................................... 108
6.3 Blend of Cellulose acetate with organic additive Polyaziridine (PEI) for Dialysis
Application ............................................................................................................................. 109
6.3.1 Introduction................................................................................................... 109
6.3.2 Results and discussions ................................................................................ 110
6.3.2.1 SEM Analysis ............................................................................................ 110
6.3.2.2 AFM Results .............................................................................................. 111
6.3.2.3 FTIR Analysis ............................................................................................ 112
6.3.2.4 Contact Angle and water uptake measurement ......................................... 113
6.3.2.5 Porosity % .................................................................................................. 115
6.3.3 Permeation Testing ....................................................................................... 116
6.3.3.1 MWCO and Pure Water Flux: ................................................................... 116
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6.3.3.2 BSA rejection% ......................................................................................... 117
6.3.3.3 Urea clearance % ....................................................................................... 118
6.3.4 Conclusion .................................................................................................... 119
6.4 Investigation of effect of solvent on cellulose acetate membranes blended with
Polyethylene imine ................................................................................................................. 120
6.4.1 Introduction................................................................................................... 120
6.4.2 Results and Discussions ................................................................................ 121
6.4.2.1 SEM Analysis ............................................................................................ 121
6.4.2.2 Effect of solvent on morphology ............................................................... 122
6.4.2.3 AFM Results .............................................................................................. 123
6.4.2.4 Contact Angle ............................................................................................ 123
6.4.3 Permeation Testing ....................................................................................... 124
6.4.3.1 Pure Water Flux ......................................................................................... 124
6.4.3.2 BSA rejection % ........................................................................................ 125
6.4.3.3 Urea clearance % ....................................................................................... 125
6.4.4 Conclusion .................................................................................................... 126
6.5 Comparison of all fabricated membranes ..................................................................... 127
6.5.1 Contact angle of fabricated membranes ....................................................... 127
6.5.2 Pure water flux of fabricated membranes ..................................................... 127
6.5.3 BSA rejection % of fabricated membranes .................................................. 128
6.5.4 Urea clearance % of fabricated membranes ................................................. 129
6.6 Biocompatibility Test ...................................................................................................... 130
6.6.1 Cell Viabilities .............................................................................................. 131
6.6.2 Cellular attachment ....................................................................................... 134
6.7 Blood Mimic Testing ........................................................................................................ 137
6.7.1 Density of Blood Mimic fluid ...................................................................... 137
6.7.2 Permeation testing using Blood mimics fluid ............................................... 137
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References.............................................................................................................. 141
Conclusion .............................................................................................................................. 143
Future recommendations ...................................................................................................... 144
List of Tables
Table 1.1: membrane preparation methods and membrane prepared ...................... 10
Table 1.2: Classification, properties and uses of tubular module or membranes .... 13
Table 1.3: Classification, properties and uses of Flat sheet membranes. ................ 14
Table 1.4: Separation processes and their properties............................................... 18
Table 2.1: Various polymers used for Dialysis membrane synthesis. ..................... 26
Table 2.2: Concentration of water soluble and protein bound uremic toxins in healthy
human blood ............................................................................................................ 29
Table 2.3: acetyl values and solubility of cellulose acetate in various solvents ...... 36
Table 3.1: Specifications of cellulose acetate .......................................................... 42
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Table 4.1: Composition of Cellulose acetate/PEG blended membranes ................. 64
Table 4.2: composition of Cellulose acetate/PEG/Glycerol blended membranes ... 64
Table 4.3: Tabular illustration of results shown by CA/PEG and CA/PEG/Glycol
blended membranes ................................................................................................. 70
Table 5.1: Composition of CA/PEG/Glycol and HA blended membranes ............. 76
Table 5.2: Surface roughness study of CA/PEG/Glycol and HA blended membranes
................................................................................................................................. 78
Table 6.1: CA/sericin blended membrane composition .......................................... 92
Table 6.2: Composition of CA/PVP blended membranes ..................................... 101
Table 6.3: Composition of CA/PEI blended membranes ...................................... 110
Table 6.4: Composition of CA/PEI membranes prepared with different solvents 120
Table 6.5: Composition of blood mimic fluid ....................................................... 137
List of Figures
Figure 1.1: Presentation of Hemodialysis procedure ................................................. 3
Figure 1.2: Schematic presentation of membrane filtration. ..................................... 4
Figure 1.3: Classification of membranes based on origin, material and morphology.5
Figure 1.4: Schematic representation of symmetric membranes ............................... 6
Figure 1.5: Anisotropic membranes .......................................................................... 7
Figure 1.6: Graphical representation of membrane classification on the basis of
structure ..................................................................................................................... 8
Figure 1.7: Diagrammatic representation of phase inversion method ....................... 9
Figure 1.8: Schematic of tubular membrane module............................................... 11
Figure 1.9: Schematic of hollow-fiber membrane module ...................................... 12
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Figure 1.10: Flat sheet membrane module .............................................................. 14
Figure 1.11: Dead-end filtration .............................................................................. 16
Figure 1.12: Cross-flow filtration ............................................................................ 16
Figure 1.13: Schematic diagram of Osmosis and Reverse Osmosis process .......... 17
Figure 2.1: Location and Cross-sectional image of human kidney [4].................... 22
Figure 2.2: Hemodialysis process [9] ...................................................................... 25
Figure 2.3: Structure of cellulose acetate [15] ......................................................... 26
Figure 2.4: Diffusion transport ................................................................................ 27
Figure 2.5: Osmosis mechanism .............................................................................. 28
Figure 2.6: Ultrafiltration process............................................................................ 28
Figure 2.7: Adsorption phenomena ......................................................................... 29
Figure 2.8: Structure of Cellulose acetate polymer ................................................. 32
Figure 2.9: Structural presentation of PEG.............................................................. 33
Figure 2.10: Structure of Monosodium glutamate ................................................... 34
Figure 2.11: Molecular Structure describing D-glucose monohydrate ................... 34
Figure 2.12: Chemical formula of Chitosan ............................................................ 35
Figure 2.13: Structural formula of Glycerol ............................................................ 35
Figure 3.1: The structural representation of cellulose acetate ................................. 42
Figure 3.2: Chemical formula and model of Glycerine ........................................... 43
Figure 3.3: Chemical Formula of Polyethylene Glycol ........................................... 43
Figure 3.4: Branched Poly-Ethylene Imine ............................................................. 44
Figure 3.5: Structural formula of PVP ..................................................................... 45
Figure 3.6: Structural formula of Sericine fibrinogen ............................................. 45
Figure 3.7: Structure of Hydroxyapatite .................................................................. 46
Figure 3.8: Membrane fabrication procedure. ......................................................... 48
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Figure 3.9: Diagrammatic and schematic representation of scanning electron
microscopy ............................................................................................................... 49
Figure 3.10: Representation of AFM instrument and principle ............................... 50
Figure 3.11: Diagrammatic and schematic presentation of FTIR instrument ......... 51
Figure 3.12: Contact angle illustration and properties studied through Contact angle
................................................................................................................................. 51
Figure 3.13: porosity and water uptake experiments ............................................... 53
Figure 3.14: Dead-end filtration set up for pure water flux, molecular weight cut-off
and BSA rejection calculations ................................................................................ 54
Figure 3.15: Diffusion setup for BSA rejection, Urea clearance and Blood mimic
testing ....................................................................................................................... 56
Figure 3.16: MC3T3-E1 Cells after 24 hrs .............................................................. 57
Figure 3.17: 5x5 mm squares pieces and sterilization of test samples using 70%
ethanol...................................................................................................................... 57
Figure 3.18: Experimental sequence of cell viability test (MTT) ........................... 58
Figure 4.1: SEM Surface images showing the influence of PEG on M0 to M4 and
influence ofcombination of PEG and glycerin on the morphology of membranes M4
to M8 ........................................................................................................................ 66
Figure 4.2: AFM scans of CA/PEG and CA/PEG/Glycol blended membranes ...... 68
Figure 4.3: FTIR spectrum of selected CA/PEG and CA/PEG/Glycol blended
membranes ............................................................................................................... 69
Figure 4.4: Graph presenting Fluxes of pure water, urea and glucose solutions by
CA/PEG ................................................................................................................... 71
Figure 4.5: Graph presenting Fluxes of pure water, urea and glucose solutions
byCA/PEG/Glycol ................................................................................................... 72
Figure 4.6: Selectivity of fabricated membranes ..................................................... 73
Figure 5.1: Surface morphology of CA/PEG/Glycol and HA blended membranes 77
Figure 5.2: Cross-sectional view of CA/PEG/Glycol and HA blended membranes 77
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Figure 5.3: AFM analysis of CA/PEG/Glycol and HA blended membranes .......... 78
Figure 5.4: FTIR spectrum of CA/PEG/Glycol and HA blended membranes ........ 79
Figure 5.5: Contact angle measurement of CA/PEG/Glycol and HA blended
membranes ............................................................................................................... 79
Figure 5.6: Water flux measurement of CA/PEG/Glycol and HA blended membranes
................................................................................................................................. 80
Figure 5.7: BSA rejection calculations of CA/PEG/Glycol and HA blended
membranes ............................................................................................................... 81
Figure 5.8: Urea clearance calculation of CA/PEG/Glycol and HA blended
membranes ............................................................................................................... 81
Figure 5.9: Cell viability after 1 day ........................................................................ 82
Figure 5.10: Cell viability after 2 days .................................................................... 83
Figure 5.11: Cell viability after 6 days .................................................................... 83
Figure 5.12: Cell viability after 9 days .................................................................... 83
Figure 5.13: Cell viability after 13 days .................................................................. 84
Figure 5.14: Cellular attachment of NTC at four times ........................................... 85
Figure 5.15: Cellular attachment of CA/PEG/HA at four times .............................. 86
Figure 5.16: Graphical presentation of cellular attachment of CA/PEG/HA at four
times ......................................................................................................................... 87
Figure 6.1: The surface and cross-sectional SEM micrograph of pure and CA-sericin
blend membranes ..................................................................................................... 93
Figure 6.2: AFM micrograph of pure and CA-sericin blend membranes................ 94
Figure 6.3: FTIR spectrum of pure and CA-sericin blend membranes ................... 95
Figure 6.4: Contact angle and water uptake of CA-sericin blended membranes .... 96
Figure 6.5: Molecular weight cutoff and pure water flux of CA-sericin blended
membranes ............................................................................................................... 98
Figure 6.6: Porosity % of CA-sericin blended membranes ..................................... 98
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Figure 6.7: BSA rejection % of CA-sericin blended membranes ............................ 99
Figure 6.8: Urea clearance % of CA-sericin blended membranes ......................... 100
Figure 6.9: Difference in appearance of membranes ............................................. 102
Figure 6.10: Surface morphology (upper row) and cross-sectional view (lower row)
of CA/PVP blended membranes ............................................................................ 103
Figure 6.11: FTIR spectrum of CA/PVP blended membranes ............................. 104
Figure 6.12: Contact angle and water uptake of CA/PVP blended membranes .... 105
Figure 6.13: Effect of PVP concentration and evaporation time on CA/PVP blended
membranes ............................................................................................................. 106
Figure 6.14: BSA rejection measurement of CA/PVP blended membranes. ........ 107
Figure 6.15: Urea clearance % of CA/PVP blended membranes .......................... 108
Figure 6.16: Surface morphology of CA/PEI blended membrane using SEM...... 111
Figure 6.17: Cross sectional view of CA/PEI blended membrane using SEM ..... 111
Figure 6.18: AFM micrographs of CA/PEI blended membrane using SEM ......... 112
Figure 6.19: FTIR spectrum CA/PEI blended membrane using SEM .................. 113
Figure 6.20: Hydrogen bonds formation between the NH group of PEI and the OH
group of CA ........................................................................................................... 113
Figure 6.21: Contact angle and degree of swelling of CA/PEI blended membranes.
............................................................................................................................... 115
Figure 6.22: Graphical representation of porosity trend in CA/PEI blended
membranes ............................................................................................................. 116
Figure 6.23: Pure water flux and solute rejection % of CA/PEI blended membranes
............................................................................................................................... 117
Figure 6.24: BSA rejection presentation by CA/PEI blended membranes ............ 118
Figure 6.25: Urea Clearance trend of CA/PEI blended membranes ...................... 119
Figure 6.26: Surface (a) and cross-sectional (b) image of fabricated membranes 122
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Figure 6.27: AFM micrographs of CA/PEI membrane fabricated with different
solvents .................................................................................................................. 123
Figure 6.28: Contact angle measurement of CA/PEI membranes casted with different
solvents .................................................................................................................. 124
Figure 6.29: Pure water flux measurement of membranes fabricated with variable
solvents .................................................................................................................. 124
Figure 6.30: BSA rejection % of CA/PEI membranes fabricated with different
solvents .................................................................................................................. 125
Figure 6.31: Urea clearance % of CA/PEI membranes fabricated with different
solvents .................................................................................................................. 126
Figure 6.32: Combined Contact angle values of all prepared membranes ............ 127
Figure 6.33: Combined Pure Water Flux of all prepared membranes ................... 128
Figure 6.34: Combine BSA rejection % of all prepared membranes .................... 129
Figure 6.35: Combine Urea clearance % of all prepared membranes ................... 130
Figure 6.36: Cell viability on C.A+ F.A+PEI after 1 day ..................................... 131
Figure 6.37: Cell viability on C.A+ F.A+PEI after 2 days .................................... 132
Figure 6.38: Cell viability on C.A+ F.A+PEI after 6 days .................................... 132
Figure 6.39: Cell viability on C.A+ F.A+PEI after 9 days .................................... 133
Figure 6.40: Cell viability on C.A+F.A+PEI after 13 days ................................... 133
Figure 6.41: Cellular attachment at NTC at four points ........................................ 134
Figure 6.42: Cellular attachment at CA-PEI-FA at four points ............................. 135
Figure 6.43: Graphical representation of cellular attachment at CA-PEI-FA at four
points ..................................................................................................................... 136
Figure 6.44: Permeation setup ............................................................................... 138
Figure 6.45: BSA Standard curve using UV-Vis spectrophotometer .................... 138
Figure 6.46: Urea Standard curve using UV-Vis spectrophotometer .................... 139
xxv
Figure 6.47: BSA rejection trend through C.A+ F.A+PEI membrane during blood
mimic testing ......................................................................................................... 139
Figure 6.48: Urea clearance trend through C.A+ F.A+PEI membrane during blood
mimic testing ......................................................................................................... 140
1
Chapter 1 : General Introduction to Membranes
Vis-a-vis Biomedical Applications
A membrane is a thin, flat sheet that can be described can be as a barrier which permits
selective transportation of species under the impact of a driving force. Membrane
technology is extensively accepted and utilized in industrial and medicinal areas.
Membrane filtrations predominantly are identified for their fast process, robust
performance for marketable products, modest operation and multi-purpose application in
biomedical ground. A wider spectrum includes aspects from purification of fresh media
of fermentations, separation of biopharmaceuticals, polishing of protein products [1-3].
Purification and separation using microfiltration and ultrafiltration are being studied
intensely due to the versatile nature of the research and good accessibility in research
facilities. The past period has seen an immense growth in biopharmaceutical industry and
currently in developing countries efforts are also being made to introduce latest medicinal
technologies in their health and medicines area. The large sized molecular products from
bio-industry received constant attention in membrane filtration area. Nevertheless,
purification via membrane is overlooked as most attention goes into innovative
functionalities, better preparation techniques, combinatorial applications and up-to-date
explorations. An equivalent effort must be made for all kinds of applications out of the
competence from membrane filtration as it has a lot more to be developed. Membrane
technology is linked with nanotechnology, bio-medication and analysis. So, it should be
accepted as more than samples preparation. Membranes for biomedical and analytical
applications are prospected to have a growing future [4].
Polymeric membranes are widely used as part of drug delivery systems, medical implants,
biosensors, diagnostic assays etc. [5]. Membrane based operations are applicable
efficiently for treating patients with illness including gas exchange with blood (e.g. blood
oxygenation) or for the excretion of toxic components from blood (e.g. Hemodialysis).
Membranes having appropriate molecular mass cut-off are used as discriminating barriers
to avoid the immune system constituents from contacting with implants and letting
metabolites and nutrients to pass freely to and from cell. Due to vast production of
2
commercialized polymeric membranes since 1980, membrane separation/filtration has
promptly become a competitive separation technique. The lack of mechanical parts in
membrane filtration makes this very easy and attractive for variable applications [6].
Renal failure or kidney failure is among the most important health problems being faced
by many people all over the world. These patients choose either transplantation or
undergo hemodialysis treatment. Latest research has shown that 28% people suffer from
renal failure all over the world. Patients who choose hemodialysis have to go through it
regularly [7-8].
During hemodialysis treatment, the patient’s blood is pumped to the blood
chamber of a dialyzer/dialysis unit. This chamber comprises of a stack of semi-permeable
membranes. Patient’s blood passes through the membranes surrounded by dialysate,
surplus wastes from blood goes to dialysate via diffusion and convection mechanism.
Later, purified blood is returned to the patient [9]. Throughout the process, the most
significant part of dialyzer is membrane.
3
Figure 1.1: Presentation of Hemodialysis procedure
1.1 Introduction
Membrane science has gone through an extensive historical advancement in laboratory.
Its first important industrial application was recognized in the 1960s. During these 5
decades of 5 fast developments, membrane-based operations have grown tremendously
within industrial and medical areas thus, have countless benefits to improve human life
[10-12]. Membrane technology has various applications like ultrafiltration, nanofiltration,
reverse osmosis, gas separation operations, pervaporation and electrodialysis [13-15]. The
membrane is a basic part of above mentioned applications. The capability of a membrane
to permit the passage of chemical entities is the key property which forms the base of
these applications.
4
1.2 Fundamental Concepts
Membrane is well-defined delicate barrier between two phases which allow transfer of
some constituents but keeps others. Constituents are separated on the basis of their
physical and chemical characteristics. Fig 1.2 shows the permeation through membrane.
Figure 1.2: Schematic presentation of membrane filtration.
Membrane’s technology is valuable because of the facts that the systems are convenient
to install and operate. Membranes processes are flexible and cost-effective. They are
environmental friendly. Operations and applications involving membranes are compact
and can be efficiently scaled up. These operations are continuous and can be operated at
atmospheric/ moderate conditions. They are more preferred as are easy in handling. High
flow rates, pressures and feed compositions can be affectively handled during procedure.
Systems that use membranes can be molded according to the specific task. Membrane
technology can be combined with other separation techniques to make the process more
proficient.
1.3 Classification of Membranes
Membranes are categorized into various classes based on
Type of material used for fabrication
Morphology of membrane
Membrane structure
5
Manufacturing technique used to prepare the membrane
Figure 1.3: Classification of membranes based on origin, material and morphology.
1.3.1 Classification of Membranes Based on Membrane Materials
Various materials are used for the preparation of membranes. Based on the type of
material used, membranes are categorized as organic (polymer membranes) and inorganic
or ceramic membranes.
a) Organic polymer membranes
These membranes are synthesized using polyvinylidene fluoride (PVDF),
polyethersulfone (PES),polyether rimide(PEI), polyacrylonitrile
(PAN),polyphenylsulfone (PPSu), and polyamide (PA) [16]. Currently, polymeric
membranes are in use principally because an appropriate polymer can be selected
for the specific separation task from the existing variety. Similarly, in comparison
to other materials, polymeric membranes are inexpensive. For purification
operation, the structure and behavioral properties of polymers, like thermal,
mechanical, chemical stability, and the permeability are vital.
b) Inorganic membranes
These membranes are fabricated from materials like ceramics, aluminum, fiber
reinforced carbon and high grade steel. These are used when employment of
6
polymeric membranes is not possible due to the features and properties of the feed
supply or the polymeric membrane has to be washed periodically due to the feed
utilization. Inorganic membranes have high resistant against heat and chemical
shocks as compared to polymer membranes. They also exhibit slow aging rate and
long active lives. Disadvantages of in inorganic membranes are expensive
fabrication and module construction.
c) Composite membrane
These are identified as “hybrid or mixed matrix membrane”, this includes
polymeric plus inorganic membranes. The surface which behaves as an active
separating layer is coated on a support which certifies selective or specific species
to pass across.
1.3.2 Classification of Membranes Based on Morphology and Structure
On the basis of morphology, membranes are classified as symmetric or asymmetric.
Membranes can be molecularly homogeneous i.e., totally unvarying in composition and
structure or variable in their chemical and physical nature [17-19]. The thickness of
symmetric membranes varies from 10–200μm, and this characteristic dictates the
resistance offered by membrane to mass transfer. Fig 1.4 shows structure of symmetric
membranes.
Figure 1.4: Schematic representation of symmetric membranes
An asymmetric membrane is composed of a thin, delicate (0.1-1.0 μm) sheet spread on
very porous (100-200 μm) substructure. The barrier layer is prepared through interfacial
polymerization technique [18-19].The upper thin layer behaves as the selective
7
membrane. Separation abilities of this membrane depend on the nature of the material
used for membrane preparation and pore radius. The mass transfer rate is mostly
dependent on thickness of upper skin. The porous layer is a support for the thin, active
layer and put little influence on the separation mechanism.
Figure 1.5: Anisotropic membranes
The membrane classification on the basis of membrane structure can be graphically
represented using Fig 1.6.
8
Figure 1.6: Graphical representation of membrane classification on the basis of structure
1.4 Preparation of Porous Polymeric Membranes Different methods are reported for membranes preparation in literature. Using the listed
methods organic, inorganic and composite membranes can be synthesized. These methods
includes
1.4.1 Phase Inversion Technique
Many commercial membranes are fabricated using phase inversion method. It is very
useful method for membranes synthesis with different topographic properties. Various
factors are involved in controlling the morphology of prepared membrane. This method
for membrane fabrication comprises dissolution of the polymer in an appropriate solvent,
which is then extruded in the preferred alignment or module. Later, the polymer is
precipitated using phase transition which is carried out either by changing temperature or
by a changing the constituents of the casting or bathing solution [20]. The phase inversion
method is diagrammatically represented in Fig 1.7.
9
Figure 1.7: Diagrammatic representation of phase inversion method
1.4.2 Precipitation by Solvent Evaporation
This method involves polymer dissolution in a suitable solvent that results in formation of
casting solution which is later casted on a proper support. When casting is complete, the
solvent from the wet casted film is evaporated in an inert atmosphere to yield
homogeneous membrane with dense structure.
1.4.3 Precipitation from the Vapor Phase
Another technique applied for the fabrication of porous membrane is precipitation from
vapor phase. The wet casted sheet or membrane is placed in a vapor phase having non-
solvent concentrated with solvent molecules. The amount of solvent added in the vapor
phase inhibits the vaporization of solvent from the casted layer. Movement of the non-
solvent molecules into casted layer results in generation of porous structure.
1.4.4 Precipitation by Controlled Evaporation
This technique includes a blending solvent and non-solvent together. Solvent used is
much volatile than non-solvent and is used to dissolve the polymer. Composition of the
membrane changes during evaporation, as non-solvent and polymer constituent increases
to higher level. This leads to polymer precipitation, resulting in development of porous
asymmetric membrane.
10
1.4.5 Thermal Precipitation
In this method, a polymer dissolved in single or mixture of solvents is cooled to assist
phase separation. Vaporization of the solvents molecules permits production of thin,
asymmetric membrane.
1.4.6 Immersion Precipitation
This technique includes casting of polymer solution on an appropriate support. This is
later followed by immersion of casted film in coagulation bath which is filled with a non-
solvent media [21]. Precipitation phenomena occur due to interchange of solvent and non-
solvent molecules among the casted membrane. These membranes often exhibit
asymmetric structure.
Membranes prepared during this research involve phase inversion technique. This
technique permits the formation of asymmetric membranes having desired pore size and
homogenous pores distribution on the surface.
1.4.7 Coating
Composite membranes are significantly fabricated using coating technique. Various
processes including dip-coating, plasma polymerization and interfacial polymerization are
applied for coating an ultrathin layer over a spongy substrate
Table 1.1: membrane preparation methods and membrane prepared
Membrane preparation
Process
Types of membranes synthesized
Phase inversion Reverse osmosis, microfiltration,
ultrafiltration, dialysis membranes
Sintering 0.1-20 microns pore size
Coating Composite membranes
Extrusion/activation Silicon rubber
Track etching Radioactive source exposure and then
etch with acids
Controlled stretching 0.1-5 microns pore size
Phase separation Glass or ceramics membranes
11
1.5 Different Membrane Modules Different membrane modules utilized in industrial and medical applications are given
below.
1.5.1 Tubular Membrane Modules
Tubular membranes exhibit a rough structure comprising of durable polymeric materials,
which allows easy processing of suspended solids and concentrated feed efficiently and
recurrently towards high finish-point concentration with no blockage. Tubular membranes
are applicable for pressure driven separation operations.
Figure 1.8: Schematic of tubular membrane module
Hollow Fiber and Capillary Membrane Modules
Hollow fiber and capillary membranes are often categorized as types of tubular
membranes. Hollow fibers (HF) are extremely applicable and low cost substitutes for
traditional filtration processes. Feed is allowed to pass from outside to in or inside to out.
The feed keeps moving through the cavity of membrane and permeate moves out via
walls of the fiber. Fig. 1.7 represents the phenomena clearly.
12
Figure 1.9: Schematic of hollow-fiber membrane module
HF unit intends to handle huge volumes for convenient flow during processes. Depending
upon the structural reliability and manufacturing, HF membranes have the ability to bear
permeate back pressure, that results in flexible system design and processing. Capillary
membranes are like HF membranes for their design and operation but differ only in
dimensions of the membranes.
The practical utilization, advantages and drawbacks of all types of tubular membrane
modules are listed in Table 1.2.
13
Table 1.2: Classification, properties and uses of tubular module or membranes
Tubular Form of Membrane
HFM CM TM
Inner diameter 0.1 -0.5 mm 0.2- 5.5 mm 5.5 -25 mm
Arrangement of
separation layer
Inside/outside Inside/outside Inside
Operating mode Cross-flow/Dead-
end
Cross-flow/Dead-
end
Cross-flow
Applications Waste water
treatment, industrial,
Biotechnology,
food, beverage,
diary etc
Waste water
treatment, food,
diary, wine industry
Treatment of waste
water contaminated
with oil, grease,
heavy metal and
suspended solids,
effluents with wide
pH and temperature
range.
Advantages High membrane
surface area,
specificity, low cost,
pressure resistant,
compact
High membrane
surface area,
specific membrane
cost
Hardly susceptible
to blockage, low
fouling, easy
cleaning, easy to
replace
Drawbacks Susceptible to
blockage, pressure
loss
Low pressure
resistance
High capital cost,
low packing
density, high
pumping cost,
HFM = Hollow fiber membranes, CP= Capillary membranes, TM = tubular membranes
1.5.2 Flat Sheet Membranes
These membranes are prepared by casting the polymeric solution on a glass plate or a
rigid support. Later, precipitation of the polymer occurs. Flat membranes can be placed as
stacked sheet or in spiral wound form. These membranes when placed in modules are
separated using spacers that cause a little surface to volume ratio.
These membranes are comparable to a filter paper as they are positioned in a sandwich-
like pattern with their active sides facing each other as shown below Fig. 1.10.
14
Figure 1.10: Flat sheet membrane module
In this model, a set of membranes with specified surface area are provided vacuum
distributors, sealing gaskets, and two end plates consisting of one plate and frame. The
feed solution is allowed to circulate within stacked membranes. These membranes can be
arranged spirally for spiral wound module as well. This module is applicable in series as
well as parallel. Few properties regarding two types of flat sheet membranes are listed in
Table 1.3 below.
Table 1.3: Classification, properties and uses of Flat sheet membranes.
Flat Membrane
Spiral wound module Plat or frame module
Arrangement of separation
layer
Outside Out side
Operating mode Cross-flow/ Dead-end Cross-flow
Applications Desalination of sea water,
Brackish water treatment,
water softening, diary, food,
pharmaceutical, applications
Treatment of high viscous
effluents
Advantages High surface area, volume,
energy
Easily replaceable, hardly
susceptible to blockage
Disadvantages Can be blocked easily,
pressure loss
High capital cost, low surface
area
1.6 Membrane Based Separation Processes
Commonly used separation processes utilizing membranes are microfiltration (MF),
ultrafiltration (UF), Nanofiltration (NF) and Reverse osmosis. These four operations
employ a hydrostatic pressure difference as driving force and are correlated to one
another. They can separate variable components depending upon their particle size.
15
1.6.1 Microfiltration
Removal and separation of suspended solids from the feed stream is carried out using
microfiltration. Various natural and synthetic polymers utilized for the production of
membranes for this application includes CA, PVDF, PTFE, PA, PS, PC, PP etc.
Membranes for this application possess uniform porosity of approximately 80%. The
mechanism followed in MF is sieving although filtration is affected by the interactions
among the solution and active surface area of membrane [22].
1.6.2 Ultrafiltration
Ultrafiltration (UF) is operational under the influence of pressure. It is applied for the
removal of particles size ranging from 0.001- 0.02 μm [23]. The membranes used for UF
permit the passage of solvents and low molecular weight salts but molecules with large
size are rejected. The prime use of the UF operation is a filtration of macromolecules, but
is also supporting mining operations to separate and recover flotation agents, surfactants
and organometallic complexes. UF is applicable as pretreatment for different operations
including NF or RO. UF membranes have low osmotic affect and a high working pressure
of 1-7 bar is needed to eliminate the resistance offered by liquid permeation through the
membrane’s pore. UF is engineered depending on two operating arrangements:
1. Dead-end filtration
2. Cross-flow filtration
1.6.3 Dead-end filtration
The filtration method in which feed is forced across the membrane and retained material
is collected on the surface, is termed as dead end filtration. It is a batch process.
Accumulated matter on the membrane lessens the filtration ability, due to clogging or
plugging. Additional stage is needed to eradicate the collected material. This technique
can be used to concentrate different compounds.
16
Figure 1.11: Dead-end filtration
1.6.4 Cross-flow filtration
In this filtration, feed flow is continuous along the membrane surface which inhibits
material build upon the filtering surface. The pressure applied to the feed stream to pass
across act as driving force for filtration operation. It also provides fast flow to generate
turbulence. This alignment is named "cross-flow", as the flow of feed and filtration is at
90 degrees angle with each other. This process is an outstanding way to separate filterable
components from liquids.
Figure 1.12: Cross-flow filtration
1.6.5 Nano-filtration
Nano-filtration (NF) system operates at low pressures as compared to RO. It possess
higher flux values but quality of permeate is inferior then that of permeate received with
RO. Selectivity of NF is not conceivable with RO. Less pressure is needed to operate NF
so, it has lower energy demand than conventional RO systems. NF is applied for the
filtration of multivalent ions and materials such as sulphate, phosphate, Mg and Ca,
17
according to the structure and arrangement of ion. The molecular weight cutoff value for
NF membranes is about 200 Daltons.
1.6.6 Reverse Osmosis (RO)
Removal of ionic components, metals as well as large molecules from water streams such
as industrial wastewater and mine water is possible by using reverse osmosis, etc [24].
Water is the only material that can flow through RO membrane leading to the elimination
of all dissolved and floating organic and inorganic components from feed. The RO works
at pressure relative to the osmotic pressure exerted by solution. The osmotic pressure
ranges from 15-80 bar normally. In RO, species are separated depending upon their shape
and size, their ionic charge, the material used for membrane fabrication and its interaction
with species under operation.RO is mostly helpful in desalination of seawater and
brackish water [25]. The eventual purpose of RO is to recollect components from feed as
product, to yield uncontaminated filtrate or decrease wastewater volume. Fig 1.13 shows
mechanism of osmosis and reverse osmosis.
Figure 1.13: Schematic diagram of Osmosis and Reverse Osmosis process
18
Table 1.4: Separation processes and their properties
Microfiltration Ultrafiltration Nanofiltration Reverse
Osmosis
Operation
mode
Cross-flow and
Dead end
Cross-flow and
Dead end
Cross-flow Cross-flow
Separating
mechanism
Sieving Sieving Diffusion and
exclusion
Diffusion and
exclusion
Membrane
type
Symmetric
Polymeric or
ceramic
Asymmetric
polymer,
Composite or
ceramic
membranes
Asymmetric
polymer,
Composite
membranes
Asymmetric
polymer,
Composite
membranes
Module
type
Spiral wound,
hollow fiber,
tubular, plate or
frame modules
Spiral wound,
hollow fiber,
tubular, plate or
frame modules
Spiral wound,
hollow fiber,
tubular, plate or
frame modules
Spiral wound,
hollow fiber,
tubular, plate or
frame modules
Permeate
flux
High High Medium Low
1.7 Transport mechanism
The separation using membrane technology may be due to variances in size and shape of
the permeating species, or because of the membrane’s interaction with separating
constituents. Other factors like chemical nature of solute or electrical charges on them and
vapor pressure of various components in a blend also affect membrane’s separation
ability. The membrane can be installed in liquid and gaseous phases. The driving force
essential for movement of components across the membrane is transmembrane pressure
difference, an activity difference of components or may be an electrical potential slope or
a temperature difference [26].
1.8 Research Objectives
The basic objective of research was to fabricate pure and modified polymeric membranes
for dialysis application. Objectives of this research work are listed
Polymeric membrane fabrication
Utilization of fabricated membrane for dialysis purpose
Establishment of an experimental set up for performance investigation of
fabricated membrane.
19
In this study, suitable polymeric material was utilized as matrix, whereas organic and
inorganic materials were used as additives. These organic and inorganic materials were
incorporated in polymer matrix to study their effects on morphology and performance of
fabricated membrane when utilized in dialysis operation. Synthesized membrane was
tested on diffusion set up to find its efficiency for dialysis operation. The interactions of
organic and inorganic additives were also examined for their effect on urea and BSA
rejection during dialysis.
20
References
[1] P.G. Kerr, L. Huang, Review: Membranes for haemodialysis, Nephrology 15 (2010)
381–385.
[2] W.R. Clark, R.J. Hamburger, M.J. Lysaght, Kidney international 50(1999) 2005 –
2015.
[3] H. Waheed, A. Hussain, Sara Farrukh, DESALIN WATER TREAT (2016).
[4] P.G. Kerr, L. Huang, Review: Membranes for haemodialysis, Nephrology 15 (2010)
381–385
[5] G. Arthanareeswarana, D. Mohanb, M. Raajenthirenb, , J. Membr. Sci 350(2010) 130
-138.
[6] Willium R.Clark, Day Ong Gao: Properties of membranes used for hemodialysis,
2002.
[7] US Renal Data System, Annual data report: atlas of chronic kidney disease and end
stage renal disease in the United States. 2009
[8] Hemodialysis Membranes: J Am Soc Nephrol 13: S62–S71, 2002
[9] Bingel M, Lonnemann G, Koch KM, Dinarello CA, Shaldon S:. Nephron 50: 273–
276, 1988
[10] S.V. Wroblewski, Wied. Ann. Phys 8 (1879) 29.
[11] S. Loeb, S. Sourirajan, Report No. 6060, UCLA, (1960).
[12] S. Loeb, S. Sourirajan, Adv. Chem. Ser 38 (1962) 117.
[13] S. Darvishmanesh, A.Buekenhoudt, J. Degreve, B.V. Bruggen, J. Membr. Sci 334
(2009)43–49.
[14] Z.Q. Huang, K. Chen, S.N. Li, X.T.Yin, Z. Zhang, H.T. Xu, J. Membr. Sci 315
(2008)164–171.
[15] K.S. Roelofs, T. Hirth, T. Schiestel, J. Membr. Sci 346 (2010) 215–226.
[16] Deshmukh SP, Li K , J Membr Sci 150(1998) 75–85
[17] P. Bernardo, G. Clarizia, Chemical energy transactions 32 (2013)1999-2004
[18] J.M.S. Henis, M.K. Tripodi, J. Membr. Sci. 8 (1981) 233.
[19] T.S. Chung, Polym. and Polym. Comp.4 (1996) 269.
21
[20] R.E. Kesting and A. Menefee, Role of formamide in preparation of celluloseacetate
membranes, KoUoid Z. Z. Polym., 230(2) (1969) 341.
[21] H. Strathmann , P. Scheible, R. W. Baker,Journal of applied polymer science
15(2004).
[22] J.C. Schipper, J. H Hannimaayer, C. A. Smolders, A. Costense, Desalination 38
(1981) 339-348
[23] N. Sing, M. Cheryan,Journal of food engineering 38(1998) 57-67.
[24] Sourirajan, reverse osmosis Academic press. 1970.
[25] Guo-dong. Kang, Yi-ming. Cao, Sciverse science direct 46(2012) 584-60
[26] RW Hamilton, Principles of dialysis: diffusion, convection and dialysis machine,
Atlas of Diseases of the Kidney, 1999
22
Chapter 2: Background of Polymeric Membranes for
Dialysis
Kidneys perform an important role in maintaining human health. Water balance and
minerals level, removal of acidic metabolic waste and effective working of the endocrine
system are all due to kidney. However, when kidneys are incapable to clear waste
materials from the body, medical actions are needed to keep the patient alive.
Hemodialysis is the most common treatment for patient. Normally patients undergo
hemodialysis treatment for some time before kidney transplant.
2.1 Kidneys
Kidneys are bean-shaped organs having length, width and thickness of 11 cm, 5–6 cm,
and 3–4 cm respectively. Each kidney weighs about 120–160 g [1-2]. The function of a
normal kidney is to regulate the blood volume, remove waste products, control blood
pressure and acid-base balance, regulation of sodium and potassium level beside
endocrinal functions [3].
Figure 2.1: Location and Cross-sectional image of human kidney [4]
23
2.1.1 Kidney failure
Kidneys are the vital component to maintain the balanced environment within human
body. Any chronic disease, infection or damage to any of the two kidney will limits its
performance or in some case the kidney will be completely non-functional. Along with
keeping water, metabolic wastes or excretements and toxins balanced in body, kidney
also performs other function as hormonal secretion (erythropoietin, calcitriol, and renin),
regulation of ions concentration, regulation of pH and ions production to control blood
pressure and red blood cell production.
Malfunctioning of kidney may lead to misbalancing of body, disturbed water balance,
trouble in sleeping, fatigue, blood in urine, reduce appetite, muscle cramps and fatigue
along with less concentration level. Accumulation of high metabolic wastes within human
body and excess water concentration may lead to severe health problems or may be death.
The kidney damage or failure may be small or whole kidney. It will lead the patient’s
body to be unable to remove excess water and metabolic wastes which afterward effect
blood contents, blood pressure and blood volume inside body. There are two types of
renal failure depending upon the cause of failure.
1. Acute renal injury (ARI)
2. Chronic renal failure (CRF)
Acute kidney failure (AKI) or acute renal injury (ARI) is “a sudden decrease in kidney
function. In medical terminology can be defined as an unqualified elevation in serum
creatinine, more than or equal to 0.3 mg/dl or lessening urine output (documented oliguria
of less than 0.5 mL/kg per hour for more than six hours). It is also described as
percentage increase in serum creatinine of more than or equal to 50% (1.5-fold from
baseline)[5]. Chronic kidney failure (CRF) is defined by the National Kidney Foundation
as either kidney damage because of some accident or a glomerular filtration rate below 60
mL/min/1.73 m2 body surface areas for at least three months.
24
2.1.2 Medical treatments
There are two possible treatment methods for patients with kidney failure. The most
common of these is hemodialysis. The other is transplantation of kidney, but due to lack
of donor kidneys this possibility is limited and expensive in comparison to hemodialysis.
Hemodialysis is performed as a “bridge to transplant” for affected personas it sustains
patient’s life before transplant.
2.2 Hemodialysis, membranes and principles
Hemodialysis is the procedure of eliminating metabolic waste and surplus water from
renal patient’s blood. It is applied as an artificial replacement to renal failure patient.
Figure 2.2 shows a hemodialysis process. During hemodialysis, the blood from patient’s
body is driven to the blood chamber of a dialyzer. This section of dialyzer is consists of
pack of semi-permeable membranes. When blood flows inside the membranes, the
dialysate flows in space adjoining membranes. Thus, waste from blood goes to the
dialysate via diffusion and convection and cleaned or purified blood is pushed back to the
patient’s body [6-8]. In this process, membrane is the most significant part, as the pore
size of the membrane decides which molecules can permit through it. Dialysate is the
solution to pull toxins from blood during procedure. It generally consists of pure water,
sodium chloride, sodium bicarbonate or sodium acetate, calcium chloride, potassium
chloride and magnesium chloride.
25
Figure 2.2: Hemodialysis process [9]
2.2.1 Commonly used hemodialysis membranes
A hemodialysis membrane is a semipermeable barrier which is used to separate patient’s
blood from dialysate during hemodialysis. The pore size of membrane governs selective
permeability of molecules as it defines the molecules to be rejected or permitted via a
membrane during process. In addition, it affects the biological response of the patients
during and after the hemodialysis treatment [10]. Universally available hemodialysis
membranes can be classified into three groups: regenerated cellulose substituted cellulose
and synthesized polymers.
26
Table 2.1: Various polymers used for Dialysis membrane synthesis.
Regenerated
Cellulose
Substituted
Cellulose
Synthetic Polymers
Cuprophane Cellulose acetate Polyacrylonitrile (PAN)
Cellulose diacetate Polymethylmethacrylate
(PMMA)
Cellulose triacetate AN69
Hemophan Polysulfone
Vitamin E coated Polyethersulfone
a) Cellulose
In 1960s Cellulose membranes were one of the most commonly used membranes as they
are inexpensive and have uniform porosity with minimal thickness [10]. Attachment of
large number of free hydroxyl groups on cellulose monomer make it hydrophilic by
nature. Their immune-reactivity is the disadvantage it faces [11, 13] and only being
available in low-flux form, so they were progressively replaced by substituted cellulose
and synthetic membranes.
Figure 2.3: Structure of cellulose acetate [15]
b) Substituted cellulose
Substituted cellulose membranes are parallel to cellulose membranes, but the free surface
hydroxyl groups are replaced by acetyl residuals from acetate or triacetate [16-18].
Vitamin E [19] and polyethylene glycol grafted [20] cellulose membranes have also been
developed to enhance the biocompatibility of cellulosic membranes. The substituted
cellulose membranes possess better bio-compatibility, but they still have low permeability
for large solutes.
27
c) Synthetic membranes
Synthetic membranes available since 1970s include membranes prepared from AN 69,
polysulfone (PS), polyamide (PA), polyacrylonitrile (PAN), polymethylmethacrylate
(PMMA), polyethersulfone (PES) and polycarbonate [21]. They are collectively called
synthetic membranes. These polymeric membranes can be fabricated with various pore
sizes and required molecular weight cut-off, as well as high-flux ability. These
membranes also have excellent biocompatibility. These membranes are mostly
hydrophobic which leads to adsorption of cells and proteins on the surface of membrane.
2.2.2 Basic principles of hemodialysis
The transport mechanism in hemodialysis is principally diffusion and convection [22-23].
Osmosis, Ultrafiltration, Adsorption can also occur during process, and they are all
explained as follows.
1. Diffusion
Diffusion is the spontaneous passive movement of solutes from region with higher
concentration to lower concentration. The process is illustrated in Figure 2.4. In
hemodialysis, diffusion is the movement of water soluble toxins from blood to dialysate.
The rate of transport depends on diffusion coefficients of the solutes in blood, in
membrane and in dialysate. The movement is also affected due to the concentration
gradient across the membrane and the active area of membrane [24].
Figure 2.4: Diffusion transport
2. Convection
Convection process in hemodialysis is the instantaneous transference of water and solutes
from blood to dialysate across membranes due to pressure gradient (flowing fluid). The
rate of convection is based upon the hydraulic permeability, the concentration of solutes
28
in blood and the pressure gradient across the membrane and sieving coefficient of solutes,
membrane area.
3. Osmosis
The transport of solvent molecules via semi-permeable membranes towards a region of
higher solute concentration in order to attain concentration balance is called osmosis. The
driving force in osmosis is the concentration difference. Figure 2.5 shows the movement
of solvent molecules during osmosis. Osmosis in hemodialysis refers to the movement of
water across membranes into blood plasma or interstitial fluid.
Figure 2.5: Osmosis mechanism
4. Ultrafiltration
The process of removing surplus water into the dialysate due to difference in pressure is
ultrafiltration. The pressure in the blood chamber is higher, so the water moves from a
blood’s side with higher pressure to other side with lower pressure, i.e. the dialysate. A
typical ultrafiltration process is represented in Figure 2.6.
Figure 2.6: Ultrafiltration process
5. Adsorption
It is the phenomena of adhesion of ions, molecules or atoms from a gas, liquid, or
dissolved solid onto a solid surface. During Hemodialysis adsorption happens when
29
uremic toxins adhere to the surface of semi-permeable membrane or into the adsorbents
inside the membranes.
Figure 2.7: Adsorption phenomena
2.3 Uremic toxins
Uremic toxins are contaminants that gather in chronic renal failure patients. These toxins
display several cytotoxic activities in the blood, possess different molecular weights and
some of them are adhered to other proteins, primarily to albumin. These toxins are
normally divided into three groups: water-soluble toxins, protein-bound toxins and large
toxins. Their concentration in healthy people and affected patients are related in Table
2.2.
Table 2.2: Concentration of water soluble and protein bound uremic toxins in healthy human blood
Solute CN/µM CU/µM Cmax/µM Group
Urea <6700 38,333 ±18,333 76,667 Carbamides
(water soluble solutes)
Uric acid < 400 496 ± 265 873 Purines
(water soluble solutes)
Creatinine < 106 1204 ± 407 2124 Guanidines
(water soluble solutes)
p-Cresole 5.6 ± 9 186 ± 41 377 Phenols
(protein bound solutes
Indoxyl sulfate 2.4 ± 22 211 ± 365 940 Indoles
(protein bound solutes
CN= Healthy person, CU = Renal patient, Cmax= Literature Values ,µM= micro molar [25]
2.3.1 Water-soluble toxins
Toxic components with molecular weight (MW) less than 500 Da are water soluble. Urea
and creatinine belongs to this category [26]. These are highly water soluble and do not
adhere to proteins, hence easily removed by hemodialysis. These molecules do not
30
necessarily have toxic activity [27]. The increment of creatinine in serum is typically the
result of uremic retention, but it can also be caused by muscle breakdown. Morbidity and
mortality in hemodialysis patients are certainly associated to their serum creatinine level
[28].
2.3.2 Protein-bound toxins
Protein-bound solutes are a grouped as toxins that bound to proteins, like albumin. They
are usually overlooked, as hemodialysis adequacy is characteristically benchmarked by
urea removal. Research has exposed that protein-bound toxins are relevant to the
advancement of chronic kidney disease (CKD), along with aggravation of cardiovascular
disease [29-30].
2.3.3 Large toxins
Large toxins are molecular weight greater than 500 Da. β-microglobulin is an example.
Buildup of molecules is unconventionally associated with an increased mortality risk [31-
32]. These molecules can only be cleaned by hemodialysis membranes with pore sizes
larger enough for these molecules to cross.
2.4 Properties of Hemodialysis Membranes
Clinical performance of a hemodialyser is vital subject for renal failure patients. Choice
of the dialysis membrane must be cautiously made to promising well-being as well as
efficacy throughout operation. Life of patient should not be endangered [33]. To certify a
positive hemodialysis operation, the membrane utilized must possess some vital
properties. Along with extraordinary permeability and blood compatibility, the chosen
membrane must have characteristics which are mentioned below [34].
a. The pore radius must be of appropriate size.
b. The distribution of pore size must be narrowed to get clear molecular weight cut-
off for dialysis membrane.
c. The smallest pores with minimum diameter have to be on the innermost surface to
avoid blocking of solutes within pores.
31
d. The studies have revealed that membranes with good hydrophilicity have better
biocompatibility because of decrease protein adsorption on active surface
The surface characteristics of membrane are directly linked to the chemical composition.
To fabricate membrane with desired surface properties, polymer selection is important.
Potential hemodialysis membranes must not adsorb any proteins or cells and must possess
high permeability for toxic solutes present in the blood.
2.5 Membrane preparation techniques
Loeb and Sourirajan or phase inversion method for preparation of asymmetric membranes
has a great influence on developing of membrane technology. Asymmetric membranes
comprises of a delicate, moderately dense skin layer reinforced by a much porous
sublayer. The thin surface controls the permeability and imparts high selectivity while the
porous lower layer offers mechanical strength only. Structural features of membrane, like
size and shape of pores, fraction of the compact top layer and porous sublayer, are
determined by the membrane fabrication parameters [35].
Polymeric asymmetric hemodialysis membranes are prepared via phase inversion
technique which can be attained through four basic methods.
1. Thermally-induced phase separation,
2. Vapor-induced phase separation,
3. Immersion precipitation (wet phase inversion),
4. Dry phase inversion.
All mentioned methods involve a homogeneous polymer solution which separates into
less polymeric and high polymeric phases. The polymer-rich phase forms membrane,
while the phase with low polymer concentration is rich in solvents and non-solvents.
In this research, polymeric membranes have been prepared using immersion precipitation
(wet phase inversion). Phase inversion via immersion precipitation is highly applicable
membrane fabrication process. A polymer is dissolved in solvent and is casted on glass
slab. Later it is submerged in a coagulation bath having non-solvent. Due to solvent and
non-solvent exchange, precipitation takes place which subsequently form membrane that
leaves the glass surface immediately.
32
The viscosity, temperature of polymeric casting solution and the temperature of non-
solvent coagulation bath control final structure of membrane.
A recent study has shown various factors affect the structure of fabricated membrane.
These factors include composition of the casting solution, temperature and air velocity,
thickness of casted membrane and the relative humidity in the atmosphere [36].
2.6 Cellulose acetate Membranes
Cellulose Acetate (CA) is a polymers utilized for preparation of membranes required in
separation applications since mid-1980s. The first cellulosic membrane was fabricated by
Loeb and Surirajan for reverse osmosis. The cellulose based membranes are principally
made up of pure cellulose acetate, cellulose di acetate and cellulose triacetate or blend of
these polymers.
The most significant feature of cellulose acetate membrane’s performance is their degree
of acetylation. The molecular structure of CA is given in Fig 2.8. The substitution of
hydroxyl group with acetyl groups shows degree of acetylation. These functional groups
vary from each other in sizes which affects the packing of polymeric chains [37-38]. The
flexibility and mobility of chains are also affected by the replacement of functional
groups; as intermolecular hydrogen bonding is lessen with it.
Figure 2.8: Structure of Cellulose acetate polymer
Synthesis method is another factor which can affect the effectiveness of membrane.
Mainly the evaporation rate of solvent and water can result in damage to porous structure
of membrane due to capillary force which leads to formation of dense membranes [39].
To prevent this damage, membranes are dried by solvent exchange methods.
The cellulose acetate polymer was not that much explored. Keeping in mind all above
discussion and facts, organic and inorganic additives were blended with cellulose acetate
33
polymer and their effect was studied on dialysis membrane performance. Based on our
best knowledge these studies reported for the first time.
2.7 Additives
The incorporation of additive, binder or filler as a constituent to polymeric casting
solution has been one of the practices used for modification of prepared membrane. The
purpose of additive was described as a pore-generating agent that improves permeation
characteristics [40-42]. Additives may be organic or inorganic and have lesser volatility
as compared to solvent and exhibit appropriate solubility for the casting medium. It is
more practical to explain the organic and inorganic additive effect on the membrane
fabrication in terms of coagulation value, viscosity, precipitation type, precipitation rate,
diffusion coefficient, etc. Literature shows numbers of organic and inorganic additives
used for membrane fabrication process. Some of these additives are given below,
2.7.1 Poly ethylene glycol (PEG)
Polyethylene is a polyether compound with molecular formula C2nH4n+2On+1as shown in
Fig 2.9. It has wide range of applications including industrial and medicinal [43]. In
membrane technology PEG is used as porogen (pore forming agent) to induce uniform
and appropriate pore formation. This additive is easily available and is biocompatible
therefore utilized in membrane formation excessively with various polymer like cellulose
acetate, polyether sulphone, polyether imide etc. [44].
Figure 2.9: Structural presentation of PEG
2.7.2 Monosodium glutamate (MSG)
Monosodium glutamate (MSG) is identified as a food additive. The structural formula is
represented in Fig 2.10. Presence of hydroxyl group increases its hydrophilic properties
34
which make it more acceptable to be used in biomedical applications. Secondly it is cheap
[45].
Figure 2.10: Structure of Monosodium glutamate
2.7.3 D-glucose monohydrate
D-glucose monohydrate is a monohydrate from D-glucose. It belongs to sugar family i-e
natural saccharides and carbohydrates as shown in Fig 2.11. It increases the
hydrophilicity as well as creatinine and urea clearance when used with cellulose acetate
as additive [46].
Figure 2.11: Molecular Structure describing D-glucose monohydrate
2.7.4 Chitosan
Chitosan is a linear polysaccharide and has number of industrial and biomedical uses.it is
excessively used in membrane technology because of its high ability to bind toxic metal
ions from medium. This is because of lone pair of electron on its amino group which
makes an active site for toxic ion adsorption [47]. Keeping this in mind it is now used
frequently as additive in membrane technology. The repeated monomers for chitosan is
shown in Fig 2.12.
35
Figure 2.12: Chemical formula of Chitosan
2.7.5 Glycerol
Glycerol, glycerine or 1,2,3-propanetriol with molecular formula C3H8O3 is the simplest
polyol. It is utilized as plasticizer additive in membrane fabrication process. Incorporation
of glycerol enhances the membrane hydrophilicity due to presence of –OH functional
group as presented in Fig 2.13 [48]. It also gives flexibility to the membrane structure
[49].
Figure 2.13: Structural formula of Glycerol
2.7.6 Lithium Additives
Lithium additives including LiCl, LiBr and LiF are hygroscopic salts and are used in
membrane fabrication as additive and porogen [50-53]. Literature shows that membranes
fabricated using these additives have more porosity and smaller pore size, therefore are
more suitable for Ultrafiltration, Nano-filtration membrane distillation and gas
permeation applications.
2.7.7 Oxides
Oxides of aluminum, graphene, zinc etc are utilized in fabrication of membranes for gas
separation processes [54-56]. Incorporation of these additives result in the better pore
generation and higher hydrophilicity of fabricated membranes. These additives also led to
36
improved porosity and membrane thickness. They are used in membrane fabrication
utilized in pervaporation, ion separation and filtration processes.
2.8 Solvents
Cellulose Acetate being organic in nature tends to dissolve in organic solvents. The
solubility of Cellulose Acetate in a solvent is subject to its acetyl value [57-59]. CA is
soluble in variety of solvents including Aniline, Diethanolamine, Cyclohexanone,
Tetrachloroethane, Acetic Acid, Formic Acid, Acetone, Formic acid, Tetrahydrofuran,
N,N- Dimethylacetamide (DMAC), 1-Methyl-2-pyrolidone (NMP) etc [60]. Different
acetyl values and solubility of Cellulose Acetate in various solvents are given below
Table 2.3: acetyl values and solubility of cellulose acetate in various solvents
Solvent 51%
Acetylation
55%
Acetylation
61%
Acetylation Acetone X Cyclohexanone X Methyl ethyl ketone X X Methyl formate ∆
Methyl acetate ∆ ∆
Ethyl acetate X X Dimethyl formamide ∆
N-Methyl pyrolidone
Methyl glycol ∆ X Methyl glycol acetate X Tetrahydrofuran X Methylene chloride X
Chloroform X ∆
Dimethylsulfoxide ∆
Propylene carbonate X X = Soluble, X = Insoluble, ∆ = partially soluble
37
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40
Chapter 3: Experimental Procedures & Materials
3.1 Introduction
The experimental work in this research is comprised of two main sections.
Fabrication of polymer - organic material blended membrane.
Fabrication of polymer - inorganic material blended membranes.
3.1.1 Polymer- Organic Additive Blended Membranes for Dialysis
This section of work is dedicated to synthesis, characterization and performance testing of
pure polymeric and polymer- organic additive blended membranes. Pure polymeric
membrane was synthesized using pure polymer and solvent acid. However, for blended
membranes, organic additives were incorporated in polymer matrix (polymer plus
solvent). The characterization and performance testing of all fabricated membrane was
done. Different steps carried out for the aimed research are listed below.
Synthesis of pure polymeric membrane.
Synthesis of blended membranes with organic additives.
Characterization of all fabricated membranes.
Performance testing of all fabricated membranes.
3.1.2 Polymer- Inorganic particles Blended Membranes for Dialysis
This portion of research is focused on fabrication of polymeric membranes doped with
inorganic filler. Here again, pure polymeric membrane was synthesized using pure
polymer. Later, inorganic particles were incorporated within polymer matrix for
modification. The sequence of work is given below.
Synthesis of pure polymeric membrane.
Blending of inorganic particles in polymeric dope solution.
Characterization of all fabricated membranes
41
Dialysis efficiency testing of all fabricated membranes.
Above mentioned work is discussed briefly in following chapters. This chapter contains
all the specifications regarding materials involved in the synthesis of membranes,
preparation methods for particles and membranes, the techniques to characterize
membranes and the setups for pure water flux calculations, molecular weight cut-off
measurements, BSA rejection and urea clearance measurement studies.
3.2 Materials
3.2.1 Selection of Polymer
The selection of polymeric material is vital. CA polymer was targeted here due to its
cheap cost, easy availability, comfortable handling and non-toxicity. The Cellulose
Acetate is the most applied polymer, which has been in various biomedical applications.
Cellulose Acetate membranes make 80% of membrane’s market in the world [1]. The
famous for biomedical membrane supply includes SURGENEX®, BIOVANCE®,
Viscofan Bioengineering ®, TEXTURED cytoflex ® etc.
The extensive medical application of Cellulose Acetate membrane is because of its
renewable, biodegradable and biocompatible nature. It also offers better physical,
mechanical and chemical properties [2-3]. Cellulose Acetate is easy in handling, low in
cost, non-toxic and is easily available. It is soluble in number of solvents like Acetone,
Formic Acid, Acetic Acid, Tetra Hydro Furan etc. The structural formula is represented in
Figure. 3.1.
42
Figure 3.1: The structural representation of cellulose acetate
The commercially available Cellulose Acetate with mol. weight 50,000 with 39%
acetylation was purchased from Sigma Aldrich Germany. The polymer was dried at 70 °C
in oven over night before utilization for synthesis of membranes. The properties of
Cellulose Acetate are tabulated in Table 3.1.
Table 3.1: Specifications of cellulose acetate
Mol wt. 50,000 Dalton by GPC
Extent of
acetylation
39.7 wt %
Impurities ≤ 3.0% water
Refractive index N20/D 1.475 (lit)
Density 1.3 g/mL at 25C (lit)
3.2.2 Organic filler
To fabricate Cellulose Acetate polymeric membranes with organic additives various
chemicals were utilized including Glycerin, Polyethylene Glycol (200), Sericin,
Polyvinyl-Pyrolidone (PVP) and Poly-Ethylene Imine (PEI).
a) Glycerine
Glycerine, 1,2,3-Propane Triol or glycerol (C3H8O3) belongs to polyol group of
compounds [4]. It is colorless, odorless thick liquid that tastes sweet and is non-
toxic. Glycerol is hygroscopic and hydrophilic in nature due to the presence of
three hydroxyl group. Presence of –OH group is responsible for the dissolution of
43
Glycerol in water. Addition of glycerine to cellulosic membranes dope solution
enhances their hydrophilicity and also adds flexibility to membrane [5].
For the research purpose Glycerine with % purity of 99.99% was purchased from
sigma Aldrich. Structural formula of glycol is given below in Fig 3.2.
Figure 3.2: Chemical formula and model of Glycerine
b) PEG
Polyethylene glycol commonly represented as H-(O-CH2-CH2)n–OH belongs to
polyether family. It is also known as polyethylene oxide (PEO) or
polyoxyethylene (POE). PEG is often used in medicinal application because of its
hydrophilic nature and biocompatibility. It is an organic porogen because of its
organic composition [6]. Addition of PEG results in small surface area and pore
volume. Molecular weight of PEG dictates the porosity. This additive has the
ability of protein separation when used as additive in polymeric membranes [7].
Polyethylene glycol (PEG) 400 by Panreac was utilized in this research.The
Structural formula is shown in Fig 3.3.
Figure 3.3: Chemical Formula of Polyethylene Glycol
44
c) PEI
Poly-Ethylene Imine (PEI) or Polyaziridine, Poly [imino (1,2-ethanediyl)] is a
member of branched chain polymers with several amine groups and two carbon
aliphatic spacer attached to the main structure as shown in Fig 3.4. It is widely
used to modify membrane surface [8]. Using various methods of chemical
modification, PEI is incorporated into membranes [9-12]. PEI enhances uniform
pore-size distribution and imparts mechanical rigidity to membrane. It possesses
characteristics such as chemical reactivity and is capability of selectively
adsorbing biological macromolecules (protein).
Figure 3.4: Branched Poly-Ethylene Imine
Polyetyleneimine/ polyaziridine (PEI) branched with average molecular weight of
25,000 was purchased from Aldrich for the completion of experimental work.
d) PVP
Polyvinylpyrrolidone (PVP) also known as polyvidone or povidone.it is
hydrophilic additive which enhances the hydrophilic behavior of CA membranes
as shown in Fig 3.5. It is a water-soluble and increases the porosity of membranes
as well [13-14]. Literature shows that it has good blood compatibility as well [15].
PVP with average molecular weight of 30,000 was purchased from Fluka to carry
out the experiments for task completion.
45
Figure 3.5: Structural formula of PVP
e) Sericin
Sericin a natural silk protein is chosen as additive to blend in CA matrix. Sericin is
very hydrophilic macromolecule and is derived by cocoons of silkworm by a
method called degumming. Bombyx mori cocoon is well known to produce sericin
protein, which is composed of 18 amino acids. The molecular weights of sericin
protein range from 24 to 400 kDa. The polar side chains of hydroxyl, carboxyl and
amino groups in sericin assist in easy cross-linking, copolymerization and
blending with other polymers to form value-added biodegradable materials [16].
For this research Sericin powdered was purchased from Sigma Aldrich. The
structure of sericine fibronigen is shown in Fig 3.6. It is problematic to make a
pure sericin membrane, but few are formed having sericin in a cross-linked form,
blended with other polymers, or copolymerized with other substances are reported
previously.
Figure 3.6: Structural formula of Sericine fibrinogen
46
3.2.3 Inorganic Additives
In order to investigate the effect of inorganic additive blending on membrane’s
morphology and performance, Hydroxyapatite (HA) was blended with cellulose acetate
polymer. The HA particles were synthesized using method described in paper.
a) HA
Hydroxyapatite (HA) appealed considerable attention as a carrier for
biomoleculesbetween other nano sized fillers.Its excellent bioactivity and
biocompatibility make it appropriate inorganic additive. It possesses betterprotein
adsorption.HA isprimarily applied for repairing bone tissue [17-18]. Adsorption
chromatography also utilizes HA and it’s also usedas a column in a high
performance liquid chromatography for separating various proteins [19]. HA
owns two different binding sites in its structure, i.e., Ca or calcium sites which
are rich in calcium ions or positive charge which bind to acidic groups of proteins
and the P sites which adhere to basic groups of proteins due to lack of positive
charge as shown by Fig 3.7 [20, 21]. Here it is used as inorganic filler and was
prepared in lab. Later was crushed by ball mill to attain the required size of
particles.
Figure 3.7: Structure of Hydroxyapatite
3.2.4 Solvents
To prepare cellulose acetate polymeric membranes with various organic and inorganic
additives, we used acetic acid as solvent or polymer additive blending medium.at the end
effect of various solvent on modified cellulose acetate membrane was studied using
Formic Acid,N-Methyl-2-Pyrolidone(NMP) and N,N-Dimethylacetamide (DMAC).
47
3.2.5 Others
BSA with mol. Weight 66,000 Dalton, Urea having mol. Weight 60.06, PEG with
variable mol. Weight of 10,000 and 35,000 along with Dragondorf Reagent was
purchased from Sigma Aldrich.
3.3 Fabrication Processes
Different methods applied for the membrane preparation have been discussed
before.Regardless of the method used, the membrane design criteria listed below should
be kept in view.
Membrane fabrication methods should be easy.
Additive used in the membrane synthesis should be stable and nontoxin in
biological environment. They should also selectively adsorb uremic toxins
and must reject biological molecules.
Pore size of membranes should be big enough for toxins to pass and small
enoughto prevent albumin to pass.
Membranes should be biocompatible.
Membranes should have suitable permeability.
Membranes should be able to remove both water soluble toxins and
protein boundtoxins.
During this research, membranes were synthesized using phase inversion method
(diffusion induced phase separation). During the method, polymeric film is contacted
with vapor or liquid to initialize demixing (solvent and non-solvent exchange), which
modifies the local composition of membrane [22-23].
To synthesize the pure Cellulose Acetate membrane, the polymer was heated in the oven
for 24hrs at 70˚C to remove any water if present. Later, the polymer- solvent mixture
having 1.5g of CA and 10ml Acetic Acid was stirred for 24 hrs. After stirring, the
solution was sonicated for 2hrs to get rid air bubbles if formed. The resulting solution was
casted on glass slab having wet membrane thickness of 200µm. The casted membrane
was allowed to evaporate for about 30 sec.
Later membrane was placed in coagulation bath containing non solvent solution (Water)
with temperature about 25˚C. Later the prepared membrane was washed several times
48
with water and was placed in water for 24hrs prior to testing. The synthesis procedure is
presented in Figure 3.8.
Figure 3.8: Membrane fabrication procedure.
For each set of membranes the organic or inorganic additives were added in the dope
solution and solution was allowed to stir long enough to dissolve the additive. All the
procedure was similar for casting all membranes samples.
3.4 Characterization Techniques
3.4.1 Scanning Electron Microscopy
To study cross section and surface morphology of fabricated membranes, scanning
electron microscopy (SEM) was utilized. In this technique, the membrane surface is
analyzed by electron beam. The beam of electron is generated via electron gun with
prerequisite energy in keV. The electron beam is focused on the surface of sample by
means of electronegative lenses. The rectangular shaped cut sample first dipped in liquid
nitrogen to freeze the membrane structure. This N2 treated sample is then sputtered with
gold particles to make the membrane surface responsive to striking electron beam. The
electrons strike at the sample surface and interact with the atoms. This results in back
scattering or emission of secondary electron, which are detected by detectors. These
secondary electrons reveal surface morphology of sample under investigation. The signal
obtained is weak so amplifiers magnify the signal. An improved highly contrasting image
of the sample surface is obtained with nanometer resolutions. The dissimilarity in number
and speed of electrons reflected from different regions of sample resulted in grey scale
images [24].
49
In this work, Scanning Electron Microscopy (JSM 6409A, Jeol Japan) Fig 3.9 was used to
investigate the surface morphology of pure and blended membranes. Double sided carbon
tape was used to stick the sample on copper stubs. These samples after sputter coating
with thin gold film were placed in SEM to get both surface and cross sectional images of
membranes.
Figure 3.9: Diagrammatic and schematic representation of scanning electron microscopy
3.4.2 Atomic Force Microscopy
Roughness of membrane surfaces were studied by the Atomic Force Microscopy (AFM).
In this technique, a cantilever with a sharp tip taps on sample surface to evaluate the
surface morphology, roughness and porosity [25].
The tip or cantilever interacts with the sample surface due to its raster scanning motion.
The side to side and up/down movements of the tip as it examine the surfacelaterally; the
surface is scanned via the “beam deflection method”. This method comprises of a laser
that is reflected off towards back end of the cantilever and guided to a position sensitive
detector which tracks the vertical and lateral signals of cantilever. The deflection
sensitivity of the detectors should be standardized to know how many nanometers of
motion correspond to a unit of voltage measured on the detector.
In this research, AFM, JEOL (JSPM-5200) was used to investigate the sample’s
topography, roughness and porosity Fig 3.10. In order to remove moisture trapped in
membranes, the pure and blended membranes were dried in vacuum oven after that piece
50
of membrane samples are placed on slide using two sided tape and then were examined
using AFM..
Figure 3.10: Representation of AFM instrument and principle
3.4.3 Fourier Transform Infrared Spectroscopy
Fourier Transform Infrared Spectroscopy also called FTIR Spectroscopy or FTIR
Analysis. It is an analytical procedure applied fordistinguishing organic, inorganic and
polymeric materials. The FTIR device uses infrared radiation of around 10,000 to 100 cm-
1 on a sample. Out of these radiations some are absorbed and few passed through the
sample. The absorbed radiations are transformed into rotational and/or vibrational energy
by the sample under investigation. Using these valuesof transmittance and absorption,
structural explorations of polymers and chemical compounds are made. The spectrum is
generated at the detector end using these signals. This spectrum typically ranges from
4000 cm-1
to 400cm-1
. FTIR confirm the presence of functional groups attached in
polymers or other materials [26].
In this work FTIR Spectrum 100 PerkinElmer, MID-IR instrument was utilized. Small
pieces of the pure and blended membranes were cut and positioned in a pallet holder for
examination. The membrane sampleholderwas then mounted in an FTIR instrument
(PerkinElmer). The used wave number range for FTIR was 450–4000 cm-1 at room
temperature with 1 cm-1 resolution in transmission mode. The FTIR spectrometer is
depicted in Fig 3.11.
51
Figure 3.11: Diagrammatic and schematic presentation of FTIR instrument
3.4.4 Contact Angle Measurement
Contact angle is calculated by placing a liquid drop on a surface of membrane. The angle
formed between the solid/liquid is denoted as the contact angle. The mostly used
technique used for contact angle measurement is looking at the image of the drop and
reading the two-dimensional angle formed between the surface and the drop with the
vertex at the three-phase line as shown below.
In this research, Hydrophilicity or hydrophobic nature of the membrane was investigated
under Tantec Contact Angle Meter [27-28]. Single water drop was endorsed to rest on
membrane. Goniometer was aligned and then focused on membrane water interface and
contact angle was recorded. For every sample, 8 readings were taken to get the mean
value of angle Fig 3.12.
Figure 3.12: Contact angle illustration and properties studied through Contact angle
52
3.4.5 Porosity of Membrane
Ration of Volume of pores on membrane surface to the total volume of membrane defines
membrane’s porosity [29]. The symbol for the porosity is ∈ and was calculated by dry–
wet weight method. Porosity of membrane is obtained by using equation
𝑷𝒐𝒓𝒐𝒔𝒊𝒕𝒚(∈) =
𝑾𝒘𝒆𝒕− 𝑾𝒅𝒓𝒚
𝜹𝒘𝑾𝒘𝒆𝒕− 𝑾𝒅𝒓𝒚
𝜹𝒘 +
𝑾𝒅𝒓𝒚
𝜹𝒑
(1)
Where Wwet and Wdry are the weight in gram of wet and dry membranes,whereasδw is the
density of pure water (g/cm3) and𝛿𝑝is density of polymer respectively
3.4.6 Water Absorption Measurements (Degree of swelling)
Swelling is a property of interaction of a polymer with solvent. Addition of liquid in a
polymer matrix results in its swelling. The water uptake or swelling can be reversible or
irreversible [30]. In some cases, polymers have the ability to absorb moisture which leads
to alter the related properties. Total moisture removal from polymer matrix is not
possible, so it become part of polymer matrix. This is also termed as water uptake or
water absorption.
For water uptake/absorption or degree of swelling calculations, the membrane sample (2
cm x 2 cm) was cut and dried in an oven at 60 °C for 12 h. it was weighed (Mdry) Fig
3.13. The preweighed membrane under testing was waterlogged in deionized water at
ambient temperature for next 24 h. The soaked membrane was taken out and after getting
rid of water from the surface with tissue paper, sample was weighed again (Mwet). The
water uptake of membrane was calculatedby equation given below. In equation,Mwet and
Mdrysymbolize the wet and dry weights of membrane samples correspondingly.
𝑾𝒂𝒕𝒆𝒓 𝒕𝒂𝒌𝒆 𝒖𝒑 =𝑴𝒘𝒆𝒕− 𝑴𝒅𝒓𝒚
𝑴𝒅𝒓𝒚 × 𝟏𝟎𝟎(2)
The thickness of sample was measured by digital screw gauge and measurements were
made at 5 different positions of the membrane under analysis[31].
53
Figure 3.13: porosity and water uptake experiments
3.5 Methods to Test Membrane dialysis efficiency
3.5.1 Pure water flux (PWP)
Pure water flux is the measurement of the hydraulic permeability of a membrane. It is a
basic parameter investigated before membranes application [32]. PWP was calculated for
all membrane samples fabricated during this study using the same procedure. During
experiments, feed utilized was distilled water. All tests were carried out at ambient
temperature (30±3℃) keeping the pressure limits between 0.1 to 3 bars.
Experimental setup includes the dead-end filtration cell. Flat sheet hemodialysis
membrane was mounted on the membrane sieve and the sieve was fitted at its position.
The feed compartment was filled with water (feed) and was closed using lid which was
connected to a nitrogen cylinder via pipes. The nitrogen gas flow provides the pressure at
which PWP calculations are to be made. The pressure control valves are adjusted to
provide the required pressure which pushed the feed through the membrane. Permeate is
collected at the other end. Figure 3.14 shows the experimental setup for measuring PWP.
The volume of permeate within specific time is noted. Experiments were carried out for
each membrane to confirm the reproducibility of results. The PWP was measured using
the equation below.
𝑭𝒍𝒖𝒙 (𝑱) =𝑸
∆𝒕 ×𝑨(3)
54
Where J signifies the permeation flux (Lm-2
h-1
) of pure water, Q is the volume of
permeate solution (L), Δt is the time taken for permeation (h) and A represents the active
area of testing membrane.
Figure 3.14: Dead-end filtration set up for pure water flux, molecular weight cut-off and BSA rejection
calculations
3.5.2 Molecular weight cut-off measurement (Solute transport)
The fabricated flat membranes were examined with non-ionic macromolecules (PEG of
varying molecular weights 10–60 kDa) to find out the lowest permeation limit of sample.
This study was carried out before exposing the membrane to final testing. This test of
finding the lowest weight of molecule that will be 90% retained by the membrane is
called molecular weight cutoff (MWCO). During experiment, the solutions of variable
PEG molecular weights were prepared and were filtered using membrane in dead end
filtration setup as shown above in Fig 3.14.The PEG concentration in feed and permeate
was studied under UV-Visible Spectrophotometer. Later, the extent of solution separation
was calculated using equation (4)
𝑹𝒆𝒋𝒆𝒄𝒕𝒊𝒐𝒏(𝑹) = (𝟏 − 𝑪𝒑
𝑪𝒇) × 𝟏𝟎𝟎% (4)
Here Cp and Cf are the solute concentrations in permeate and feed, separately [33].
55
3.5.3 BSA rejection
Along with pure water flux test and MWCO, Dead end filtration setup was also utilized
for Bovine Serum Albumin (BSA) rejection investigation [34]. The 1000ppm BSA
solution was filled in the feed chamber and was filtered. Concentration of BSA molecules
within feed and permeate was found using UV-Vis spectrophotometer UV mini 1240,
Shimadzu. BSA rejection % was calculated using equation (5).
% 𝒓𝒆𝒋𝒆𝒄𝒕𝒊𝒐𝒏 = (𝟏 −𝑪𝒑
𝑪𝒇) × 𝟏𝟎𝟎 (5)
Where, R is the rejection of solutes (%), p and r are the BSA mass concentration in
permeate and feed, respectively (g/L).
3.5.4 Urea Clearance
The dialysis membrane’s performance was estimated in terms of urea clearance capacity.
Urea should be removed during dialysis. Its accumulation causes amyloidosis-disabling
illness [35-36]. For urea clearance measurement, 1 mg/ml solution of urea was prepared
by dissolving 1g urea in 1000ml water. From this stock solution 50ml was placed on the
donor side of diffusion setup and 2 L deionized water was poured on the receiver side.
Diffusion was allowed across the synthesized membranes for 210 minutes. The dialysis
cell or diffusion setup is shown in Fig 3.15. The variation in concentration on each side
was measured using TOC instrument after every 30 minutes. Urea was determined by
Total Organic Carbon Analyzer (TOC-500, Shimadzu). The concentration of urea was
determined by the equation (6) where Ci and Cf are initial and final concentration at time t
respectively
𝐔𝐫𝐞𝐚 𝐜𝐥𝐞𝐚𝐫𝐚𝐧𝐜𝐞 % = 𝐂𝐢 −𝐂𝐟
𝐂𝐢× 𝟏𝟎𝟎(6)
56
Figure 3.15: Diffusion setup for BSA rejection, Urea clearance and Blood mimic testing
3.5.5 Biocompatibility test
Biocompatibility tests were carried out to find the cytotoxicity and cellular attachment of
fabricated membrane via cell culture. Biocompatibility was tested by culturing murine
fetal calvarial MC3T3-E1 cells (RIKEN Bio Resource Center, Tsukuba, Ibaraki, Japan)
directly on the test materials (direct contact test), using polystyrene well plate as a
control according to ISO 10993-5 [37]. Complete growth medium was prepared using
Dulbecco’s Modified Eagle medium (DMEM D5523) with supplements including 10%
Fetal Calf Serum (FCS, Gibco ®), 1% penicillin–streptomycin (Gibco ®), 2 mM
glutamine (Gibco ®), Sodium Carbonate, 2 mM L-ascorbic acid (Sigma Aldrich),
calcium chloride and monobasic sodium phosphate as recommended by the suppliers of
the cell line. Cultures were kept in an incubator with 5% CO2 and 100% humidity at 37
°C, with culture media being replenished every 2 days. Cells were tested every 3–4 days
[38]. After achievement of 70–80% confluence on the culture flasks (Corning® T-25
flask), MC3T3-E1 cells were detached using 0.25% (w/v) Trypsin - 0.53 mMol EDTA
(Gibco ®) and seeded in 24 well plates (Corning®) with initial cell density 1 × 104
cell/cm2) and maintained in growth medium under conditions mentioned previously.
After 24 hours, a uniform mat of cells was achieved in the 24 well plates.
57
Figure 3.16: MC3T3-E1 Cells after 24 hrs
Samples of the test material were prepared for the direct contact test according to ISO
10993-5. 5 x 5 mm squares were prepared and sterilized in 70% ethanol in a laminar flow
hood for fifteen minutes after which they were washed with PBS, dried and placed in a
24-well plate at one sample per well. Media was changed after every 48 hours.
Figure 3.17: 5x5 mm squares pieces and sterilization of test samples using 70% ethanol
Observations were made on five occasions: day 1, day 2, day 6, day 9 and day 13.
Cellular response was evaluated qualitatively using inverted microscope (Zeiss Axio
Observer Z1, Zeiss, USA); cells were observed for changes in the morphology; signs of
cell death like detachment, cell lysis and any improvement in cellular appearance were
also noted, including cellular attachment to samples and integrity of the cellular network.
Quantitative cell viability was calculated using the methyl tetrazolium (MTT) assay. After
observing the plates under a microscope and taking visual records, the entire plates were
58
treated with MTT (0.005g per ml) solution.10μl was added to each well of the plates
using a multi-channel micropipette to ensure identical treatment of each well (Figure
3.22). These plates were then placed in the incubator for four hours. After four hours,
crystals of insoluble purple formazan were visible under the microscope. These were
solubilized by treating 10% SDS (Sodium Dodecyl Sulphate) in 0.01 m HCl for 24 hours.
The experimental sequence is shown below in Fig 3.18.
Figure 3.18: Experimental sequence of cell viability test (MTT)
The plates were placed in Platos® plate reader to read absorbance at 650nm. After
subtraction of background absorbance, values were compared with the images. Cell
viability was calculated according to the following equation 7.
% 𝐂𝐞𝐥𝐥 𝐕𝐢𝐚𝐛𝐢𝐥𝐢𝐭𝐲 = (𝐌𝐞𝐚𝐧 𝐀𝐛𝐬𝐨𝐫𝐛𝐚𝐧𝐜𝐞 𝐠𝐢𝐯𝐞𝐧 𝐛𝐲 𝐒𝐚𝐦𝐩𝐥𝐞
𝐌𝐞𝐚𝐧 𝐀𝐛𝐬𝐨𝐫𝐛𝐚𝐧𝐜𝐞 𝐠𝐢𝐯𝐞𝐧 𝐛𝐲 𝐂𝐨𝐧𝐭𝐫𝐨𝐥) × 𝟏𝟎𝟎 (7)
According to ISO standards, decrease of cell viability by more than 30% is a cytotoxic
effect. All the experiments were performed three times to ensure the results. Data was
presented as mean and standard deviation was also done. Results were also checked by
One-Way ANOVA and Post-hoc Tukey tests and p<0.05 was considered significant.
3.5.6 Blood Mimic testing
Blood mimic testing was done to investigate the performance of best fabricated
membrane when the solute components were summed together in feed solution called as
blood mimic [38]. This solution was having the composition and viscosity similar to that
of human blood. This feed solution was utilized in the diffusion cell that was already
utilized in testing for urea clearance measurements. The mixture was then analyzed using
59
UV-Vis Spectrophotometer to find feed (blood mimic) and permeate concentrations of
samples. This testing confirmed the applicability of fabricated membrane for dialysis
application.
In this testing, Blood mimic fluid was made comprising of 22% glycerol dissolved in
780ml of water. Later the composition was altered using BSA and urea but the density
was kept constant for whole blood. This mixture was having the actual density of
1.046g/ml which is in agreement with blood density values (1.043 -1.060g/ml) reported in
literature [39].
60
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63
Chapter 4: Optimization of Polymeric Material
4.1 Introduction
With the advancement in membrane technology; concentration, separation and
purification have turn out to be industrially workable unit operations as they have
extraordinary separation efficiency. Other properties like less energy needs, low space
requirements, ease of operation via modern compact modules make this tool a best
choice. Reutilization and reprocessing of chemicals and water make membrane processes
as favorable methods in separation operations [1]. Membrane being an important part of
the process performs a vital role in uttering productivity and effectiveness of the
operation. Membranes are being increasingly used in laboratory as well as industries
since five decades.
Dialysis membranes are characterized by their material used their for synthesis such as
Cellulose Acetate, poly-methyl methacrylate (PMMA), poly-acrylonitrile (PAN),
polysulfone (PS), ethylene vinyl alcohol (EVAL) copolymer and polyamide [2]. Various
authors have stated that different materials used for dialysis membrane fabrication have
different aptitude on biocompatibility and performances efficiency during application.
Cellulose acetate is frequently used material for making dialysis membranes. This is
because of its tremendous properties like biocompatibility, better desalting, high flux, and
low cost [3]. The first formed Cellulose Acetate (CA) membranes had low flux. These
were much liable to bacteriological as well as chemical agents [4]. The CA performance
can be upgraded by blending it with suitable additives. This alteration will fulfill new
requirements and supplementary membrane properties for operations.
In this work, CA membranes were fabricated for Ultrafiltration purpose. The PEG (MW
400) and Glycerol were incorporated as a plasticizer and pore-forming agents. This work
has opened new ways for simplistic structural improvisations in CA matrix by using
plasticizer and pore forming agents. Phase inversion technique was followed to synthesize
membranes. The synthesized membranes were characterized by SEM, FTIR and AFM
64
analysis techniques. The permeation study of water, urea and sugar via these membranes
resulted in interesting results, which are discussed here.
4.2 Results and Discussions
The membranes were synthesized first with 10 wt. % of CA, variable weight % ages of
Polyethylene glycol (PEG), Acetic Acid along with distill water. In this set PEG weight
composition was varied as 6%, 8%, 10% and 12% respectively.
Table 4.1: Composition of Cellulose acetate/PEG blended membranes
Element Weight percentage %
M0 M1 M2 M3 M4
Cellulose acetate 10.2 10.2 10.2 10.2 10.2
Acetic acid 82.3 76.1 74.4 72.9 71.5
Polyethylene glycol 0.0 6.2 7.9 9.9 11.5
Distill water 7.5 7.5 7.5 6.9 6.8
Net total 100 100 100 100 100
Later the best (PEG 6% by weight) among this set of membranes was further modified
with glycerol. Variable composition of modified membranes is given below.
Table 4.2: composition of Cellulose acetate/PEG/Glycerol blended membranes
Element Weight percentage %
M5 M6 M7 M8
Cellulose acetate 10.4 10.2 10.2 10.2
Acetic acid 72.5 71 70 69
Polyethylene glycol 6.2 6.2 6.2 6.2
Glycerol 4.3 6.1 8.1 10.1
Distill water 6.8 6.4 5.5 4.5
Net total 100 100 100 100
65
4.3 SEM Analysis
SEM images were used to study the changes occurred due to addition of PEG and
Glycerol in ultrafiltration dialysis membrane. Fig 4.1 represents the SEM surface images
of all fabricated membranes. In case of M0 (pure polymeric membrane) a compact
spongy structure with thick asymmetric upper layer is seen. The macro-void formation is
enhanced by the addition of hydrophilic additive PEG in dope solution as shown by the
SEM images of M1 to M4 membranes. The membrane morphology changes from thick
dense skin to porous asymmetric structure. Presence of small quantity of hydrophilic
additive promotes instantaneous demixing which enhance pore formation and sugar flux
of produced membranes. The hydrophilic additive PEG, also acting as non-solvent boost
phase inversion mechanism from delayed demixing to instantaneous demixing resulting
in the generation of pores in membranes structure. It is assumed here that PEG played an
important role in modifying the characteristics of cellulose acetate permeability in
dialysis operation. The presence of small amount of PEG caused speedy formation of
nuclei having PEG rich phase in comparison to diffusion of non-solvent into polymer
solution so that nuclei with high concentration of solvent are present and hence promotes
opening or macrovoids formation [5,6]. Additive also aids in rise of the membrane
content i.e increased membrane’s density or higher number of ingredients of membrane
recipe. This results in enhanced degree of swelling. Former research in Ultrafiltration [7,
8] proved that suitable concentration of additives improves creation of macrovoids and
pores whereas high volume of non-solvent lowered the development because of delayed
demixing at growth stage. Dialysis membranes fabricated in this research showed that
increasing amount of additive enhances the viscosity of dope solution and results in
difficulty in membrane casting. Secondly, it produces membranes with low flux.
Lowering the quantity of hydrophilic additive causes the surface layer’s polymer particles
to swell in vertical direction which produces space between additive’s molecules thus
generating pores and in case of high amounts of additive the particles will be overlapped
and will cause swelling in horizontal direction resulting in the production of dense
membranes with low flux. Hence it is concluded that the CA membranes with PEG will
give better flux for water and sugar clearance.
66
In case of membranes M5 to M8, the addition of Glycerol in dope solutions resulted in
uniform distribution of nano-sized pores and also better appearance. The uniform pore
distribution is an essential parameter for constant flux. Although rest of the membranes
have nano-sized pores but in case of M8, the pores were uniformly distributed that bring
about high sugar clearance rate and reasonable flux measurement.
Figure 4.1: SEM Surface images showing the influence of PEG on M0 to M4 and influence of combination
of PEG and glycerin on the morphology of membranes M4 to M8.
67
4.4 Atomic Force Microscopy analysis
The pure and PEG/ Glycerol modified CA membranes were examined under Atomic
Force Microscopy (AFM) in tapping mode. Three dimensional AFM images of top
surfaces of all samples having scanning area of (10µm×10µm) are shown in Fig 4.2. The
light areas in AFM micrographs are related to height and dark areas indicate depression.
From CA/PEG/ Glycerol micrographs, it can be inferred that pure CA membrane has
smooth surface. However incorporation of PEG at variable concentration somehow
enhances surface roughness in comparison to pure CA membrane. An obvious
interpretation for increased roughness is formation of micro and nano pores in
membranes, which generates somewhat heighted features. In the membranes M5 to M8,
Glycerol was included to regulate pore size in membranes and the roughness of
membranes was more in M5. However, a decreasing trend in surface roughness was
observed with increasing concentration of glycerol from M6 to M8. However, uniformity
in nano pore size was observed in membrane (M8) with maximum glycerol wt. %.
68
Figure 4.2: AFM scans of CA/PEG and CA/PEG/Glycol blended membranes
4.5 FTIR Spectroscopic Analysis
FTIR spectra of the selected membranes are collectively shown in Fig 4.3. The FTIR
spectrum of Mo (unblended CA membrane) is related to membranes having different wt.
% of PEG in casting solution (M1 and M3) and membranes with constant PEG and
varying Glycerin wt% in casting solution.
69
In spectrum of pure Cellulose Acetate membrane Mo, the peak at 3417 cm-1
is credited to
stretching vibrations of carboxylic acid group (-COOH). At 1790cm-1
, the prominent peak
represents carbonyl group (C=O). Another peak shows presence of alkyl group (C-CH3)
and at 1235cm-1
the peak is because of asymmetric stretching of ether group (C-O-C). In
case of Polyethylene glycol the characteristic peaks are found at 3410cm-1
, 2869cm-1
and
1100cm-1
represent the –OH, -CH2 and C-O-C respectively. FTIR spectrum of membrane
M1 and M3 the broadening of peaks in –OH region displays confirmation of interaction
between CA and PEG.
In case of Glycerin added membranes M5 and M8, the presence of peak at 3339cm-1
show –OH bond stretching and C-H bond vibration is shown at 2935cm-1
. The small peak
at 2879cm-1
is because of alcoholic stretching in Glycerol. The broadening of –OH peak
and C-H stretching peak shows the perfect blending of PEG and Glycerol with Cellulose
Acetate.
Figure 4.3: FTIR spectrum of selected CA/PEG and CA/PEG/Glycol blended membranes
4.6 Membrane Performance
Prepared membranes were tested to examine their performance. In table given below, it is
illustrated that the membranes were synthesized using Cellulose Acetate polymer having
70
hydrophilic additives like PEG and Glycerol in formulation recipe. Addition of
hydrophilic additive like PEG affected the properties of fabricated membrane and hence
the characteristics changed accordingly [9]. In the reported work, there is comparison of
polymeric membranes having only PEG and the polymeric membrane having variable
quantity of Glycerol but keeping PEG wt.% constant in dope solutions. The result showed
that M1 having 6.2 wt.% PEG is showing good sugar selectivity i.e 11.5 wt. %,however
the addition of glycerol in membrane formulation increased the sugar selectivity up to
15%.
Table 4.3: Tabular illustration of results shown by CA/PEG and CA/PEG/Glycol blended membranes
Results Synthesized Membranes
M1 M2 M3 M4 M5 M6 M7 M8
Pure Water Flux
(Lit/hr.m2)
639.91 254.93 941.6 367.82 743.82 1280.8 772.89 693.86
10% Urea Soln Flux
(Lit/hr.m2)
278.22 246.13 901.5 329.86 522.6 1142.2 694.4 584.8
10% Sugar Soln Flux
(Lit/hr.m2)
55.46 135.29 771.38 227.73 132.3 666.67 260.6 45.6
Pure Water Permeance
(Lit/hr.m2.KPa)
7.86 3.13 11.58 4.52 9.14 15.75 9.50 8.53
10% Urea Permeance
(Lit/hr.m2.KPa)
3.42 3.03 11.08 4.05 6.43 14.04 8.54 7.19
10% Sugar Permeance
(Lit/hr.m2.KPa)
0.68 1.66 9.48 2.80 1.63 8.2 3.20 0.56
Sugar Selectivity 11.5 1.19 0.12 1.62 5.62 1.92 2.96 15.21
71
Figure 4.4: Graph presenting Fluxes of pure water, urea and glucose solutions by CA/PEG
Fig 4.3: Graph presenting Fluxes of pure water, urea and glucose solutions by CA/PEG
In case of 1st set of membranes i.e membranes having PEG hydrophilic additive in dope
solution resulted in formation of membranes having small pores ranging in micro and
nano size range. As shown in Fig 4.3, the fluxes of water, Urea and sugar via M1, M2,
M3 and M4 showed same trend. The fluxes of water and urea are high as compared to
sugar solution. However, the membrane M1 has revealed interesting results. In this
membrane water flux was much higher as compared to sugar and urea fluxes. The non-
uniform pore distribution causes blockage of nano sized pores while micro pores cannot
stop the flow of sugar molecules across the membrane and hence the membrane offered
less hindrance to the flow and resulted in low permeability of sugar. Also the amount of
hydrophilic additive effected the formation and distribution of pore. Increasing weight
percentage of additive resulted in larger pore diameter and increased permeability. M1
showed good pore diameter and distribution hence resulted in better sugar permeability in
comparison to other three membranes.
The properties and performance of M1was further enhanced by adding Glycerol in the
dope solution. The addition was made gradually and at 10.1 weight % addition of
Glycerol, the membranes were formed with uniform distribution of nano-sized pores. Fig
4.4 presents similar trend of water, Urea and sugar solution as represented by CA/PEG
blended membranes. However, M8 shows lowest sugar flux in comparison of water and
72
urea solution fluxes. This means that M8 offer resistance to the flow of sugar molecules
through Cellulose Acetate modified membrane and thus reduces the concentration of
sugar molecules in the filtrate.
Figure 4.5: Graph presenting Fluxes of pure water, urea and glucose solutions by CA/PEG/Glycol
Fig 4.5shows that M8 membrane is with highest sugar selectivity 15.21 in correlation to
that of water. The accumulation of glycerol to PEG/CA dope solution give rise to
nonporous membrane formation which hinders the passage of sugar molecules. The
molecular weight cut-off for nano-filtration is 200 – 1000 Dalton, whereas the molecular
weight of sugar molecule is 342.29 Dalton. The separation in case of nano porous
membranes is because of size difference, therefore the sugar molecules can’t pass through
the nonporous membranes.
73
Figure 4.6: Selectivity of fabricated membranes
4.7 Conclusion
In this section, the consequences of the adding PEG and Glycerin as additives to
Cellulose Acetate ultrafiltration membrane were investigated. Membrane’s morphology
and performance were examined. The glucose clearance was expressively increased by
the concentration of Glycerin in dope solution having PEG. High amount of glycerin i.e
up to 10 wt.% in PEG dope solution heightened urea clearance. The increment of glycerin
more than 10 wt.% lowered the performance efficiency of membrane. Combination of
glycerin and PEG resulted in the formation of hemodialysis ultrafiltration membranes that
can separate glucose efficiently due to uniform nano- sized pore distribution on
membrane surface.
74
References
[1] G. Arthanareeswaran, P. Thanikaivelan, K. Srinivasn, D. Mohan, M. Rajendran,
Elsevier, 40 (2004 ) 2153-2159
[2] Sakai, K. 2. Dialysis Membranes.J. Membr. Sci, 11(1994) 91-130.
[3] H. Schiffl, S.M. Lang, A. Konig, T. Strasse, M.C. Haider, E. Held, Lancet, 344 (1994)
570–572.
[4] T.F. Parker III, R.L. Wingard, L. Husni, T. Ikizler, R.A. Parker, R.M. Hakim, Kidney
Int, 49 (1996) 551–556.
[5] J.H. Kim, K.H. Lee, J. Membr. Sci, 138 (1998) 153–163
[6] B. Chakrabarty, A.K. Ghoshal, M.K. Purkait, Journal of Membrane Science, 309
(2008) 209–221
[7] Toraj Mohammadi, Ehsan Saljoughi, Desalination, 243 (2009) 1–7
[8] Ehsan Saljoughi, Mohammad Amirilargani, Toraj Mohammadi, Desalination, 262
(2010) 72
[9] Ani Idris, Lee Kuan Yet, Journal of Membrane Science 280 (2006) 920–927
75
Chapter 5: Result and Discussion of membranes with
In-organic Additive
5.1 Inorganic Additives
Various inorganic additives have been used previously for dialysis application including
lithium chloride (LiCl), Aluminum chloride (AlCl3), Titanium oxide (TiO2), Silver
nanoparticle etc. In this section Hydroxyapatite (HA) is used as an inorganic additive
blended with Cellulose Acetate. HA is an inorganic hydrophilic molecule with good bio
activity, biocompatibility and protein absorption. Keeping all in view this material is
selected to be blended with CA.
5.2 Blend of cellulose acetate/PEG with Hydroxyapatite inorganic
additive) for dialysis
5.2.1 Introduction
In previous work [1] Cellulose Acetate was used blended with Poly Ethylene Glycol
(PEG) and glycerol as plasticizer as well as pore generating agent. Pure water flux, urea
selectivity and sugar selectivity was made to study fabricated membranes. In this work, it
was desired to enhance the mechanical properties of already synthesized membranes
using Hydroxyapatite (HA) molecular formula Ca5(PO4)3(OH) an inorganic
nanoparticles. HA possess good bio-activity and bio-compatibility and is good adsorbent
for protein [2]. HA used was having particle size of 30 -50nm. All these properties make
this more applicable for dialysis membranes. Phase inversion method was used for the
fabrication of flat sheet membranes. Various characterization techniques including SEM,
AFM, FTIR, XRD and EDS were done to understand the effect of in-organic HA additive
in already prepared composition.
76
Table 5.1: Composition of CA/PEG/Glycol and HA blended membranes
Element Weight percentage %
M0 M1 M2 M3
Cellulose acetate 15.0 15.0 15.0 15.0
Acetic acid 75.0 70.0 65.0 60.0
Polyethylene glycol 6.2 6.2 6.2 6.2
Glycerol 2.0 2.0 2.0 2.0
Hydroxyapatite 0 5.0 10.0 15.0
Distill water 2.0 2.0 2.0 2.0
Net total 100 100 100 100
5.1.2 Results and Discussions
5.1.2.1 SEM Analysis
SEM images show the topography of formed membranes. It displays the appearance of
the surface whether it’s porous or dense. SEM also presents the shapes of pores. In Fig
5.1(a) it is shown that already present PEG in the composition, acts as porogen and
facilitates the pore formation but addition of Hydroxyapatite effects the pore radius and
distribution of pores throughout the membrane. The particle size of HA (40 - 60 nm)
dictate pore radius of membranes during phase inversion [3]. Gradual increase of HA
weight percentage increases the porosity of membrane and reduces pores diameter too. It
heightens uniform distribution all over the membrane and also results in macro-voids
formation in vertical direction as shown in Fig 5.1 (b) HA % up to certain limits improves
the required properties but increment in weight % beyond optimum level disturbs the
characteristics of synthesized membranes by increasing pore radius and membrane
roughness which inversely affect the membrane’s performance.
77
Figure 5.1: Surface morphology of CA/PEG/Glycol and HA blended membranes
Figure 5.2: Cross-sectional view of CA/PEG/Glycol and HA blended membranes
5.1.2.2 AFM Analysis
The 3D-AFM micrographs of upper surfaces of all membranes having area of 6 x 6 µm2
under scan are shown in Fig 5.2. The roughness parameters that describe the surface like
arithmetic mean roughness (Ra) and the square average roughness (Rq) were analyzed for
all samples under AFM instrument and are tabulated in Table 2. Light areas in
micrographs relate to elevations, whereas the dark regions indicate depressions. The
presented results lead to the conclusion that the incorporation of various concentration of
HA one way or another improved the roughness of surface in relation to pure CA
78
membrane except 15 weight % HA loading in doped solution. The observable
clarification for the changed morphology was the presence of HA in CA modified
composition, which spreaded homogeneously and generated better features.
Table 5.2: Surface roughness study of CA/PEG/Glycol and HA blended membranes
Synthesized membranes Rq(nm) Ra(nm)
M0 87.7 69.8
M1 53.6 43.0
M2 48.3 39.2
M3 36.4 26.6
Figure 5.3: AFM analysis of CA/PEG/Glycol and HA blended membranes
5.1.2.3 FTIR Results
The FTIR spectra of pure CA/PEG membranes and CA/PEG blended with HA are
summed up in Fig 5.3. FTIR results of CA/PEG membrane was compared with that of the
modified CA/PEG/HA membranes. In the spectrum of pure CA/PEG, peak at 3417 cm-1
was credited to stretching vibration of the carboxylic acid group (--COOH). The strong
peak around 1790 cm-1
was allocated due to stretching mode of the C=O bond, and
another peak at 1370 cm-1
indicated bending of C-CH3 group. The distinctive peak around
1235 cm-1
was present due to asymmetric stretching of ether C-O-C vibration. The
broadening of OH peak in M1, M2 and M3 is because of enhanced OH functional group
79
by hydroxyapatite addition [4-5]. This feature expresses homogenous dispersion of HA in
doped solution.
Figure 5.4: FTIR spectrum of CA/PEG/Glycol and HA blended membranes
5.1.2.4 Contact angle measurement
Contact angles values (θ) of all prepared membranes, including CA/PEG and
CA/PEG/HA modified were taken using sisile drop method on using Tantec Contact and
are displayed in Fig 5.4 [6-7].
Figure 5.5: Contact angle measurement of CA/PEG/Glycol and HA blended membranes
80
5.1.3 Permeation Testing
5.1.3.1 Pure water flux
Reasonable water permeation is required for all dialysis membranes as they will avoid the
excessive loss of water from the patient’s body [8-9]. In this case, it was measured using
Dead end filtration cell. The results revealed that M2 shows the average value of
169L/hr.m2. Graphical representation of prepared membranes PWP is also given in Fig
5.5.
Figure 5.6: Water flux measurement of CA/PEG/Glycol and HA blended membranes
5.1.3.2 BSA rejection%
An ideal dialysis membrane must avoid albumin loss during its application. Fig 5.6 shows
the % BSA rejection of all CA/PEG and CA/PEG/HA blend membranes measured. All
membranes except pure CA/PEG membrane have more than 90% rejection of BSA while
membrane M0 has 43.7% rejection. This rejection value was reasonably attractive for all
dialysis membranes to inhibit albumin loss [10]. The membrane M2 with 96.8% rejection
gives optimum properties concerning dialysis. All results were measured using using
Shidmazu UV mini 1240 at 280nm.
81
Figure 5.7: BSA rejection calculations of CA/PEG/Glycol and HA blended membranes
5.1.3.3 Urea clearance %
60% urea reduction is the essential parameter for commercial dialysis membrane
[11].Here, urea concentration was measured using diffusion setup and concentrations
were recorded using Total Organic Carbon Analyzer (TOC-500, Shimadzu).
Fig.5.7shows the urea reduction measurements of all fabricated membranes. Membrane
M2 possess highest urea reduction value of 55.2 in contrast to the CA/PEG membrane
with 49.4% urea reduction, which is higher than required commercial parameters for
applicable dialysis membrane as reported by Eknoyan [12].
Figure 5.8: Urea clearance calculation of CA/PEG/Glycol and HA blended membranes
82
5.1.4 Biocompatibility testing
CA/PEG/HA membranes were the only membranes prepared using inorganic additive. Its
biocompatibility was tested via cytotoxicity assy. MTT assay (3-(4,5-Dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) was done to study the cell viability and cell
proliferation on membrane surface. These tests illustrate whether the membrane material
is toxic or not and will it favor cell replication when living cells come in contact with
membrane.
In vitro testing was carried out for CA/PEG/HA and was compared to the non-treatment
control (NTC) and commercial dialysis membrane. NTC is non-treated control i.e the
cells treated with vehicle solvent in which your membrane is placed for testing or
untreated cells. Dialysis tubing was provided in dry, roll by med lab Islamabad. It was
made up of Cellulose Acetate having flat sheet width of 1.3”, with typical molecular
weight cut-off value of 14,000 dalton.
The samples were tested for cell viability after variable intervals like 1 day, 2 days, 6
days, 9 days and 13 days. The results of testing are graphically represented below.
Figure 5.9: Cell viability after 1 day
83
Figure 5.10: Cell viability after 2 days
Figure 5.11: Cell viability after 6 days
Figure 5.12: Cell viability after 9 days
84
Figure 5.13: Cell viability after 13 days
From all above figures i.e Fig 5.8 to Fig 5.12, it is seen that when living cells i.e cell
culture is brought in contact with the CA/PEG/HA membrane the cells survive even for
the long duration of 13 days. This indicated that the membrane material is nontoxic and
will not degrade or no cellular breakdown will occur when this material would be used in
living medium. As this material will not kill living cells so it is biocompatible.
Cellular attachment
Non treatment control (NTC) was used as a standard to compare the cellular attachment
of CA/PEG/HA membranes. The cellular attachment for NTC at four points is shown
pictorially in Fig 5.13.
85
Figure 5.14: Cellular attachment of NTC at four times
CA/PEG/HA
The fabricated membrane was treated with the cell culture and the cell attachment was
observed at four different time intervals i.e day 2, day6, day 9 and day 13. Photographic
results are shown in Fig 5.14 below.
86
Figure 5.15: Cellular attachment of CA/PEG/HA at four times
Cell viabilities on all four time points (Day 2, day 6, day 9 and day 13) were comparable
with the NTC. There was no significant difference found between the viabilities on any
87
day as shown in figures above. This material i.e CA/PEG/HA is therefore nontoxic and
will not damage living cells.
Appearance of the cells on all four time points was compared to NTC in Fig 5.14. It is
seen that the cells remained adhered to the tissue culture plastic, displaying development
of cells connections. Properly spread networks of the cell are visible. There were no
indications of cellular breakdown like granularity around the nucleus or detachment of the
cells from the membrane. At the end of the experiment (day 13), healthy cells were
visible in the treated membrane which was comparable to the untreated control.
Cells were observed to be attached to the membrane surface. As the test advanced
towards the end the cluster kept on growing as the cells were replicating. On day thirteen,
the membrane seems surrounded by cells. This cell attachment test determines that the
membrane CA/PEG/HA is highly biocompatible as it has shown higher viabilities than
dialysis tubing and NTC. This result was also presented in graphical form in Fig 5.15
below.
Figure 5.16: Graphical presentation of cellular attachment of CA/PEG/HA at four times
All above biocompatibility test including cell viability and cellular attachment proved that
the membrane CA/PEG/HA is biocompatible but the urea clearance and BSA rejection
88
found was not good enough to be used for dialysis application. Therefore the blood mimic
testing was not performed for CA/PEG/HA membrane.
5.1.5 Conclusion
The assimilation of HA to CA/PEG resulted in the production of membranes having
better surface modification and performance. Synthesized membranes were tested for
contact angle, water flux, BSA rejection and urea clearance %. SEM, EDX, AFM and
FTIR characterization was carried out to inspect the morphology of membranes.
Cytotoxicity test were carried out to find out the biocompatibility of fabricated
membrane. The FTIR spectra show that CA/PEG/HA blend membranes possess high
polarity and strong hydrogen bonding. The incorporationof HA in CA/PEG altered the
organization of void structures, the tear and sponge-like bodies converted into finger-like
regular channels. The pore radius also decreased as seen in SEM images in Figure5.2
(a,b). The blended membranes have developed hydrophilicity and protein resistance
confirmed by contact angle and protein rejection tests respectively. The BSA rejection
was increased from 43.7% in case of unmodified CA/PEG membranes to 96.8% for
CA/PEG/HA modified membranes. These modified membranes showed appreciable
uremic waste clearance relative to the unmodified CA membranes. The percentage urea
clearance was increased to 55.2% from already reported value of 52%. The
biocompatibility of CA/PEG/HA was found good enough as it showed much better cell
viability and cellular attachment than NTC. Categorically, the presented study has an
extensive impact on utilization in dialysis.
89
References
[1] H. Waheed, A. Hussain, Sara Farrukh, DESALIN WATER TREAT (2016).
[2] A. Hayder, A. Hussain, A.N. Khan, H. Waheed, Polym. Bull. (2017)
[3] H. Ehrlich, B. Krajewska, T. Hanke, R. Born, S. Heinemann, C. Knieb, H. Worch, J.
Membr. Sci. (2006) 124 -128.
[4]M. S.Albernaz, G. Weissmuller, A. L. Rossi, A. M. Rossi, R. S.Oliveira, jdit (2015)
0210 -012.
[5] H. Zhang, Y. Yana, Y. Wanga, S. Li, Mater. Res. Bull.6(2002) 111 – 115.
[6] Z. Chen, M. Deng, Y. Chen, G. He, M. Wu, J. Wang, J. Membr. Sci. 235 (2004) 73 -
86.
[7] Y. Baek, J. Kang, P. Theato, J. Yoon, Desalination 303 (2012) 23–28
[8] A.Idris, K. Y. Lee, H. K. HingJurnal Teknologi, 42(2005) 35–46.
[9] M. Sivakumar, D. R. Mohan, R. Rangarajan,J. Membr. Sci. 268 (2006) 208–219.
[10] Y. Iwasaki, H. Yamato, T. Nii-Kono, A. Fujieda, M. Uchida, A. Hosokawa, M.
Motojima, M. Fukagawa, J. Bone Miner.Metab.24(2006)172–175.
[11] R.C. Vanholder, R. V. De Smet, S. Ringoir, Clin.Chem.38(1992) 1429–1436.
[12] G. Eknoyan, G. J. Beck, A. K. Cheung, J. T. Daugirdas, T. Greene, J. W. Kusek, M.
Allon, J. Bailey, J. A. Delmez, T. A. Depner, N Engl. J. Med. 347(2002) 2010–2019.
90
Chapter 6: Result and Discussion of Dialysis via
Polymer-Organic Material Blended Membranes
Organic Additives
Cellulose Acetate being organic polymer was blended or modified using organic
additives, binders or porogens. To overcome the biocompatibility issue in CA, Fushimi et
al. [1] grafted polyethylene glycol (PEG) chains into the cellulosic membranes.
Diamantoglou et al. [2] also synthesized hemocompatibledialysis membrane using
cellulose by dropping cellulose linters with modified cellulose. They achieved low c5a
activation by blending cellulose linters with cellulose dodecenylsuccinate. In Japan, Ye et
al.[3] proposed that accurate surface modification of CA could make it more suitable for
applications in the field of medicine and biology. He coated the PMB80 (poly (2-
methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate) on CA hollow
fiber during phase inversion process. The CA/PMB80 coated membrane show less protein
adsorption as well as reasonable permeability. Advance research on phospho lipid
modified CA membrane is ongoing and Ye et al. have studied the potential of the
synthesized membrane in hemo purification operation [4]. Echanova et al.[5] found that
cellulose-based polymer shows interesting features that make it perfect for biomedical
purposes. Hoeniehet al.[6] suggested that appropriate alteration on cellobiosic unit
assisted in preparing biocompatible, cellulose based membranes that are as good as to
synthetic membranes.
6.1 Blend of Cellulose acetate-Sericin (organic additive)
6.1.1 Introduction
Sericin a natural silk protein is chosen as additive to blend in CA matrix. Sericin is
very hydrophilic macromolecule and is derived by cocoons of silkworm by a method
called degumming. Bombyx mori cocoon is well known to produce sericin protein, which
is composed of 18 amino acids. The sericin protein’s molecular weights range from 24 to
91
400 kDa with serine (40%), glycine (16%), glutamic acid, aspartic acid, threonine and
tyrosine as major amino acid groups. The sericin molecules possess polar side chains of
hydroxyl, carboxyl and amino groups which assist in easy cross-linking,
copolymerization as well as amalgamation with other polymers to produce value-added
biodegradable materials [7]. Sericin occurs primarily in an amorphous random coil and in
β-sheet structure. The randomly coiled structure is convertible to the β-sheet structure.
This is doneby repeated moisture absorption and mechanical stretching [8]. Moreover, it
binds fibroin fibers together. Nowadays, it is successfully applied in many applications
like medical biomaterials, degradable biomaterials, compound polymers, functional
biomembranes, hydrogels, and functional fibers and fabrics [9]. It is problematic to
fabricate pure sericin membrane, but few membranes from sericin in a cross-linked form,
blended with other polymers, or copolymerized with other substances are reported
previously.
The purpose of this research is to synthesize hemodialysis membrane which
possesses highest BSA rejection and urea clearance properties. For this purpose, sericin is
blended with CA. The modifications in the structure and performance efficiency of
CA/sericin blend membranes were analyzed accordingly. All pure and blended
membranes were made by applying the phase inversion technique. Several analytical
techniques, including Fourier Transform Infrared Spectroscopy(FTIR), Scanning Electron
Microscopy (SEM), and Atomic Force Microscopy(AFM), were utilized for
characterization of the pure and blend membranes. Influence of adding different weight
percentages of sericin in CA membrane has been examined in terms of BSA rejection and
urea clearance. To date, no report is published previously with this working idea,
therefore the presented study is novel and significant in biomedical applications.
The composition of cellulose acetate blended with sericin fibrinogen is tabulated below
92
Table 6.1: CA/sericin blended membrane composition
Membrane CA
Wt%
Sericine
Wt%
Acetic acid
Wt%
M-0 20 -- 80
M-2.5 20 2.5 77.5
M-5.0 20 5.0 75.0
M-7.5 20 7.5 72.5
M-10 20 10.0 70.0
6.1.2 Results and Discussion
6.1.2.1 SEM Analysis
The comparison of SEM images, surface and cross section of pure and CA/sericin blend
membranes are displayed in Fig. 6.1. The M-0is micro-porous membrane and cylindrical
pores can be viewed through its cross section. However, successive rise in sericin
concentration in M-2.5, M-5.0, and M-7.5membranes generates nano-porosity and
compact pores in cross section. The presence of nano pores and their uniform distribution
is bit lower in case of M-2.5. M-5.0shows higher uniformity and porosity and this trend
keep on increasing further in M-7.5and M-10. The possible explanation for this behavior
is the addition of hydrophilic additive that stimulates instantaneous demixing and ultimate
increases in pore formation. Herein, sericin acts as hydrophilic additive and improves
phase inversion mechanism by instantaneous demixing that creates nano-porosity in CA
matrix. Furthermore, presence of sericin also increases the degree of swelling of these
membranes[10-11]. Finally, M-7.5 membrane with uniform pore distribution and
appropriate pore dimensions shows better pure water flux, improved urea clearance,
higher BSA rejection and enhanced hydrophilicity.
93
Figure 6.1: The surface and cross-sectional SEM micrograph of pure and CA-sericin blend membranes
94
6.1.2.2 AFM Analysis
The comparison of AFM images of all pure and sericin blended membranes is displayed
in Fig 6.2. It is observed that M-0hassmooth surface relative to all blend membranes. This
is because of no additive which leads to homogenous mixing and finally smooth surface
generation. The incorporation of sericin at varying concentration increases the surface
roughness infabricated membranes. In case of M-2.5roughness is less due to low
concentration of sericin additive whereas M-5.0 andM-7.5show higher values of
roughness parallel to high additive concentration. MembraneM-10shows little bent
towards smooth surface because of compactness and dense nature in membrane structure
as a result of increment of high sericin concentration in casting solution. Some of the
sericin protein is leached out during the washing procedure and little amount is
incorporated within the polymeric membrane which induces membrane roughness. These
outcomes are also in agreement with previously described SEM results. Since sericin
generates nano-porosity in CA membrane and thereby enhances its roughness.
Figure 6.2: AFM micrograph of pure and CA-sericin blend membranes
95
6.1.2.3 FTIR Analysis
Fig 6.3 shows the spectra of pure and blends membranes. The FTIR of pure CA
membrane [12] is studied in comparison to membranes having altered wt% of sericin in
casting composition. The pure and CA-sericin blend membranes are symbolized with M-
0, M-2.5, M-5.0, M-7.5 and M-10, respectively in order of increasing serine wt.% in
each membrane.
In spectrum of M-0, the peak at 3417 cm-1
is due to stretching vibrations of hydroxyl
group (-OH) [13]. Visible broadening of this peak is because of additional amine group
(N-H) that also absorbs in range of 3300-3350 cm-1
. At 1790 cm-1
, the prominent peak
depicts the bending vibration of carbonyl group (C=O). Moreover, the peak at 1235 cm-1
illustrates bending vibration of an ether group (C-O-C). The obvious broadening of –OH
peak in the spectrum of each M-2.5, M-5.0, M-7.5 and M-10 membranes relative to M-0
clearly confirms the successful blending of sericin in CA.
Figure 6.3: FTIR spectrum of pure and CA-sericin blend membranes
6.1.2.4 Contact angle and water uptake
Contact angle of fabricated membranes were calculated using Sessile drop. Measurement
of water absorption capacity was also done to categorize the membrane as hydrophobic or
96
hydrophilic[14, 15]. Increased water uptake and small contact angle values designate low
hydrophobicity. Contact angle and water absorption or water uptake values of pure and
CA-sericin blend membranes are shown in Fig 6.4.Itwas found that addition of sericin
protein lowers the contact angle values from 76.3ᵒ to 52.0ᵒ and augmented water
absorption from 323%to 396%.Inthemembranes,M-2.5 to M-7.5, the reduction of contact
angle and raised water absorption were due to addition of sericin. Whereas on increasing
the sericin wt% beyond 7.5 as in M-10, the densities and compaction of prepared
membrane increases which results in increase of contact angle measurement and lowering
of water uptake. The contact angle measurement and water uptake of M-7.5show that it is
highly hydrophilic and can absorb maximum water.
M-0 M-2.5 M-5.0 M-7.5 M-1050
60
70
80
90
Conta
ct
angle
Membranes
M-0 M-2.5 M-5.0 M-7.5 M-10200
250
300
350
400
450
Wate
r A
bso
rp
tio
n (
%)
Membranes
Figure 6.4: Contact angle and water uptake of CA-sericin blended membranes
6.1.3 Permeation Testing
6.1.3.1 Pure Water Flux and MWCO
The PWF of all synthesized membrane was studied and it confirms that addition of sericin
in CA reduces pore size, which results in the lowering of flux from 67 L/h.m2 in case of
M-0membrane 49.9L/hr.m2. Fig 6.5 shows that M-2.5 was having flux value of
62L/hr.m2,M-5.0 have flux value 57L/hr.m
2 and M-7.5 were with 52L/hr.m
2. M-10
showed lowest flux values. The lowering of flux may also be attributed to the compaction
of membrane casting solution as density of doped solution is also increased due to high
97
weight% of sericin added. For dialysis, moderate water flux is required which will
prevent the excessive loss of water from the patient’ body to outside, so in that case,
membrane M-7.5 shows the average value of 52 L/hr.m2and was chosen as the
appropriate one .
In terms of MWCO, fabricated membranes were examined by using different molecular
weight PEG and BSA. Fig 6.5 below shows that pores radius in case of M-0 is large
which results in excessive loss of PEG and BSA molecules. On increasing sericin weight
% the pore radius tends to be lower which make these membranes good for PEG and BSA
molecules rejection. In other words, membranes M-2.5, M-5.0, M-7.5 and M-10 showed
better MWCO. M-7.5showed Solute rejection or MWCO values above 90% and is
selected as the best one among prepared membranes for dialysis application
10000 20000 30000 40000 50000 60000 700000
20
40
60
80
100
So
lute
Rej
ecti
on
(%
)
Molecular Weight cutoff (Dalton)
M-0
M-2.5
M-5.0
M-7.5
M-10
98
M-0 M-2.5 M-5.0 M-7.5 M-1048
52
56
60
64
68
PW
P (
L/h
.m2 )
Membranes
Figure 6.5: Molecular weight cutoff and pure water flux of CA-sericin blended membranes
6.1.3.2 Porosity
The addition of sericin not only enhances the protein rejection but also acts as porogen
and resulted in increased porosity. In this case, Fig 6.6shows that membrane porosity is
enhanced from 77% in case of M-0(pure CA) to 83% in M-7.5. Higher the amount of
sericin higher is the porosity of the membrane because more amount is leached out during
washing procedure. But the increment of sericin beyond 7.5 wt% resulted in highly
viscous solution and compact membranes formation which results in the formation of
dense membranes with low porosity as in case of M-10.
M-0 M-2.5 M-5.0 M-7.5 M-1077
78
79
80
81
82
83
84
Por
osit
y (%
)
Membranes
Figure 6.6: Porosity % of CA-sericin blended membranes
99
6.1.3.3 BSA rejection %
Dialysis patients undergo albumin loss illness if albumin (≈67 kDa) is lost during dialysis
operation [16]. Ideal dialysis procedure should avoid albumin loss, elimination or
decrease during operation. Fig 6.7showsthe%rejection of pure and CA/sericin blend
membranes. All membranes except pure CA membrane have more than 70% rejection of
BSA while membrane M-7.5 has 95.2% rejection, which was essential for all dialysis
membranes to avoid albumin loss [17]. The addition of sericin protein results in the
binding of protein from feed solution due to amino linkage among protein-protein
molecules and hydrogen bonding between protein molecules and other components
present within feed which results in rejection and stoppage of protein molecules from
feed. Conclusively, protein nature of sericin additive forms the basis of high protein
rejection of fabricated membranes.
M-0 M-2.5 M-5.0 M-7.5 M-10
20
40
60
80
100
BS
A R
ejec
tion (
%)
Membranes
Figure 6.7: BSA rejection % of CA-sericin blended membranes
6.1.3.4 Urea clearance %
Uremic toxics are the collection of compounds within patient’s body, which exist in urine
under normal conditions [18]. When the amount of uremic toxins is elevated beyond the
normal range, undesirable effects occur and this was termed as uremic syndrome. Urea is
the main component of toxic component present in urea and is generally used to mark
100
quality of hemodialysis membranes. For an operational hemodialysis membrane, urea
reduction must be at least 60%. Fig.6.8 explains the urea reduction of various membranes.
Membrane M-7.5 presents the uppermost value for urea reduction of 65.3% in contrast to
the pure CA membrane with 52.1% urea reduction, which is higher than parameters for a
good dialysis membrane as reported by Eknoyan [19].
M-0 M-2.5 M-5.0 M-7.5 M-1051
54
57
60
63
66
Ure
a R
edu
ctio
n (
%)
Membranes
Figure 6.8: Urea clearance % of CA-sericin blended membranes
6.1.4 Comparison Study
The concept of incorporation of sericin to CA resulted in the production of
membranes having better surface modification and performance. The presence of sericin
in CA membranes altered the alignment of pore structures. The tear and spongy bodies
transformed into finger-like regular channels and the pore radius decreased as shown by
SEM images. The blended membranes have better hydrophilicity and protein resistance as
confirmed by contact angle and protein rejection experiments respectively. The BSA
rejection increased from 23% in case of unmodified CA membranes to 96% for modified
membranes in case of M-7.5. Membranes M-0, M-2.5, M-5.0, M-7.5 and M-10 (modified
membranes) have shown appreciable uremic waste clearances relative to the unmodified
CA membranes. The percentage urea clearance increased to 60% in M4. Conclusively,
the presented study has a wide impact on utilization in dialysis.
101
6.2 Blend of Cellulose acetate-polyvinyl pyrolidone PVP (organic
additive)
6.2.1 Introduction
In previous research it was found that CA/sericin blended membranes presented better
results in comparison to pure CA and CA/PEG/Glycerol blended membranes. Although
hydrophilicity, molecular weight off , pure water flux are of required level but still there
is margin to improve the BSA rejection % and urea clearance % from 96% and 60% to
higher level respectively.
Keeping in mind the good water solubility, hydrophilicity and good blood compatibility
Polyvinyl pyrolidone or povidone was used as organic additive. It also possesses the
ability of generating good porosity when used as porogen.
In this work CA was blended with PVP 30,000 and its effect on membrane morphology
and permeation was tested. PVP was incorporated in CA membrane using diffusion
induced phase inversion method. Various membranes of CA polymer were prepared with
weight % of PVP varying as 1, 3, 5 and 7% respectively and were nominated as Mpvp0,
Mpvp1, Mpvp3, Mpvp5 and Mpvp7.
Table 6.2: Composition of CA/PVP blended membranes
MEMB CA WT % PVP WT % ACETIC ACID WT %
Mpvp0 18.5 --- 81.5
Mpvp1 18.5 1.00 80.5
Mpvp3 18.5 3.00 78.5
Mpvp5 18.5 5.00 76.5
Mpvp7 18.5 7.00 74.5
But when we increased the PVP concentration beyond 5 % the membranes formed were
having visible pore and the casting was not possible as it was for normal membranes. The
visible physical difference in membranes with concentration less than 5% and beyond 5%
is shown in Fig 6.9 below.
102
6.2.2 Results and Discussions
6.2.2.1 SEM Analysis
SEM investigations were carried out to study effect of adding PVP to CA matrix. Surface
and cross-sectional views of CA/PVP blended membranes are displayed in Fig 6.10.
Addition of PVP to CA matrix leads to the generation of asymmetric membranes having
dense upper layer and porous sub layer having finger like structures across the membrane.
From these images, it is seen that elevating PVP concentration at first from 0 to 3 wt.%
caused development of macro voids and much porous structure with good water flux in
case of Mpvp1 and Mpvp3. However, further increment of PVP concentration beyond 4
wt.% resulted in formation of large visible pores making membranes not functioning for
filtration, purification and dialysis operations as shown by Mpvp5. It can be said that the
existence of PVP with non-solvent characteristics resulted in immediate demixing in
coagulation bath and macro voids generation in membrane’s structure when added up to 3
wt.%[22]. Whereas due to low affinity between PVP and CA polymer, large pores were
formed when wt.% was increases beyond 4wt.% .
It is observed that adding a hydrophilic additive PVP, to the CA polymer solution
induces dual effect on the membrane morphology. Actually, the final membrane
construction is subjected to the dominance of instantaneous or delayed demixing which is
because of PVP addition in CA cast solution. Increasing PVP concentration, initially from
Figure 6.9: Difference in appearance of membranes
103
0 - 3 wt.% causes formation of macro voids in membranes. However, further increase of
PVP concentration in CA matrix results in formation of larger finger like projections and
highly permeable structures. Hence, it appears that little addition of PVP (up to 3 wt.%)
makes instantaneous demixing predominate, while further addition of PVP (up to 7
wt.%), and low affinity effect, makes formation of membranes with large and visible
pores.
Figure 6.10: Surface morphology (upper row) and cross-sectional view (lower row) of CA/PVP blended
membranes
6.2.2.2 FTIR Analysis
Addition of polyvinyl pyrolidone (PVP) to Cellulose Acetate matrix resulted in the
broadening and shifting of bands and peaks in FTIR spectrum Fig 6.11. FTIR spectrum of
CA/PVP blended membranes presented prominent carbonyl group ( ̶C=O) stretching
vibration of at ~ 1734 cm-1
and stretching vibration of ̶ CH group at ~ 2960 cm-1
, which
are defining peaks of CA [21]. Along with these few characteristic peaks of PVP were
also shown by Mpvp1, Mpvp3 and Mpvp3 such as stretching vibration of (C=O ̶ N)
amide carbonyl group at ~ 1656 cm-1
, bending vibration of ̶ CH2 ̶group at ~ 1498 cm-1
.
C ̶ N group also exhibited stretching vibration of tertiary at ~ 1295 cm-1
[22]. It is visible
from the spectrum that as wt. % concentration increases the peaks are becoming sharper.
Mpvp5 in the fig shows the same trend as Mpvp1 and Mpvp3 but the values were small
and not as prominent as in case of other two membranes. So, presence of PVP in CA
matrix is confirmed by the bands and peaks shown by Mpvp1, Mpvp3 and Mpvp5.
104
Figure 6.11: FTIR spectrum of CA/PVP blended membranes
6.2.2.3 Influence of PVP concentration on hydrophilicity and water uptake
Contact angle and water absorption or water uptake capacities arenormally used to
estimate relative hydrophilic or hydrophobic properties of the membrane. Small contact
angle and increased water absorption characterize extraordinary hydrophilicity of
membrane. Contact angle and water absorption for cellulose acetate and modified
membranes is displayed in Fig 6.12. It is seen membrane Mpvp0 is having higher contact
angle whereas membranes with increasing concentration i.eMpvp1,Mpvp3 Mpvp1Mpvp5
have trend of lowering contact angle values. The addition of PVP resulted in increased
hydrophilicity of the fabricated membranes. The hydrophilicity of membranes is parallel
to water uptake or degree of swelling of prepared membranes. Higher the hydrophilicity,
higher will be the water uptake of membrane. This inclination is prominent in graph
showing water uptake % of Mpvp0, Mpvp1,Mpvp3 andMpvp5.
105
Figure 6.12: Contact angle and water uptake of CA/PVP blended membranes
6.2.3 Permeation Testing
6.2.3.1 Effect of PVP concentration and evaporation time on pure water flux
Along with composition of casting solution, there are other parameters that affect the
morphology and performance of synthesized membranes. Time given for evaporation to
the wet membrane is among one of them. To study the effect of evaporation, casted
membranes were given different evaporation time like 0, 10, 20 and 30 sec. After
washing and 24 hour water treatment, the prepared membranes were tested for pure water
flux using dead end filtration arrangement and it was found that evaporation time has a
visible impact on CA/PVP blended membrane’s performance. Increasing the evaporation
time affects the inner structure and compaction of polymeric and additive’s particle
within the membrane structure. Greater the evaporation time, better is the pore formation.
Pores are distributed uniformly at the membrane surface. This is due to the fact that
longer evaporation time gave enough duration for removal of solvent and this result in the
formation of macrovoids and channels running from one side to the other. From Fig 6.13,
it is visible that the membranes formed with zero sec evaporation time gave lowest water
flux, whereas increasing evaporation time gradually increased the flux values of casted
membranes. Best membranes were casted when given thirty seconds evaporation time.
These membranes have optimum values of flux as shown.
106
Figure 6.13: Effect of PVP concentration and evaporation time on CA/PVP blended membranes
The above figure shows that when membranes were casted with 0 sec evaporation time
the membranes formed were compact with no or small pores radius. This resulted in less
water flow through membrane which leads to lower water flux. When evaporation time
was increased from 10 to 30 sec, the radius of pore was enlarged and water flux gradually
increased. All membranes casted with 30 sec evaporation time presented better water flux
values as compared to 10 and20 sec evaporation time.
6.2.3.2 BSA rejection% study of CA/PVP blended membranes
Renal failure patients would experience albumin (≈67 kDa) loss during the dialysis
treatment. For good dialysis operation, albumin loss should be avoided during operation.
Fig 6.14 represents the %BSA rejection of all pure CA and CA/PVP blend membranes.
All prepared membranes except pure CA membrane have BSA rejection more than 90%.
Membrane Mpvp1, Mpvp3 and Mpvp5 have % rejection of 96.7, 97.8 and 99.2
respectively which was relatively attractive for all dialysis membranes to avoid albumin
loss [23]. The presence of PVP makes all these membranes highly hydrophilic and
presence of pyrolidone moiety inhibit the absorption of protein on to the membrane
surface. This protein absorption inhibition rejects the protein back into the feed. This
results in the membranes with higher BSA rejection %. The membrane Mpvp1 with
96.7% rejection givesoptimum properties for example urea clearance, contact angle,
107
water uptake and water flux concerning dialysis therefore it was chosen as the best in this
group.
Figure 6.14: BSA rejection measurement of CA/PVP blended membranes.
6.2.3.3 Urea clearance study of CA/PVP blended membranes
Urea clearance study is one of the major studies made to investigate the efficiency of
hemodialysis membranes. Removal of waste including urea, excess water etc is essential
to maintain balance within patient blood. For a finest hemodialysis membrane, the urea
clearance should be at least 60% Fig 6.15 demonstrates the urea reduction of different
CA/PVP blended membranes. Membrane Mpvp1 shows urea reduction of 62% in contrast
to the pure CA membrane with 52.1%, which is higher than commercial parameters of
good dialysis membrane. Formation of finger like projection that runs across the
membrane due to the addition of PVP results in the increment in urea clearance. The PVP
molecule improves the macro-void formation and was added as porogen in CA matrix.
108
Figure 6.15: Urea clearance % of CA/PVP blended membranes
6.2.4 Conclusion
During this research, polyvinyl pyrolidone (PVP) with molecular weight 30,000 was used
as additive for the manufacture of CA/PVP flat sheet membranes using phase inversion
method. CA/PVP membranes showed compact structure with porous layer having macro
voids when viewed in cross section. SEM and AFM characterization confirmed the
uniform distribution of pores resulting in roughness at surface. FTIR studies revealed the
effective bonding between PVP and CA. Hydrophilic nature was also studied by under
taking contact angle, porosity and water uptake experiments. All these characterizations
showed that hydrophilicity increased with addition of PVP to CA matrix.
The performance testing showed that the efficiency of CA/PVP blended membranes was
enhanced in terms of PWP, BSA rejection and urea clearance. This study revealed that
PVP is an appropriate additive to enhance uremic waste clearance. Conclusively, the
CA/PVP blended membranes can be used as a potential solution for hemodialysis
treatment after some appropriate changes.
109
6.3 Blend of Cellulose acetate with organic additive Polyaziridine (PEI)
for Dialysis Application
6.3.1 Introduction
Previous research work resulted in the production of flat sheet membranes of cellulose
acetate by blending it with Poly-Ethylene Glycol, Glycerol, Sericin and Polyvinyl
Pyrolidone (organic additives). The results have shown that the increment of these
additives enhanced the porosity and hydrophilicity of membranes produced. The
permeation studies showed that the pure water flux was moderate but BSA rejection and
urea clearance are enhanced remarkably. In this approach Poly-Ethylene Imine (PEI) or
polyaziridine is added in polymer matrix to further improve the permeation efficiency of
fabricated membranes.
Polyethyleneimine (PEI) or Polyaziridine, Poly [imino (1,2-ethanediyl)] is a branched
chain polymer with number of amine groups and two carbon aliphatic spacer. It has been
broadly used to transform membrane surface [24]. PEI can be introduced in to membranes
and used as ligands by different methods of chemical modification [25]. In this work, a
mixing method was established to fabricate a modified membrane directly. It gathers the
properties of CA in membrane formation including high uniformity of the pore size
distribution and mechanical rigidity, and the characteristics of PEI like chemical
reactivity and capability of selectively adsorbing biological macromolecules (protein)
collectively.
In this study, CA membranes were modified by blending with PEI with altered
compositions. The effect of additive assimilation in the casting solution was studied by
undertaking membrane morphology, contact angle, pure water flux, water uptake,
porosity, molecular weight cut off, BSA rejection and Urea clearance. The composition of
CA/PEI blended membranes is given below.
110
Table 6.3: Composition of CA/PEI blended membranes
Membrane CA wt % PEI wt % Acetic acid wt% Water wt%
M0 15.5 --- 84.5 1
M0.5 15.5 0.50 83.0 1
M1.0 15.5 1.00 82.5 1
M1.5 15.5 1.50 82.0 1
M2.0 15.5 2.00 81.5 1
M3.0 15.5 3.00 80.5 1
6.3.2 Results and discussions
6.3.2.1 SEM Analysis
SEM surface and cross sectional micrograph of pure CA (M0) and PEI/CA MMM (M0.5,
M1.0, M1.5, M2.0, and M3.0) are shown in Fig 6.16 and Fig 6.17. M0, membrane with
no PEI is dense one with no pore generation as seen in cross sectional image. Addition of
PEI wt% in casting solution resulted in formation of asymmetric membranes with nano
sized pore in M0.5, M1.0, M1.5, M2.0 and M3.0. Addition of hydrophilic additive (PEI)
resulted in stimulation of instantaneous demixing, thus improving phase inversion
mechanism. The lost PEI played the role of porogen during washing process because of
its good solubility in water. PEI when added in small amount results in the formation of
pores with small radius. PEI also resulted in increment in pores density and reduction of
pore radius in prepared membranes [26].
111
Figure 6.16: Surface morphology of CA/PEI blended membrane using SEM
Figure 6.17: Cross sectional view of CA/PEI blended membrane using SEM
6.3.2.2 AFM Results
The AFM images were taken to study the topography of fabricated membranes. The
comparison of AFM micrographs of all pure CA (M0) and PEI/CA MMM (M0.5, M1.0,
112
M1.5, M2.0, M3.0) membranes is shown in Fig 6.18. M0 has smooth surface
comparative to all other membranes [27] as no additive was added to it. Addition of PEI
in variable wt% increases the roughness of membranes gradually. Addition of PEI beyond
1.5% increases the density of casting solution results in the production of compact
membranes having low roughness. The combination of CA and variable wt% of PEI and
its effect on the surface roughness in M0.5, M1.0, M1.5, M2.0 and M3.0is shown below.
These findings are in agreement with SEM images shown previously in Fig 6.16 and
6.17.
Figure 6.18: AFM micrographs of CA/PEI blended membrane using SEM
6.3.2.3 FTIR Analysis
Comparison of FTIR spectra of pure CA (M0) and PEI/CA MMM (M0.5, M1.0, M1.5,
M2.0 and M3.0) membranes is shown in Fig 6.19. From spectra, it can be seen that no
chemical changes occurred during blending.
The strong bands because of the –OH, –CH and C=O stretching modes were situated at
3766–3336, 2960 and 2860, and 1750 cm-1, respectively in the infrared spectra of pure
CA (M0), the C–O single bond stretching modes were situated at 1240 and 1040 cm-1
[28]. Compared with pure CA, the new absorption peaks appeared at 1580 and 1460 cm-
113
1, which corresponded to the N–H bending vibration in the PEI molecular structure in the
CA/PEI blends [29]. A strong and broader peak shifted to 3446 cm-1 was assigned to the
O–H stretching vibration as shown in Fig 6.20. This was due to the formation of
hydrogen bonds between the NH group in PEI and the OH group inCA. In addition, the
C–H stretching vibration peaks appeared at 2920 and2850 cm-1 at the C–H superposition
of CA and PEI methylene groups.
Figure 6.19: FTIR spectrum CA/PEI blended membrane.
Figure 6.20: Hydrogen bonds formation between the NH group of PEI and the OH group of CA
6.3.2.4 Contact Angle and water uptake measurement
Contact angles and degree of swelling values of pure CA and CA/PEI membranes are
illustrated in Fig 6.21. It is noticed that incorporation of PEI lowered the contact angle
114
values from 78◦ esa c nia of pure CA M0 to 69◦ in M3.0. The hydrophilic property of
PEIbecause of its –OH functional group introduces hydrophilicity to the membranes as
well. This increases the wetting characteristic of membraes and results in membranes
with lower contact angles.
The degree of swelling of membrane is directly related to its hydrophilic nature.
This means higher the hydrophilicity, higher will be the degree of swelling or water
uptake% of membrane, whereas contact angle and water uptake are inversly related to
each others. Fig 6.20 is also showing graphical presentation of CA/PEI mmbranes. It is
seen that degree of swelling increased from 440 in case of membrane with no PEI to 639
for membrane with highest wt % of PEI.
115
Figure 6.21: Contact angle and degree of swelling of CA/PEI blended membranes.
6.3.2.5 Porosity %
The addition of PEI not only improves porosity of MMM membranes but also have
significant impact on other characteristics like protein rejection and biocompatibility. Fig
6.22 shows porosity behavior of pure CA (M0) and PEI/CA MMM (M0.5, M1.0, M1.5,
M2.0 and M3.0) membranes. It can be seen that membrane’s porosity enhanced from
80.7 % for M0 to 91.3 % for M3.0. This increment is due to gradual increase in PEI wt %
in membrane casting solution. Hence, amount of PEI is in direct relation to the porosity
generated in the membrane. For dialysis operation, membrane with optimum porosity is
needed to manage the pure water flux and BSA rejection. Keeping this in view M1.0was
selected as the best one.
116
M0 M0.5 M1.0 M1.5 M2.0 M3.0
80
82
84
86
88
90
92
Poro
sity
(%
)
Synthesized Membranes
Figure 6.22: Graphical representation of porosity trend in CA/PEI blended membranes
6.3.3 Permeation Testing
6.3.3.1 MWCO and Pure Water Flux:
The molecular weight cutoff and pure water flux of all pure CA (M0) and PEI/CA MMM
(M0.5, M1.0, M1.5, M2.0, M3.0) membranes were studied to test the permeation ability
of membrane. It was done to calculate the solute size that can be retained by membrane
up to 90%. It is shown that the addition of PEI in the CA matrix reduces the pore size and
results in lowering of MWCO. The pure water flux rises from 67 L/h.m2 in case of M-0 to
81 L/hr.m2 for M1.0.Presence of abundant amine groups on PEI backbone leads to higher
affinity of CA membranes towards water. For rest of the membranes M0.5, M1.5, M2.0
and M3.0 results are shown in Fig 6.23.the decrease in the value of water flux after
increasing the PEI weight % beyond 1% is due to increased density of casting solution
which resulted in the formation of compact or somewhat dense membranes. Pore radius
was small which resulted in lowering of flux values and MWCO. For dialysis, reasonable
water flux is required which will avoid the excessive loss of water from the patient’s body
to outside, so in that case, membrane M1.0 shows the average value of
81L/hr.m2.Similarly the graphical representation of MWCO is also given in given in Fig.
6.22.
117
M0 M0.5 M1.0 M1.5 M2.0 M3.0
40
60
80
Pu
re W
ate
r F
lux
(L
it/h
r.m
2)
Synthesized membranes
10000 20000 30000 40000 50000 60000 700000
20
40
60
80
100
Solu
te R
ejec
tion (
%)
Molecular Weight Cutoff (Dalton)
M0
M0.5
M1.0
M1.5
M2.0
M3.0
Figure 6.23: Pure water flux and solute rejection % of CA/PEI blended membranes
6.3.3.2 BSA rejection%
Fig 6.24 represents the % BSA rejection of all pure CA (M0) and PEI/CA (M0.5, M1.0,
M1.5, M2.0, M3.0) membranes. All membranes except M0 have more than 90%
rejection of BSA while membrane M2.0 and M3.0 have 100.0% BSA rejection. This high
118
rejection value is because of excessive amine groups in PEI structure. These amine
groups form amine or peptide linkages with amine groups of protein. These linkages
results in binding the proteins on feed side and do not allow them to pass across the
membrane. Due to presence of PEI, all modified membranes were having BSA rejection
above 95%. The membrane M1.0 with 99.4% BSA rejection shows optimum properties
including pure water flux, hydrophilicity, water up take, MWCO etc which is good for
dialysis operation.
M0 M0.5 M1.0 M1.5 M2.0 M3.0 --0
20
40
60
80
100
BS
A R
eje
cti
on (
%)
Synthesized Membranes
Figure 6.24: BSA rejection presentation by CA/PEI blended membranes
6.3.3.3 Urea clearance %
Urea reduction of about 60% is required for a dialysis membrane to be used in practical
operation. Fig.6.25 illustrates the urea reduction of pure CA (M0) and PEI/CA
membranes. Addition of PEI increases the porosity and reduces the pore radius as stated
before so urea clearance was increased in case of M0.5, M1.0, M1.5, M2.0 and M3.0.
The highest urea reduction of 67.6% is shown by M1.0 in contrast to the M0 with 52.1%
urea reduction value. Decrease in urea clearance % is due to compaction of membrane
structure which was the result of increased PEI wt % in casting solution composition.
119
M0 M0.5 M1.0 M1.5 M2.0 M6
52
56
60
64
68
Ure
a C
leara
nce (
%)
Synthesized Membranes
Figure 6.25: Urea Clearance trend of CA/PEI blended membranes
.
6.3.4 Conclusion
In this work, PEI was used as filler for the synthesis of PEI/CA mixed matrix flat
sheet membranes through phase separation process. The PEI/CA membranes showed
compact structure with porous skin layer and macro-voids in cross section. SEM and
AFM micrographs of all membranes depicted homogenous spread of micropores that
results in rougher surface. The functional group identification of membranes was
compared with pure CA through FTIR which clearly showed effective binding in between
PEI and CA. In addition to this hydrophilicity of PEI/CA MMMs were examined relative
to pure CA membrane by water uptake, porosity and contact angle measurements. From
all these tests, it was found that the porosity and surface hydrophilicity of PEI/CA MMMs
were augmented with the addition of PEI. MWCO of membranes were carried as well to
characterize lowering of pore sizes. Finally, the performance efficiency of PEI/CA
membranes was evaluated in terms of PWF, BSA rejection and urea clearance. The
distinctive trends in these values substantially promote the proper adhesion of PEI as
filler into CA matrix. Conclusively, the PEI/CA membranes can serve as a promising
material for hemodialysis treatment.
120
6.4 Investigation of effect of solvent on cellulose acetate membranes
blended with Polyethylene imine
6.4.1 Introduction
In our previous work, CA has been used as a basic polymer and variable additives like
Polyethylene glycol (PEG), Glycerin, Sericin, Polyvinyl Pyrolidone (PVP) and Poly-
Ethylene imine (PEI) were blended to impart desired characteristic to the fabricated
membrane that include favorite pore size, biocompatibility and mechanical behavior [30].
This work is focused on testing the effect of different solvents on characteristics and
performance efficiency of already prepared CA based membranes doped with PEI.
Membranes prepared with this composition showed the highest BSA rejection and Urea
clearance%. They showed almost all desired properties that were needed in commercial
dialysis membrane including hydrophilicity, porosity, uniform pore distribution and
suitable molecular weight cutoff.
To study the solvent effect, we tested Acetic Acid (A.A), Formic Acid (F.A), N-Methyl-
2-pyrrolidone (NMP) and Dimethylacetamide (DMAC). The fabricated membrane’s
performance was tested on a dead-end filtration cell and laboratory-scale experimental
setup. Composition of all fabricated membranes using cellulose acetate and PEI in
different solvents is given in Table 6.4
Table 6.4: Composition of CA/PEI membranes prepared with different solvents
Membrane type
Solution Composition (wt. %) CBT
(°C) CA Solvent PEI D.Distl
H2O
CA+A.A+PEI 15.5 82.5(A.A) 1.0 1.0 25°
CA+F.A+PEI 15.5 82.5 (F.A) 1.0 1.0 25°
CA+NMP+PEI 15.5 82.5 (NMP) 1.0 1.0 25°
CA+DMAC+PEI 15.5 82.5(DMAC) 1.0 1.0 25°
121
6.4.2 Results and Discussions
6.4.2.1 SEM Analysis
Micrographs of all prepared membranes are presented in Fig 6.26. Upper row presents the
surface micrographs while lower row is presenting cross section of fabricated membranes.
All the images show formation of pores and macro- except membranes prepared with
CA as basic polymer, Formic Acid as solvent and PEI as additive. Membrane
CA+F.A+PEI showed the uniform distribution of pores all over the surface and the
average pore diameter was measured to be 70.3nm which is less as compared to
membranes prepared using NMP and DMAC as solvents. Whereas membranes prepared
using Acetic Acid was with lower pore diameter (49.4613nm) but it was having non-
uniform pore distribution because of formation of scaffolds.
(a)
122
(b)
Figure 6.26: Surface (a) and cross-sectional (b) image of fabricated membranes
6.4.2.2 Effect of solvent on morphology
From the SEM images given in Fig 6.26, it is obvious that solvent played a vital role in
defining the morphology, pore size and pore size uniformity in membrane. In case of
Acetic acid the pore forms are smaller and form efficient macrovoids and scaffolds in
membrane structure but uniformity of pore is not much visible. However, in case of
Formic Acid the pores generated are with appropriate size and their distribution on
membrane surface is also uniform that impart good and efficient characteristics to the
dialysis membrane formed [31- 32]. In case of DMAC and NMP, SEM image shows that
the blending was not homogeneous which resulted in the formation of irregular pores
with variable pore sizes. The cross-section also showed dense and porous patchy
membrane image.
123
6.4.2.3 AFM Results
AFM results shown in Fig 6.27They represent that membrane prepared using formic
acid and acetic acid as solvent shows less roughness. Membrane prepared with Formic
Acid is much smooth and is more suitable for hemodialysis application as with lower
surface roughness, the biocompatibility of fabricated membrane rises.
Figure 6.27: AFM micrographs of CA/PEI membrane fabricated with different solvents
6.4.2.4 Contact Angle
A sessile drop method was used to measure the contact angle. A drop of distilled water
was placed on the membrane surface (2 x 2 cm) and the contact meter was ranged and
focused on the membrane water interface. For each sample about 8 readings were taken to
calculate the average value of contact angle. Contact angle measured for all prepared
membrane as shown in Fig 6.28.
124
Figure 6.28: Contact angle measurement of CA/PEI membranes casted with different solvents
6.4.3 Permeation Testing
6.4.3.1 Pure Water Flux
Permeation experiments revealed that the membrane prepared using Formic Acid
gave optimum flux value (80Lit/hr.m2) that is suitable for dialysis operation as shown in
Fig.6.29. However, the flux value of Acetic Acid membrane was also closed to formic
acid i.e 70Lit/hr.m2. Water flux values of membrane casted using NMP and DMAC are
too high which make them inappropriate for dialysis application as they will result in loss
of water soluble useful contents of patient’s blood.
CA|+A.A+PEI CA+F.A+PEI CA+NMP+PEICA+DMAC+PEI
50
100
150
200
250
300
350
400
Flu
x (L
it/h
r.m
2 )
Membranes
Figure 6.29: Pure water flux measurement of membranes fabricated with variable solvents
125
6.4.3.2 BSA rejection %
Fig 6.30 presents the BSA % rejection of all fabricated membranes using variable
solvents. All prepared membranes have above 90% rejection of BSA while membrane
C.A+F.A+PEI has 99% rejection, which was essential for all dialysis membranes to
prevent albumin loss [33].
CA+A.A+PEI CA+F.A+PEI CA+NMP+PEI CA+DMAC+PEI
92
94
96
98
100
BS
A r
ejec
tion
(%
)
Membranes
Figure 6.30: BSA rejection % of CA/PEI membranes fabricated with different solvents
6.4.3.3 Urea clearance %
Fig 6.31 shows the urea reduction of different membranes prepared. Previously,
C.A+A.A+PEI is the membrane with 67.2% urea reduction. Membrane C.A+F.A+PEI
shows the highest urea reduction of 69.6% in contrast to all other membrane prepared
which is higher than parameters set for commercially used dialysis membrane as
described by Eknoyan [34].
126
Figure 6.31: Urea clearance % of CA/PEI membranes fabricated with different solvents
6.4.4 Conclusion
In this section, PEI was used as filler for the fabrication of PEI/CA mixed matrix flat
sheet membranes prepared through the diffusion induced phase separation process.
Various solvents are used to check their effect on membrane morphology and dialysis
performance. Acetic Acid, Formic Acid, 1-Methyl-2-pyrolidone (NMP) and N, N-
Dimethylacetamide (DMAC) were used. The results showed that using Formic Acid as
solvent forms compact structure with porous skin layer and macro-voids in cross section.
SEM and AFM images of C.A+ A.A+PEI and C.A+ F.A+PEI membranes depicted
homogenous spread of micropores that results in smooth surface.
From all the solvents used, formic acid gave the best results. The blending is
homogeneous and macro void formation is appropriate for dialysis application. The
replacement of acetic acid with formic acid (C.A+ F.A+PEI) showed hydrophilic nature
and increased the BSA rejection percentage from 95% to 100%. Urea clearance was
augmented to 69% as compared to 67%, 63% and 61% in case of C.A +A.A +PEI, C.A
+NMP +PEI and C.A +DMAC +PEI respectively. The results revealed that from all the
mentioned above solvents, formic acid is most suitable one for dialysis operation.
127
6.5 Comparison of all fabricated membranes
In order to select the best membrane, performance of all fabricated membranes was
compared graphically in terms of contact angle, pure water flux, BSA rejection % and
urea clearance. The membrane with best performance was chosen and was further tested
for its biocompatibility and blood mimic test.
6.5.1 Contact angle of fabricated membranes
Contact angle of all prepared membranes including CA-Hydroxyapatite, CA-Sericine,
CA-Polyvinyl Pyrolidone, CA blended with Poly-Ethylene-Imine in Acetic acid, CA
blended with Poly-Ethylene-Imine in Formic acid, CA blended with Poly-Ethylene-Imine
in N-Methyl-2-Pyrolidone and Dimethyl acetamide was compared in Fig 6.32. All
prepared membranes were hydrophilic as additives used were hydrophilic.
Figure 6.32: Combined Contact angle values of all prepared membranes
6.5.2 Pure water flux of fabricated membranes
Pure water flux is an important parameter defining the efficiency and performance of
dialysis membrane. An optimum flux value is required for good dialysis membrane. PWP
of all prepared membrane was grouped and compared in Fig 6.33. It is observed in the
graph that M-3 (CA-PVP) has minimum flux value and M-7 (CA+PEI+DMAC) has
maximum PWP. Rest all membranes have moderate water permeation. Membrane M-5,
128
prepared using CA,PEI and Formic Acid possess moderate hydrophilicity and pure water
permeation.
Figure 6.33: Combined Pure Water Flux of all prepared membranes
6.5.3 BSA rejection % of fabricated membranes
BSA rejection values of synthesized membranes were combined and plotted in Fig 6.34.
Nearly all membranes had BSA rejection values above 95% except M-7. BSA rejection
above 95% is the basic requirement for commercial dialysis membranes. Therefore, M-5
was selected to be the best among all fabricated membranes with highest rejection % i.e
99%.
129
Figure 6.34: Combine BSA rejection % of all prepared membranes
6.5.4 Urea clearance % of fabricated membranes
Urea clearance values of all the prepared membranes were measure using diffusion setup.
These values were combined and compared in Fig. 6.35. Literature shows that the
membrane is suitable if it has urea clearance of about 52%. Here it is found that all the
prepared membranes were having Urea clearance above 52%.Highest Urea clearance was
obtained by M-5. This was fabricated using Cellulose Acetate blended with PEI in the
presence of Formic Acid solvent. It showed the Urea clearance of 69%.
130
Figure 6.35: Combine Urea clearance % of all prepared membranes
Keeping in view the above comparison, it is concluded that M-5 is the best fabricated
membrane with optimum pure water flux and hydrophilicity. It has highest BSA rejection
of 99% and possesses maximum urea clearance of 69.6%. So, this membrane was tested
for its biocompatibility and later for blood mimics testing.
6.6 Biocompatibility Test
Biocompatibility test were carried out to study the biocompatible nature of the best
fabricated membrane i.e C.A+ F.A+PEI that was showing the highest BSA rejection of
99% and urea clearance was found to be 69.6%. This membrane was hydrophilic and was
having optimum water flux value of 80Lit/hr.m2. For this testing cytotoxicity assay was
done to find out whether the prepared membrane shows any toxicity and prevents
microbial growth or not.
In vitro toxicity of the C.A+ F.A+PEI was tested and compared to the non-treated control
(NTC) and commercial dialysis membrane. The samples were tested for cell viability
after variable intervals like 1 day, 2 days, 6 days, 9 days and 13 days.
131
6.6.1 Cell Viabilities
Cell viability or material toxicity of C.A+ F.A+PEI was tested and compared to that of
commercial dialysis tubing and NTC. The cell culture was found to be active and living
even after 13 days interval. The cell viability was tested on four different intervals and the
graphical representation is shown in figures below.
NTC Dialysis Tubing CA+PEI+F.A
100
110
120
130
140
150
160C
ell
Via
bil
itie
s
Materials tested after 1day
Figure 6.36: Cell viability on C.A+ F.A+PEI after 1 day
After1 day, all materials have shown higher cell viability than NTC. Highest numbers of
live cells were observed in CA based material. It is clear from data that C.A+ F.A+PEI
has much higher cell viability than commercial dialysis tubing available.
132
NTC Dialysis Tubing CA+PEI+F.A
96
100
104
108
112
Cel
l V
iab
ilit
ies
Materials tested after 2 days
Figure 6.37: Cell viability on C.A+ F.A+PEI after 2 days
On day two, all materials showed good cell viabilities. C.A+ F.A+PEI showed the highest
value which indicates that the microbial cells were replicating perfectly over the
membrane surface as shown in Fig 6.37.
NTC Dialysis Tubing CA+PEI+F.A
100
120
140
160
180
200
Cel
l V
iabil
itie
s
Materials tested after 6 days
Figure 6.38: Cell viability on C.A+ F.A+PEI after 6 days
Fig 6.38 shows that even after 6days, cells were alive and were replicating on C.A+
F.A+PEI membrane. The materials in the membrane compositions are nontoxic therefore
microbial cells are alive yet and the medium is appropriate for cell division or replication
that’s why the cell viability is increasing gradually.
133
NTC Dialysis Tubing CA+PEI+F.A
80
100
120
Cel
l V
iab
ilit
ies
Materials tested after 9 days
Figure 6.39: Cell viability on C.A+ F.A+PEI after 9 days
NTC Dialysis Tubing CA+PEI+F.A
100
120
140
160
Cel
l V
iab
ilit
ies
Materials tested after 13 days
Figure 6.40: Cell viability on C.A+F.A+PEI after 13 days
Fig 6.40, clearly indicate that the cells are alive after a long duration of 13 days. It is
hence evident that the C.A+ F.A+PEI membranes are ton toxic and do not kill living cells
when in contact. Hence, it is stated that this material is biocompatible.
134
6.6.2 Cellular attachment
Non treatment control was used as a standard to compare the cellular attachment of C.A+
F.A+PEI membranes. The photographic results are shown in Fig 6.41 indicating the cell
culture attachment on NTC.
Figure 6.41: Cellular attachment at NTC at four points
The fabricated membrane of C.A+ F.A+PEI was treated with the cell culture and the cell
attachment was observed at four different times. Photographic results are shown in Fig
6.42 below.
135
Figure 6.42: Cellular attachment at CA-PEI-FA at four points
Cell viabilities on all four time points (Day 2, day 6, day 9 and day 13) were comparable
with the control (NTC). There was no statistically noteworthy difference found between
136
the viabilities on any day as shown in Figures above. This material i.e C.A+ F.A+PEI is
therefore nontoxic.
Appearance of the cells on all four time points was compared to NTC. They stayed
adhered to the tissue culture support, presenting progression of cell junctions. The cells
appeared in the form of cellular networks. The pseudopodial extensions were also fairly
recognizable. No signs of cellular degradation were found. No observance of granularity
around the nucleus or detachment of the cells from the substrate was visible during test
duration. By the end of the experiment (day 13), a full mat of healthy cells was observed
in the treated cells which was comparable to the mat of untreated cells.
Cells were observed to be attached to the surface of the membrane. As the test proceeded
towards the last day, the cluster kept on growing as the cells were replicating. On day
thirteen, the membrane seems embedded in a mat of cells. It can be concluded that the
membrane C.A+ F.A+PEI is highly biocompatible as it has shown higher viabilities than
dialysis tubing and NTC as represented in Fig 6.43.
Day1 Day 2 Day6 Day 9 Day 1380
120
160
200
Cell
ula
r att
ach
men
t
Time intervals
NTC
CA+PEI+F.A
Figure 6.43: Graphical representation of cellular attachment at CA-PEI-FA at four points
137
All above biocompatibility test including cell viability and cellular attachment proved that
the membrane sample is the best suitable for dialysis and has the potential to be further
tested for blood mimic.
6.7 Blood Mimic Testing
Cellulose Acetate (polymer) doped with Poly-Ethylene Imine (organic additive) and in
Formic Acid (solvent) membrane has all essential characteristics required for dialysis
membrane like biocompatibility, nontoxicity, moderate pure water flux, hydrophilicity,
porosity, and molecular weight cutoff. Above all, for dialysis the most necessary property
is BSA rejection above 95% and urea clearance above 60% as illustrated in literature. The
above reported membrane (CA/PEI/Formic Acid) possesses all these characteristic.
This fabricated membrane was then finally incorporated into the diffusion set were
instead of urea as feed solution blood mimic fluid was used as feed solution. Test was
carried out to find the urea clearance and BSA clearance percentages.
6.7.1 Density of Blood Mimic fluid
Blood mimic fluid prepared using BSA, urea, glycol and water was tested for density so
that it can be compared to the actual density of blood. Density was calculated using
specific gravity bottle method. Table given below shows the density values of blood
mimic solution.
Table 6.5: Composition of blood mimic fluid
Wt of
sp.gravity
bottle (g)
Wt of sp gravity
bottle and BMF
(g)
Wt of BMF
(g)
Volume of
sp.gravity
bottle (ml)
Density of
BMF
(g/ml)
20.49 46.89 26.39 25.20 1.047
31.83 85.67 53.74 51.43 1.046
From the calculations it is shown that the density calculated is 1.0464g/ml which is
accurately in comparison with the density of blood given in literature (1.043 -1.060g/ml).
6.7.2 Permeation testing using Blood mimics fluid
138
The permeation test was carried out using the similar diffusion setup that was used
previously for each set of membrane. The only difference was that the feed side was
supplied with blood mimic fluid rather than feed solution. The setup is shown below in
figure 6.44.
Figure 6.44: Permeation setup
The permeate was tested by Uv-visible spectrophotometer for measuring the
concentration of BSA and urea. For BSA, λmax used is 278 nm and for urea concentration
UV-vis was operated at 190- 210 nm range. The standard curve for BSA and Urea are
shown below in Fig 6.45 and 6.46.
Figure 6.45: BSA Standard curve using UV-Vis spectrophotometer
139
Figure 6.46: Urea Standard curve using UV-Vis spectrophotometer
Furthermore, the concentrations of BSA and urea in permeate after diffusion testing is
shown graphically in Fig 6.47 and 6.48.
Figure 6.47: BSA rejection trend through C.A+ F.A+PEI membrane during blood mimic testing
140
Figure 6.48: Urea clearance trend through C.A+ F.A+PEI membrane during blood mimic testing.
When blood mimic fluid was used as feed solution against C.A+ F.A+PEI membrane and
BSA concentration was calculated using equation derived from BSA given below.
𝐁𝐒𝐀 𝐂𝐨𝐧𝐜𝐞𝐧𝐭𝐫𝐚𝐭𝐢𝐨𝐧 (𝐱) =𝐲 − 𝟎. 𝟎𝟓𝟑𝟗
𝟎. 𝟎𝟎𝟎𝟕
Where “y” is the absorbance obtained by UV-Vis spectrophotometer.
The BSA rejection % was calculated using equation given and was found to be above
98%.
𝑹𝒆𝒋𝒆𝒄𝒕𝒊𝒐𝒏(𝑹) = (𝟏 − 𝑪𝒑
𝑪𝒇) × 𝟏𝟎𝟎%
Here Cp and Cf are the solute concentrations in permeate and feed, separately.
The Urea concentration was calculated using equation
𝐔𝐫𝐞𝐚 𝐂𝐨𝐧𝐜𝐞𝐧𝐭𝐫𝐚𝐭𝐢𝐨𝐧 (𝐱) =𝐲 − 𝟐. 𝟖𝟑𝟑𝟏
𝟎. 𝟎𝟎𝟎𝟒
Later the Urea clearance percentage was calculated using equation given below and was
found to be 60%.
𝐔𝐫𝐞𝐚 𝐜𝐥𝐞𝐚𝐫𝐚𝐧𝐜𝐞 % = 𝐂𝐢 − 𝐂𝐟
𝐂𝐢× 𝟏𝟎𝟎
These results are notable and finally make the C.A+ F.A+PEI membrane best fabricated
for dialysis application in this research work.
141
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143
Conclusion
This work was aimed for the production of polymeric membranes for dialysis purpose
and to establish an experimental set up for membrane performance investigation.
a. Polymeric membranes were prepared using cellulose acetate polymeric blended
with polyethylene glycol (PEG). These membranes were not suitable for dialysis
application as they had non-uniform pore size and distribution. Along with these
limitations they also showed poor water flux BSA rejection and Urea clearance.
To overcome this matter the prepared membranes were modified using glycerol
additive. These membranes were further modified by blending the
CA/PEG/Glycerol composition with inorganic particle (additive) hydroxyl apatite.
This composition showed good hydrophilicity, porosity, biocompatibility and
water flux along with BSA rejection and urea permeation.
b. Cellulose Acetate was blended with sericine which along with enhancing other
properties improved the BSA rejection and urea clearance up to 96 and 60 %
respectively. To enhance the urea clearance, pure CA was then blended with poly
vinyl pyrolydone, which gave the visible increment of 62.4% in comparison to
that of CA/sericine membrane where it was 60%.
Furthermore, basic polymer (CA) was blended with poly ethylene imine (PEI)
which is an organic additive. Addition of PEI resulted in the fabrication of
membranes with highest hydrophilicity and required pure water flux. These
membranes were having highest BSA rejection of up to 100% and urea clearance
of 67.6%. These results lead to the modification of this membrane using various
solvents this composition. So, different solvents including Acetic Acid, Formic
Acid, DMAC and NMP were used. The characterizations and permeation testing
showed that membrane formed with formic acid as solvent exhibit the best
characteristic for dialysis operation including biocompatibility. They have BSA
rejection above 96% and urea clearance of up to 70 % besides their good water
flux, hydrophilicity, porosity and degree of swelling. The same membrane was
later tested with blood mimic solution as well.
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c. During this research an experimental set was established to find the efficiency of
fabricated membrane for dialysis operation. This set up is manual and can be fitted
any time to study the fabricated membrane’s performance.
It can be concluded from the work done that fabrication of membranes by blending
Cellulose Acetate with Poly Ethylene Imine in the presence of Formic acid leads to the
synthesis of thin, flat sheet membranes that are with most suitable hydrophilicity, water
uptake, porosity, molecular weight cut-off and surface morphology. This composition
provides a new vision for dialysis area. This C.A+PEI+F.A membrane is not only having
high BSA rejection, appropriate Urea clearance but also cost effective. Accordingly,
blood mimic tests and biocompatibility results proved these membranes to the finest
implant for dialysis operation.
Future recommendations
The organic and inorganic materials such as PEG, glycerol, sericin, PVP,PEI and
hydroxyl apatite (HA) were incorporated within cellulose acetate polymer can be further
modified for dialysis operation. All these materials can be blended with other polymers or
combination of polymers as well to be utilized for dialysis. Further most, permeation
studies can be carried out with modified polymeric membranes using complex blood
mimic fluid (having more blood components present in it) and pure blood as well.
