INVESTIGATION OF THE PHYSICAL PROPERTIES OF CHOLESTERYL

ESTER LIQUID CRYSTAL AND THE INTERACTION WITH CELLS

WAN IBTISAM BINTI WAN OMAR

A thesis submitted in

fulfilment of the requirement for the award of the

Degree of Master of Electrical Engineering

Faculty of Electrical and Electronic Engineering

Universiti Tun Hussein Onn Malaysia

JANUARY 2016

iii

For my dearest mother, father, husband and family for their encouragement and

blessing...

To my beloved friend and their support and caring......

-Never tired and give up to gain knowledge and life is a journey of learning-

iv

ACKNOWLEDGEMENT

Alhamdulillah, I am grateful to ALLAH S.W.T the most merciful and the most

compassionate for the guidance and knowledge bestowed upon me, for without it I

would not been able to come this far. Peace be upon him, Muhammad the messenger

of god.

I would like to express my gratitude to honourable Assoc. Prof. Dr Soon Chin

Fhong, my supervisor of Master’s project. Under her supervision, many aspects

regarding this project has been explored and with the knowledge, idea and support

received from her. I also would like to thank my co-supervisor Dr Hatijah Basri for

sharing her knowledge to me.

Many thanks and grateful to the Malaysia Ministry of Higher Education for

research funding support (FRGS Phase 1 Vot No. 1050) and also Universiti Tun

Hussein Onn Malaysia for providing post graduate incentive research grant (GIPS

Vot 1111).

Finally, I would like to dedicate my gratitude to my parents, my family, my

husband and friends who helped me directly and indirectly in the completion of this

project. This encouragement and guidance mean a lot to me. Their sharing and

experience foster my belief in overcoming every obstacle encountered in this project.

Guidance, co-operation and encouragement from all people above are

appreciated by me in sincerely. Although I cannot repay the kindness from them, I

would like to wish them to be well and happy always.

v

LIST OF ASSOCIATED PUBLICATIONS

Journals

1. Chin Fhong Soon, Wan Ibtisam Wan Omar, Rebecca F. Berends,

Nafarizal Nayan, Hatijah Basri, Kian Sek Tee, Mansour Youseffi,

Nick Blagden, Morgan Clive Thomas Denyer (2014) “Biophysical

characteristics of cells cultured on cholesteryl ester liquid crystal,”

Micron, Vol. 56, pp. 73-79. Published by Elsevier B.V.

Conference Proceeding:

1. Chin Fhong Soon, Wan Ibtisam Wan Omar, Nafarizal Nayan, Hatijah

Basri, Martha Bt. Narawi, Kian Sek Tee (2013) “A Bespoke Contact

Angle Measurement Software and Experimental Setup for

Determination of Surface Tension.” Proceeding of 4th

International

Conference on Electrical Engineering and Informatics (ICEEI),

Procedia Technology. Vol. 11, pp. 487-494. Published by Elsevier

B.V.

2.

Wan Ibtisam Wan Omar, Chin Fhong Soon (2014) “Critical Surface

Tension of Cholesteryl Ester Liqiud Crystal.” Proceeding of Joint

International Conference on Nanoscience, Engineering and

Management (BOND 21), Bayview Beach Resort, Batu Ferringhi,

Penang, Malaysia, 19 – 21 August 2013, Advance Materials Research.

Vol. 925, No. 4, pp. 43-47. Trans Tech Publications, Switzerland.

3. Wan Ibtisam Wan Omar, Chin Fhong Soon (2015) “The Time Based

Study of Cell Morphology Using Atomic Force Microscopy.”

Proceeding of 5th

International Conference on Biomedical Engineering

(IFMBE), Vietnam, 16-18 June 2014, Vol. 46, pp. 227-230. ©

Springer International Publishing Switzerland.

vi

LIST OF AWARDS

1. Silver Medal in Research & Innovation Festival 2012

(R & I Fest UTHM)

Chin Fhong Soon, Hatijah Basri, Kian Sek Tee, Wan Ibtisam

Wan Omar, Morgan Denyer. “Simple Apparatus and a Contact

Angle Measurement Software for Automatic Computation of

Surface Tension.”

2. Gold Medal in Persidangan dan EXPO Ciptaan Institusi

Pengajian Tinggi Antarabangsa 2013 (PECIPTA)

Chin Fhong Soon, Wan Ibtisam Wan Omar, Hatijah Basri,

Morgen Denyer. “An Integrated System For Measurement and

Mapping of Single Cell Traction Force.”

3. Silver Medal in Research & Innovation Festival 2013

(R & I Fest UTHM)

Thong Kok Tung, Wan Ibtisam Wan Omar, Rosliza Mohamad

Zin, Yap Huing Yin. “Scaffoldless Technique to Culture 3D

Keratinospheroids.”

vii

ABSTRACT

Cholesteryl ester liquid crystals (CELC) were demonstrated with application in

biosensing and microtissue regeneration. The affinity of the cells to this liquid crystal

is unclear and required further investigation. This study focused on characterising the

physical properties of CELC and interaction of human keratinocytes with CELC. The

physical properties of CELC were characterised by a custom built contact angle

measurement system and bubble pressure measurement apparatus. Other methods

such as pendant drop were applied to determine the critical surface tension of the

CELC. Then, the characterization of the CELC was continued by using Differential

Scanning Calorimeter (DSC), X-ray Diffraction (XRD), Polarising Microscopy

(POM) and Fourier Transform Infrared Spectroscopy (FTIR). Nonetheless, the

morphology of cells interaction with CELC after it reached confluency was studied

using Field Emission Scanning Electron Microscopy (FESEM) and non-contact

mode of Atomic Force Microscopy (AFM). The results showed that the critical

surface tension of the liquid crystal using contact angle was 37.5 mN/m and the

surface tension measured using pendant drop method was found to be 23.6 mN/m.

Both results indicate that the surface of the liquid crystal was moderately

hydrophobic. From the DSC, CELC was found stable at room and incubator

temperature. From XRD results, the compound of CELC interacts in cell culture

media self-assembles into lyotropic layer. POM and FTIR analysis showed CELC

after immersion in media displayed lyotropic smectic phases. The AFM and FESEM

images indicated good adhesion of cells on the CELC. This research thus showed

that the hydrophilic layers of lyotropic phase of cholesteryl ester liquid crystal were

demonstrated with biophysical properties that support the adhesion of cells.

viii

ABSTRAK

Kristal cecair kolesterlat ester (CELC) telah menunjukkan aplikasi dalam biosensor

dan pertumbuhan semula tisu mikro. Pertalian sel-sel terhadap kristal cecair ini

adalah tidak jelas dan memerlukan kajian lanjut. Kajian ini memberi tumpuan kepada

pencirian sifat-sifat fizikal CELC dan interaksi CELC dengan keratinosit manusia.

Sifat fizikal CELC dicirikan dengan menggunakan sistem pengukuran dan kaedah

pengukuran tekanan gelembung sudut yang telah direka . Kaedah-kaedah lain seperti

‘pendant drop’ telah digunakan untuk menentukan ketegangan permukaan kritikal

CELC itu. Pencirian CELC diteruskan dengan menggunakan mesin Kalorimeter

imbasan perbezaan (DSC), difraksi sinar-X (XRD), mikroskop optis berkutub (POM)

dan perubahan fourier infra-merah (FTIR). Morfologi sel selepas interaksi dengan

CELC dikaji menggunakan mikroskop elektron pengimbas pancaran medan

(FESEM) dan mod tanpa sentuh mikroskop daya atomik (AFM). Keputusan

menunjukkan ketegangan permukaan kritikal kristal cecair menggunakan sudut

sentuh adalah 37.5 mN/m dan ketegangan permukaan dengan menggunakan kaedah

‘pendant drop’ ialah 23.6 mN/m. Kedua-dua keputusan menunjukkan bahawa

permukaan kristal cecair adalah sederhana hidrofobik. Daripada keputusan DSC,

CELC didapati stabil pada suhu bilik dan inkubator. Keputusan XRD menunjukkan,

apabila CELC berinteraksi dalam sel media, ia bertukar sendiri ke lapisan lyotropic.

Analisis POM dan FTIR menunjukkan CELC selepas rendaman dalam media,

memaparkan fasa smectic ‘lyotropic’. Gambar-gambar dari AFM dan FESEM

menunjukkan sel melekat dengan baik pada CELC tersebut. Kajian ini sekali gus

telah membuktikan bahawa lapisan hidrofilik fasa lyotropic cecair kolesterlat ester

kristal mempunyai ciri-ciri biofizikal yang menyokong lekatan sel.

ix

CONTENTS

TITLE i

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENT iv

LIST OF ASSOCIATED PUBLICATION v

LIST OF AWARD vi

ABSTRACT vii

ABSTRAK viii

TABLE OF CONTENTS ix

LIST OF TABLES xiv

LIST OF FIGURES xv

LIST OF SYMBOLS xx

LIST OF ABBREVIATIONS xxii

CHAPTER 1 PROJECT OVERVIEW

1.1 Project background 1

1.2 Problem statement 2

1.3 Objective 3

1.4 Scope of project 3

1.5 Thesis organisation 4

1.6 Thesis contribution 4

x

CHAPTER 2 LITERATURE REVIEW

2.1 Introduction to liquid crystal 6

2.2 Types of liquid crystal 7

2.2.1 Thermotropic liquid crystal 7

2.2.1.1 Nematic phases 8

2.2.1.2 Cholesteric phases 8

2.2.1.3 Smectic phases 10

2.2.2 Lyotroic liquid crystal 10

2.3 Application of liquid crystal in biosensing 12

2.4 Human keratinocytes (HaCaTs) 14

2.5 Cellular adhesion 15

2.6 Surface tension measuring technique 16

2.6.1 Contact angle measurement 17

2.6.2 Bubble pressure 19

2.6.3 Pendant drop 20

2.6.4 Surface tension of liquid crystal 22

2.7 Spectrophotometer 23

2.8 Differential scanning calorimeter (DSC) 23

2.9 X-ray diffractometer (XRD) 24

2.10 Fourier transform infrared spectroscopy

(FTIR) 26

2.11 Microscopy 27

2.11.1 Phase contrast microscope 28

2.11.2 Atomic force microscopy (AFM) 29

2.11.3 Field emission scanning electron

microscope 31

CHAPTER 3 METHODOLOGY

3.1 Introduction 34

3.2 Development of a contact angle measurement

system 36

3.2.1 Experiment for validating contact

angle measurement system 44

xi

3.3 Characterising the physical properties of

cholesteryl ester liquid crystal (CELC) 45

3.3.1 Preparation of cholesteryl ester liquid

crystal (CELC) 45

3.3.2 Measuring surface tension of CELC 46

3.3.2.1 Contact angle method 46

3.3.2.2 Bubble pressure method 47

3.3.2.3 Pendant drop method 49

3.3.3 Thermal stability analysis 50

3.3.3.1 Preparation of CELC for

differential scanning

calorimeter (DSC)

50

3.3.3.2 Preparation of CELC for

optical emission spectroscopy

(OES)

51

3.3.4 Preparation of CELC for polarising

microscopy 52

3.3.5 Liquid crystal sample preparation for

x-ray diffraction (XRD) 53

3.3.6 Liquid crystal sample preparation and

fourier transform infrared analysis 54

3.4 Studying the cells interaction with CELC 55

3.4.1 Cell culture 55

3.4.2 Cell culture on the liquid crystal and

glass substrate 56

3.4.3 AFM of cells on the liquid crystal and

glass substrate 57

3.4.4 FESEM of cells on the liquid crystal

and glass substrate 58

CHAPTER 4 RESULT AND DISCUSSION

4.1 Introduction 60

4.2 The contact angle measurement system 60

xii

4.2.1 Calibration results of contact angle

measurement system 63

4.3 Physical properties of cholesteryl ester liquid

crystal (CELC) 66

4.3.1 Cholesteryl ester liquid crystal

(CELC) 66

4.3.2 Surface of cholesteryl ester liquid

crystal 66

4.3.2.1 Contact angle 66

4.3.2.2 Bubble pressure 68

4.3.2.3 Pendant drop 70

4.3.3 Thermal stability of cholesteryl ester

liquid crystal 72

4.3.4 Polarising optical microscopy (POM) 74

4.3.5 Crystallinity study of cholesteryl ester

liquid crystal 76

4.3.6 Chemistry elements in cholesteryl

ester liquid crystal 77

4.4 Cells interaction with cholesteryl ester liquid

crystal 79

4.4.1 Morphological effect and adhesion

characteristic of cells to celc 79

CHAPTER5 CONCLUSION AND FUTURE WORKS

5.1 Conclusion 89

5.2 Future works 90

REFERENCES 92

APPENDIX A Architecture of the Arduino UNO microcontroller board

APPENDIX B Specification of L293D motor driver

xiii

APPENDIX C Arduino coding

APPENDIX D Matlab coding

APPENDIX E The data of contact angle method

APPENDIX F The data of bubble pressure method

xiv

LIST OF TABLES

TABLE NO TITLE PAGE

2.1 Application of the liquid crystal as biosensor 13

2.2 Probe liquids and their interfacial tension 18

2.3 Surface tension values for liquid crystal 22

4.1 Interfacial tension value for CELC 71

4.2 Percentage of transmission calculated from

Beer’s law

73

4.3 Differences of AFM and FE-SEM for the

application in cells imaging

88

xv

LIST OF FIGURES

FIGURE NO TITLE PAGE

2.1 Basic structure of a liquid crystal molecule 7

2.2 A graphical illustration of cholesteric

phase

8

2.3 Types of cholesteric derivatives 9

2.4 Standard thermotropic liquid crystal

phases

10

2.5 Typical phases of lyotropic liquid crystal. 11

2.6 Layer of skin at epidermis consists of

keratinocytes

14

2.7 A schematic diagram of cellular adhesion 15

2.8 Contact angle measurement of a droplet of

liquid

17

2.9 Apparatus for surface tension

measurement using bubble pressure

19

2.10 A pendant drop system from AST

Products, INC

20

2.11 The pendant drop geometry 22

2.12 Light transmitted or reflected through a

sample

23

2.13 Schematic DSC heating curve of a semi-

crystalline polymer

24

2.14 PANalytical X’Pert Powder X-Ray

Diffraction

25

2.15 Setup for a scattering experiment utilizing

X-ray diffraction

25

xvi

2.16 Fourier transform infrared spectroscopy

(Perkin Elmer Spectrum 100)

26

2.17 Sample in contact with evanescent wave 27

2.18 Light path of a phase contrast microscope 28

2.19 A Nikon Eclipse TS100 phase contrast

microscope

29

2.20 Schematic diagram of an AFM 30

2.21 Atomic force microscopy XE-100 Park

System

30

2.22 FE-SEM schematic diagram 32

2.23 Field Emission Scanning Electron

Microscopy (JEOL JSM-7600F series)

32

3.1 The project flow chart 35

3.2 Flow chart of surface tension methods 36

3.3 Setup for contact angle measurement

technique

37

3.4 Circuit diagram of contact angle motor

controller

38

3.5 Flow chart to measure the contact angle of

a droplet of fluid

39

3.6 Open pushbutton in MATLAB GUI 39

3.7 Command for OPEN pushbutton 40

3.8 Distilled water interacts with PDMS 40

3.9 Command for SNAP pushbutton 41

3.10 Command for SAVE pushbutton 41

3.11 Image processing command including

Browse function

41

3.12 An enlarged image of the selected point of

a Acetone on PDMS surface

42

3.13 Tools in MATLAB GUI 43

3.14 Command for search critical surface

tension

43

3.15 Push button in GUI for Fox Zisman graph 44

xvii

3.16 Cholesteryl ester liquid crystal formulation 45

3.17 Mixture of cholesteryl ester liquid crystal 46

3.18 CELC in cholesteric phase at room

temperature

47

3.19 Differential pressure sensor ASP1400

(SENSIRION)

47

3.20 A schematic diagram of ASP 1400

pressure sensor with RS 232

48

3.21 Experimental setup for measuring surface

tension using bubble pressure method

49

3.22 Sample drop at the end of capillary tip 49

3.23 CELC sample in DSC pan 50

3.24 Q20 Differential scanning calorimeter 51

3.25 Experimental setup using OES 52

3.26 Ocean Optics HR4000 spectrophotometer 52

3.27 CELC substrate immersed in cell culture

media

53

3.28 Sample placed on the sample holder for

analysis

53

3.29 CELC sample were placed on top of ATR

crystal

54

3.30 Medium for cell harvesting 55

3.31 HaCaT cells were sub-cultured and were

plated in each petri dish

57

3.32 HaCaT were sub-culture without and with

liquid crystal

57

3.33 Image of silicon cantilever from the AFM

system over the HaCaT cell sample

58

3.34 Glass coverslips with gold sputter coater 58

4.1 The prototype models of a designed

contact angle measurement system

61

4.2 A DC motor controller circuit connected

to Arduino UNO

61

xviii

4.3 Enlaraged image for angles value at the

intersection point a droplet of Acetone

with solid surface.

62

4.4 Enlarge image of contact angles using

angle tool shows intersection point and

contact angle

62

4.5 Eight probe liquids droplets on PDMS and

Polyimide surface

63

4.6 Fluid surface tension of the eight probe

liquid generated in excel for PDMS and

Polyimide

64

4.7 CELC in cholesteric form 66

4.8 Image of sessile drops in contact with

CELC coated glass slides and their contact

angles

67

4.9 A Fox-Zisman plot for cholesteryl ester

liquid crystal

67

4.10 Hardware consists of ASP1400 pressure

sensor

68

4.11 CELC in 0.4 second, 0.8 second and 1

second before the bubble burst

69

4.12 Enlarged image of hemisphere 69

4.13 Samples reading of surface tension using

bubble pressure

69

4.14 Image of pendant drop digitized drop and

edge detection

71

4.15 DSC shows heat flow as a function of

temperature of cholesteryl liquid crystals.

No exothermic or endothermic activities

were observed at 37 °C

72

4.16 Intensity versus wavelength for CELC 73

4.17 Percentage of transmission versus

temperature

74

xix

4.18 Image comparison using phase contrast and

cross-polarizing optical microscopy.

75

4.19 A cross-polarizing micrograph of

cholesteric based lyotropic liquid crystals

shows the wide band streaks and focal

conic textures.

75

4.20 The surface of CELC after immersion in

cell culture media for 24 hours, 48 hours

and 72 hours

76

4.21 XRD for CELC and lyotropic layer of

CELC

77

4.22 The FTIR spectrum of cholesteryl ester

liquid crystals

78

4.23 HaCaT cells morphology on glass

substrate and for 24, 48 and 72 hours

79

4.24 AFM images for keratinocytes cultured on

a plain glass and liquid crystal substrates

80

4.25 Image of HaCaT cell plated on the glass

substrate and liquid crystal substrate after

24, 48 and 72 hours

82

4.26 Thickness of HaCaTs membrane cultured

with and without the presence of liquid

crystal as the substrate

83

4.27 Line profiles of cells cultured on plain

glass and liquid crystal substrate

84

4.28 Human keratinocyte cell morphology

cultured on glass on FE-SEM

85

4.29 Human keratinocyte cell morphology

cultured on cholesteryl ester liquid crystal

on FE-SEM

86

xx

LIST OF SYMBOLS

SYMBOL DESCRIPTION

a - Radius of curvature at apex drop

Å - Angstrom

Β - Drop shape factor

C - Carbon

cm - Centimetre

cm-1 - Wavenumber

°C - Degree celcius

d - Interplanar spacing

γ or σ - Surface tension

△ρ - Difference between densities

Φ - Coordinate

θ - angle

F - Free energy

H - Hidrogen

I - Intensity

l - litre

g - Gravity

N - Nitrogen

% - Percentage

O - Oxygen

P or p - Pressure

R - Radius

T - Transmission

Tg - Glass Transition

Tm - Melting points

λ - Wavelength

m - metre

xxi

mA - Mili Amp

ml - Mili litre

mg - Mili gram

mN/m - Mili Newton per metre

mM - Micro molar

kW - Kilo Watt

kV - Kilo Volt

µ - micro

xxii

LIST OF ABBREVIATIONS

SYMBOL DESCRIPTION

5CB - Pentylcyanobiphenyl

8CB - 4′-n-octyl-4-cyano-biphenyl

ATR - Attenuated Total Reflectance

AFM - Atomic Force Microscopy

CELC - Cholesteryl Ester Liquid Crystal

CST - Critical Surface Tension

DMEM - Dulbecco’s Modified Eagles’s Medium

DMSO - Dimethyl Sulfoxide

DNA - Deoxyribonucleic Acid

DSC - Differential Scanning Calorimeter

DSCG - Disudium Cromoglycate

ECM - Extracellular Matrix

EG - Ethylene Glycol

FESEM - Field Emission Scanning Electron Microscope

Fmoc - Fluorenylmethyloxycarbonyl

FTIR - Fourier Transform Infrared Spectroscopy

GND - Ground

GUI - Graphic User Interface

HaCaTs - Human Keratinocytes

HBSS - Hanks Balance Salt Solution

IPA - Isopropyl alcohol

IR - Infrared

LC - Liquid Crystal

LCD - Liquid Crystal Display

LCPA - Liquid Crystal Pixel Array

LED - Light Emitting Diode

LLC - Lyotropic Liquid Crystal

MBBA - 4-methoxybenzylidene-4’-n-butylaniline

xxiii

MMA - Methylmethacrylate

MTT - 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium

Bromide

OES - Optical Emission Spectroscopy

PEGA - Thermolysinpolyethylene Glycol Acrylamide

POM - Polarizing Optical Microscopy

PSPD - Position Sensitive Photo Diode

PWM - Pulse Width Modulation

SD - Standard deviation

TV - Television

UV - Ultraviolet

UTHM - Universiti Tun Hussein Onn Malaysia

V - Volt

Vis - Visible

XRD - X-ray Diffraction

CHAPTER 1

INTRODUCTION

1.1 Project background

Liquid crystal (LC) is a new class of biomaterial that gained attention for usage in

biomedical engineering. They provide means for label free observation of biological

phenomenon such as non-toxic thermotropic liquid crystal overlaid with matrigel that

had been used in recognizing restructuring of mammalian cells [1, 2]. Recently, a

research found that [3], cholesteryl ester liquid crystals (CELC) containing

cholesteryl moieties may have suitable biological affinity and also enable cell

adhesion and proliferation without the need for pre-treatment with extracellular

matrix molecules.

Due to the compact molecular arrangement, liquid crystal was shown to have

high resolution on sensing ability in detecting localized cell exerted force and could

be used for cell traction force sensor [4]. This is an important property that may

reveal the potential of liquid crystal in a biomaterial to function on inert surface.

Cell-compatible materials are very important in many biomedical applications [5-7].

Moreover, the cholesterol derivative in the liquid crystal was a well known

mesogenic nature with their potential to self order into liquid crystalline substances.

However, the use of liquid crystal in biological study has not been fully

explained. In 1857, Mettenheimer used a polarising light microscope and observed

double refractility and black formed cross image composed of cholesterol from

blood. Researchers proved that, occurrence of anisotropic myelin-like bodies in fatty

streaks of the atheromatous lesions of the aorta of man [8]. The anisotropic basic

structure has been interpreted as that of liquid crystal. In our living system, liquid

crystalline order of molecule occurs in degenerating tissues of liver, spleen, lung and

thyroid in the form of small spheres which appear under a conventional light

2

microscope as highly retractile and with double contours at their edges having the

inner ring brighter than the outer ring [8].

This type of liquid crystal contains cholesteryl moieties which have affinity

for cell membranes and have the abilities to change their properties. Cholesteryl

moieties homeostasis is an important element in cellular membrane traffic and cell

survival [9]. Therefore, this type of biomaterial is attractive as substrates because it

could provide universally fundamental molecule to mammalian cells. Cholesteryl

ester are considered to be easily biodegradable thus making the system suitable for

the preparation of tissue engineering that sustain cell proliferation and migration

[10]. The advantage of using liquid crystals as the biomaterial is it being non toxic

and sensitive to biological interactions [11].

In addition, in order to study the biocompatibility of a biomaterial, surface

energy property and thermal stability of a biomaterial are important factors in

attracting the adhesion of cells and may affects cell surface interface [12]. Hence,

finding surface tension of CELC was an important factor in this study. Moreover,

biomaterials are majorly reliant on the surface energy [13] and stable over certain

temperature. However, from the perspective of biophysical compability, little

publication has been studied on the physical properties of liquid crystal. Through this

study, synthesis and characterisation of surface properties CELC to interact with

human keratinocytes (HaCaT) cells were reported. Furthermore, surface properties of

CELC materials that promoted cell affinity were presented in this thesis.

1.2 Problem statement

In liquid crystal based biosensor development, an exposure to cell culture media is

unavoidable and this may alter the surface properties of the liquid crystals over

time. After immersion in cell culture media, the hydrophilic head and hydrophilic

tails of the liquid crystal molecules would reoriented to interface with the water at

the surface of the bulk liquid crystal as reported in [4]. However, the wettability of

the surface or the surface tension of these liquid crystals in attracting the adhesion

of cells have not been clearly identified and understood. Furthermore, the chemical

properties of the CELC used in cell culture remain unknown. Although new

applications of liquid crystals have been revealed [4], little is known about the

3

expression of surface proteins and effects onto morphological changes due to the

interactions with the liquid crystals. Driven by the positive reports of CELC in

supporting cell adhesion, it is necessary to further investigate the physical

properties of CELC and characteristics of cells after interaction with the CELC.

Thus, this study reveals the application of CELC that could be extended as a new

class of bio-physical relevant cells adhesion substrate which does not require pre-

conditioning with ligands.

1.3 Objectives

The main purpose of this research work was to investigate the surface properties of

CELC. To achieve the goal of this research, the following objectives had been

achieved. The objectives for this project were:

a) to develop contact angle and bubble pressured measurement systems that

enabled analysis of the surface tension of the CELC surface.

b) to investigate the physical properties of the CELC that enabled human

keratinocytes cell attachment.

c) to determine the characteristic of the human keratinocytes interaction with the

CELC.

1.4 Scopes of project

This project was divided into six scopes which involve:

a) Motorised contact angle measurement system was developed using Arduino

UNO microcontroller, L293D motor driver and MATLAB GUI.

b) Bubble pressure method used ASP1400 pressure sensor to find the surface

tension of the CELC.

c) Investigating the surface properties of the liquid crystal using a Differential

Scanning Calorimeter (DSC), Optical Emission Spectroscopy (OES),

Polarising Optical Microscopy (POM), X-ray Diffraction (XRD), and Fourier

Transform Infrared Spectroscopy (FTIR).

4

d) Use human Keratinocytes (HaCaTs) cell lines for cell interaction study.

e) Studying the cell attachment to the liquid crystal using Atomic Force

Microscopy (AFM) and Field Emission Scanning Electron Microscope

(FESEM).

1.5 Thesis organisation

This thesis is divided into five chapters. Chapter 1 provides an overview of this

project and the objectives, scopes and problem statement of the research. Chapter 2

reveals on the CELC background and its application in biosensing and generative

medicine. Chapter 3 outlines the experiment procedure of this project including the

background information on the software used for the contact angle measurement and

bubble pressure method system, preparing sample for physical properties and cells

interaction on CELC analysis. Chapter 4 presents the results and discussion of the

project. Chapter 5 was devoted for the conclusions and recommendations for future

work.

1.6 Thesis contribution

In this thesis, the experimental finding of physical properties of CELC and cell

interactions with CELC revealed a new understanding in biomaterial for biosensing

application. The contributions of this study are as listed below:

a) Fox Zisman graph automated generated using Matlab GUI

Using measured angle value, Matlab GUI can automatically generate Fox Zisman

graph thus provides the critical surface tension (CST) value of materials.

b) Surface tension of CELC

The value of surface tension of CELC were identified and clearly explain the

understanding of wettability of the surface of these liquid crystals in attracting the

adhesion of cells.

5

c) New class of biological relevant material

The physical expression of cell adhered on CELC showed biocompatibility of CELC

exploring new potentials of liquid crystals in bioengineering and biosensing.

CHAPTER 2

LITERATURE REVIEW

2.1 Introduction to liquid crystal

Liquid crystal is a state of matter in addition to the solid crystalline and liquid

phases. Liquid crystal exhibits intermediate phases in which the molecules flow like

liquids yet possess some physical properties characteristic of crystal [14]. In other

word, it is known as mesophases. The classification of the mesophases is based on

the positional and orientation order of the molecules. The molecules of the liquid

crystals are position in a lattice and orientated in a specific directions. One can

classify liquid crystal in accordance with the physical parameters controlling the

existence of the liquid crystalline phases.

The first liquid crystal was discovered in 1888 by an Austrian botanical

physiologist Fredrich Reinitzer (1865-1927) that accidentally observed that

cholesteryl benzoate changed into a cloudy liquid at 145.5 ºC and melted again into a

clear liquid at 178.5 ºC. This material has two melting points and it is quite different

from the three states of matter (solid, liquid and gas) recognised at that time. After

identifying this phenomenon, Reinitzer asked help from a physicist, Otto Lehmann

for further investigation. Lehmann examined the intermediate cloudy fluid and

reported seeing crystallites and named the new substance as ‘liquid crystal’ (LC).

Liquid crystal molecules are often aromatic and it is attached to two substituent

groups. Liquid crystals that contain benzene rings are referred to as benzene

derivatives [15].

Figure 2.1 shows the basic structure of a liquid crystal molecule of 4-

methoxybenzylidene-4’-n-butylaniline (MBBA) which can be an example of liquid

crystal molecule that contain two benzene rings as the basic structure [15].

7

Figure 2.1: Basic structure of a liquid crystal molecule [16].

2.2 Types of liquid crystal

Considering the geometrical structure of the molecules, the liquid crystal can be

grouped into several types. There are two types of liquid crystal; the thermotropic

and lytropic liquid crystals. These materials exhibit liquid crystalline properties as a

function of different physical parameters and environments. Different types of liquid

crystals have different physical properties and application. There were two processes

to enable the transition of the mesophases; one could be altered by thermal processes

and the other by the influence of solvent [17]. For example, thermotropic liquid

crystal change phases with temperature and are often composed of a single type of

molecule whereas lyotropic liquid crystals are obtained when appropriate

concentration of a material is dissolved in a solvent such as water.

2.2.1 Thermotropic liquid crystal

This type of liquid crystal changes its orientation and positional as a function of

temperature. From the form of anisotropic phase, the crystal changes into isotropic

phases gradually as the temperature increase. In isotropic phase, the physical

properties are the same in all directions, while the physical properties for anisotropic

phase are different between one direction and another. By definition, anisotropic

means the physical properties of the phases are different and depending on the

direction and orientation of the component molecules. Each molecule in thermotropic

liquid crystal is oriented in a long range order. Thermotropic liquid crystal can be

divided into three mesophases; nematic, cholesteric and smectic phases.

N CH CH3O C4H9

Substituent Substituent Aromatic group

8

2.2.1.1 Nematic phases

The simplest and least-ordered phase of liquid crystals is the nematic phase (N)

where rod-like molecules directed on average in a particular direction which is

known as the director, n [17]. The molecules possess a high degree of long-range

order with their long axes approximately parallel, but without the distinct layers of

the smectic crystal [15]. The molecules in the nematic phase are arranged parallel to

each other and slide past each other.

2.2.1.2 Cholesteric phases

Cholesterol derivatives are the most common chemical compounds that exhibit the

cholesteric and it is also known as chiral nematic phases, this is because of the first

thermotropic liquid crystalline materials exhibiting this phase were cholesterol

derivative.

Figure 2.2: A graphical illustration of cholesteric phase [18].

Chiral means the molecule cannot be transformed into their mirror image by

rotations or translations [18]. Chiral cholesteric phase has a helical structure in which

the director is twisted along the z-axis normal to the x-axis as shown in Figure 2.2.

The cholesteric phase is similar to nematic phase but chiral molecule cause a twist in

9

the nematic structure which is normal to the long axis. The strong twists in the

molecule cause cholesteric have different optical properties from nematic phase.

Figure 2.2 shows the structure of cholesteric phase where p is the pitch of helix.

Colourful textures are observed due to the selective colour reflection from the

mesogens that are lying in planes of helical structures with different pitch length. The

planar and homeotropic orientations of the cholesteric liquid crystal mesogens

portray different textures. The colour of the textures often appears dark with very

low light reflectivity in the crossed polariser when the mesogens oriented close to the

homeotropic alignment.

Figure 2.3: Types of cholesteric derivatives (a) Cholesteryl pelargonate,

(b) Cholesteryl chloride, and (c) Cholesteryl oleyl carbonate at 10× magnification.

(Scale bar: 25 µm)

Some common cholesteric liquid crystals are cholesteryl chloride, cholesteryl

pelargonate and cholesteryl oleyl carbonate. These cholesteric liquid crystals

reversibly change colour as the temperature changes. Figure 2.3 shows the

birefringence properties of the cholesteric liquid crystal.

(a) (b)

(c)

10

2.2.1.3 Smectic phase

The second common liquid crystal phase is the smectic phase, where the molecules

form single layer of thickness approximate one molecular length as shown in Figure

2.4 [17]. As the temperature increase, the liquid crystal from crystal phase change to

smectic C, smectic A, nematic, and isotropic phase. The smectic C phase (SmC)

where the director, n, at some angle with respect to the layer normal, z. The smectic

A phase (SmA) has the directors n orientates in the same direction with respect to the

layer normal, z.

Figure 2.4: Standard thermotropic liquid crystal phases arranged from left to right in

order of increasing temperature [17].

Smectic and nematic liquid crystal phases are subjected to changes in

temperature which they can change their form and their light transmission properties

splitting a beam of light into two polarised components (ordinary and extraordinary)

to produce the phenomenon of double refraction [15]. Due to the ability of the liquid

crystal in responding to magnetic field they are found useful in liquid crystal display

(LCD) which is one of the applications of thermotropic liquid crystal [15].

2.2.2 Lyotropic liquid crystal

Lyotropic liquid crystals (LLC) are forms from dissolution surfactants in a solvent

such as water [19]. An example of the LLC systems is a fluid formed by water and

amphiphilic molecules whose constituent molecules are formed from polar head

Temperature increase

Crystal Smectic C Smectic A Nematic Isotropic

n

z n n

11

group which attracts water and a nonpolar chain that avoids water [18]. The

hydrophilic group interacts strongly with water and the hydrophobic group is water

insoluble such as soaps and detergents [17]. The phase changes of lyotropic liquid

crystal are determined by the concentration of the solute material in a solvent.

Figure 2.5: Typical phases of lyotropic liquid crystal. The figure shows the

aggregation of amphiphiles into micelles and then into lyotropic liquid phases as a

function of amphiphile concentration and of temperature producing micellar cubic

phase (l1), hexagonal phase (H1), bi-continuous cubic phases (V1) and lamellar

phases (Lα) [20].

Lyotropic liquid crystal has an axial ratio of less than 15 and made up of

molecules larger than the thermotropic liquid crystal [21]. This type of liquid crystal

are mostly found in soap, food and living things such as cell membranes [14]. Figure

2.5 shows the relationship between the liquid crystalline phases of surfactants

(Surface Active Agents) and its composition concentration is expressed in a phase

diagram as a function of temperature. At 30-40wt% of amphiphile concentration, the

micellar cubic phase (l1) will be formed. Lyotropic liquid crystal turns into a cubic

lattice when the concentraton of amphiphiles get to 60% while remaining in the

isotropic phase. As the amphiphile concentration increase, cylindrical shaped that is

normal hexagonal phase (H1) was formed from the micelles in the cubic lattice. A

complex bicontinuous (V1) was formed at a very narrow range of concentrations.

12

Following by high concentration of amphiphiles, the lamellar phase (Lα) in which

the amphiphile molecules are arranged in bilayers and each bilayer is separated by

water molecules.

2.3 Application of liquid crystal in biosensing

In recent years, liquid crystals were discovered with their potential in biosensing.

Liquid crystals were applied as biosensors because they provide label free

observation of biological phenomenon. Among the applications of liquid crystal in

biosensing include the detection of distortion in bulk of lyotropic liquid crystals due

to the presence of immune complexes [22]. A study also found that a liquid crystal

can be used to discriminate fluorescence signals on a biosensor array using liquid

crystal pixel array (LCPA) [23]. Moreover, nematic liquid crystal were found to

detect and amplifies the presence of immune complexes [24] and used as protein and

deoxyribonucleic acid (DNA) separators [25].

The LCD based detection of protease for example trypsin, elastase and

thermolysin that cleaved off the Fluorenylmethyloxycarbonyl (Fmoc) peptide

backing by a thermolysinpolyethylene glycol acrylamide (PEGA) hydrogel support

and specific detection or protease function [26]. Previous study also found that

lyotropic liquid crystal was used as biosensor for the compatibility of lyotropic liquid

crystal with virus. Most of the technique exploited the optical properties of the liquid

crystal in sensing changes in biological interactions [27]. Furthermore, bacterial

detection also one of the application use liquid crystal as biosensor. Research found

that, use of liquid crystal provided infectious disease diagnostics in clinical specimen

[28].

Application of liquid crystal by using 4-cyano-4’-pentylbiphenyl (5CB) in

biosensor also had been developed for urea detection in human body [29] and

glucose biosensor [30]. In addition, by using nematic liquid crystal, 5CB, a novel

highly-sensitive liquid crystal (LC) biosensing approach based on target-triggering

DNA dendrimers (branched molecules) was developed for the detection of p53

mutation gene segment at the LC aqueous interface [31]. Recently, cholesteryl ester

liquid crystal (CELC) was discovered for its new application in detecting single cell

traction forces [4]. Table 2.1 shows the application of liquid crystals as biosensors.

13

CELC was found to support cell adhesion and response locally to cell exerted

forces because of its compact molecular arrangements. As a biosensor, liquid crystals

are very often used in the presence of cell culture media or interact with a fluidic

environment. In this environment, amphillic molecules of liquid crystals could

Types of

Liquid Crystal

Compound Application References

Lyotropic liquid

crystal

Disodium

cromoglycate

(DSCG), water

Detecting distortion in bulk of

lyotropic liquid crystals due to

the presence of immune

complexes.

[22]

Liquid crystal pixel

array (LCPA)

LCPA device from

Ferroelectric liquid

crystal (FLQ devices

(part c LV- 1300P)

and the FLC driver

(part LV-1300P)

Discriminate fluorescence

signals on a biosensor array.

[23]

Nematic liquid

crystal

Disudium

cromoglycate

(DSCG)

Detect and amplifies the

presence of immune complexes.

[24]

Nematic liquid

crystal

5CB Monitor the presence of a

targeted biological species for

rapid screening of solution

conditions leading to

crystallization of proteins at

interfaces.

[26]

Lyotropic liquid

crystal

Tetradecyldimethyl-

amineoxide C14AO,

Decanol, water.

The compatibility of LLC with

virus.

[32]

Nematic liquid

crystal

5CB Virus detection. [27]

Nematic liquid

crystal

5CB Screening the urea level in the

human body.

[29]

Nematic liquid

crystal

5CB Screening glucose in the human

body.

[30]

Nematic liquid

crystal

5CB Signal-enhanced LC biosensing

method based on target-

triggering DNA dendrimers for

the label-free and sensitive

detection of p53 mutation gene

segment.

[31]

Cholesteric ester

liquid crystal

Cholesteryl

pelargonate

C36H62O2,

Cholesteryl chloride

C27H45Cl,

Cholesteryl oleyl

carbonate C46H80O3.

Detecting single cell traction

forces.

[4]

Table 2.1: Application of the liquid crystal as biosensor.

14

reoriented due to the interaction with the water molecules. Therefore, it is important

to investigate the surface tension of the CELC.

2.4 Human keratinocytes (HaCaTs)

Human keratinocytes (HaCaTs) cells were used in this project because of long term

aim such as for pharmacology study.

Figure 2.6: Layer of skin at epidermis consists of keratinocytes.

Figure 2.6 shows the layer of skin consist of keratinocytes. This cell is one of

the major cell types from the epidermis layer of a skin which is at outermost layer of

skin. At the epidermis layer, stratum corneum is the outermost layer of the skin

serves as waterproof barriers of the epidermis protects from dirt. Selected

keratinocytes is at stratum garnulosum. Stratum spinosum is the layer where

keratinocytes become larger, flatter and contain less water as they travel to the

surface of the skin. These cells ultimately flatten and lose their nuclei, thereby

forming the stratum corneum and eventually replacing select cells that migrate to the

skin surface and die [33].

Epidermis

15

2.5 Cellular adhesion

Multicellular organisms such as keratinocytes require adhesion for cells to adhere to

each other and the extracellular matrix. The binding of a cell to an inanimate surface,

cell-cell or the extracellular matrix are called cellular adhesions [34]. Adhesion

occurs from the action of proteins such as integrins which interact with ligands

including extracellular matrix. This process participates in cell adhesions in a large

number of physiologically important processes such as wound healing [35]. Figure

2.7 shows schematic diagram of cell adhesion in order to understand the biological

role of adhesion junctions.

Figure 2.7: A schematic diagram of cellular adhesion.

Cell adhesion is mediated by cell junctions such as desmosomes, adherens,

tight and gap junctions. Desmosomes (bond) is the intermediate filaments anchor at

the extracellular junctions, whilst hemidesmosome (half) have a different function

which is to connect the intermediate filaments of a cell to the extracellular matrix

that is a substances secreted by cells lying outside the cell membrane [36]. They can

be found in skin such as muscle cells. Adherens junctions are microfilaments anchor

the plaque that occurs under the membrane of each cell. It is a complex proteins

16

found at the sites of cell-cell adhesion [37]. Transmembrane proteins, calcium

dependent, mediate cell to cell attachment at the adherens junctions.

Tight junctions or zonula occludens are formed from membrane proteins such

as occluding, claudins and e-cadherin that strongly couple the adjacent cell

membranes [37]. Gap junctions are formed from membrane spanning proteins called

connexins [38]. Each gap junction formed from six connexins molecules. It allows

direct communication between cells such as open and closed, formed from the

channel that passes through the membrane of both cells. They are found in heart

muscle, smooth muscle electrical and chemical integration as a single functional unit

and also in embryonic development. Focal adhesions are the link at the outside of the

extracellular matrix through transmembrane proteins (integrins) with the cell

cytoskeleton (actin microfilaments). They mechanically bind the cell membrane to

the extracellular matrix via specific transmembrane receptors.

2.6 Surface tension measuring technique

The study of surface tension has wide application in applied surface science.

Adhesion and morphological of cells mostly depend on the wettability or surface

energy of a biomaterial [39].

Surface tension is related to the surface energy of a surface. Surface tension,

γ, is the force, F, per unit length, L, (equation (2.1)) that must be applied parallel to

the surface so as to counterbalance the net inward pull and has the units of dyne/cm

or mN/m.

𝛾 =𝐹

𝐿 (2.1)

Greater surface tension reflects higher intermolecular force of attraction, thus

increase in hydrogen bonds induced increase in surface tension. Many techniques

used in finding surface tension of biomaterials have been produced. In this work,

surface tension of CELC was studied by using different techniques such as contact

angle, pendant drop and maximum bubble pressure methods.

17

2.6.1 Contact angle measurement

Contact angle measurement is often used to evaluate the surface and liquid

cleanliness and the effects of surface treatments developed as a part of fundamental

research in surface science as well as for industrial applications [40]. A contact angle

is the macroscopic representation of microscopic phenomena such as surface

roughness, surface energies of the materials involved and surface coatings play a role

in the wettability of a material. By definition, a contact angle is the interior angle

formed by the substrate being used and the tangent to the drop interface at the

apparent intersection of all three interfaces which is called contact line. A static

contact angle on a flat surface is defined by Young’s equation, which is related to

interfacial surface tension between solid, vapour and liquid.

Figure 2.8: Contact angle measurement for a droplet of liquid.

The Young’s equation is essentially a force which balanced in the horizontal

direction. The contact angle may also be directly determined by the ratio of

interfacial surface tension of the unknown interfacial surface tensions using equation

(2.2).

𝑐𝑜𝑠 𝜃 = γsv− γsl

γlv (2.2)

where, γsv, γsl and γlv are the interfacial force of surface and vapour, interfacial

force of liquid and surface, interfacial force of liquid and vapour, respectively. γlv

and cos θ can be determined from the experiment. θ is the contact angle formed by

18

the substrate being used and the tangent to the drop interface at the apparent

intersection of the three phase boundary (solid, liquid and vapour). This intersection

is call contact line. Fox-Zisman method was applied to determine the critical surface

energy (γc). According to Zisman, the value of γc of a solid is equal the value of γl of

a liquid being in contact with this solid and for which the contact angle is zero [41].

Based on the theory, γc value consists of the contact angle measurements for the

studied solid and liquids of a homologous series of compounds.

In Figure 2.8, it shows the contact angle measurement where a liquid was

place on top of a surface of a solid. It is assumed that the liquid does not react with

the solid and that the solid surface is smooth. In contact angle measurement,

selecting the suitable probe liquids to interface with a surface is an important part.

One must ensure that the liquid does not react or swell with the surfaces. Hence, it

can be considered a viable probe liquid for a dynamic contact angle. Different probe

liquids formed different contact angles.

Using the value of the cos θ and the known interfacial tension of each probe

liquid, Fox-Zisman graph can be plotted corresponding to the cosine values of the

contact angle at the y-axis and surface tension of different probe liquids at the x-axis.

The intersection point at which the best fit linear regression line of the data set

intercept with cos θ = 1 indicates the critical surface tension [42]. Selecting several

suitable probe liquids to interface with a surface is an important decision for contact

angle measurement. Fox-Zisman theory suggested the use of at least four probe

liquids in order to obtained enough data points for plotting the Fox-zisman graph

[43]. A liquid can be considered a viable probe liquid for a dynamic contact angle

measurement if it does not swell or react with the solid surface.

Table 2.2: Probe liquids and their interfacial tension.

Liquid Interfacial tension (mN/m) References Isopropyl alcohol (IPA) 21.7 [44]

Acetone 23.0 [44]

Methyl-methacrylate (MMA) 30.6 [45]

Mineral Oil 35 [46]

Dimethyl sulfoxide (DMSO) 42.90 [47]

Ethylene glycol (EG) 47.3 [47]

Glycerol 64 [44]

Distilled water 72.8 [44]

19

For selecting the probe liquid, using a syringe, a drop of known volume of

liquid is dropped to a test surface and observe what happened to the droplet. If it is

absorbed into the solid or completely spreads out across the surface, the probe liquid

is not suitable to be used. This is to make sure that no additional forces and thus

affects the contact angle calculation. Polar liquid is selected as probe liquid for this

test. Some standard test liquid that can be used as probe liquids and their interfacial

tensions are shown in Table 2.2. They are selected in such a way that linear

regression line on the cos θ versus surface tension graph can be obtained over a

wider range of surface tension.

2.6.2 Bubble pressure

Another technique for determining surface tension is bubble pressure method. A

simple experiment apparatus from [48] was propose in finding surface tension using

bubble method based on the Laplace Young equation [49]. While, in [50], bubble

pressure method was used to measure the surface tension of smectic liquid crystal.

Figure 2.9 shows the apparatus for measuring surface tension using bubble pressure

method.

Figure 2.9: Apparatus for surface tension measurement using bubble pressure [50].

20

From Figure 2.9, two capillaries with same diameter were used in order to

reduce the errors in pressure measurement with differing surface tension using

different materials drawn on both openings. R1 and R2 were referred to the radius of

two inflate materials respectively. After that, the materials were deflected to a bubble

by injecting the syringe. Images were recorded by a video camera perpendicular to

the capillary axes. A sensitive pressure gauge was used to determine the pressure

differences. However, this method also can be done using single capillary.

Surface tension can be measured using the straight forward Young Laplace as

in equation (2.3) [48]:

𝑝 =4𝜎

𝑅 (2.3)

where 𝑝 is inner excess pressure, R is the radius of a spherical bubble and σ is the

surface tension [51]. This method provides a direct simple access to σ and also

suitable for study of small surface tension differences.

2.6.3 Pendant drop

The apparatus for finding surface or interfacial tension by pendant drop image

analysis through video imaging digitisation and numerical curve-fitting, VCA

Optima, developed from AST Products, INC is as shown in Figure 2.10.

Figure 2.10: A pendant drop system from AST Products, INC.

21

This system applies Laplace’s equation of capillarity in order to find the value

of surface tension. This pendant drop apparatus consist of illuminating system for

improving image quality, viewing system to visualise the drop image and data

acquisition system to find the interfacial tension from the pendant drop profile. From

the image taken, extraction of the drop contour to determine the radius of curvature

at the apex is necessary for the calculation of interfacial tension. Next, shape

comparison between the theoretical and experimental drop to get interfacial tension

value. The shape of the drop hanging on the needle is determined from the balance of

force which include the surface tension of that liquid. The values of surface tension

of unknown samples are automatically generated.

Based from Laplace’s equation, the equation of Bashforth and Adam [52]

relates the drop profile to the interfacial tension through a nonlinear differential

equation which is given in equation (2.4) and (2.5).

1𝑅1

𝑎

+ sin 𝛷

𝑥

𝑎

= −𝛽𝑧

𝑎+ 2 (2.4)

where β, the shape factor of the drop is given by

𝛽 =𝑎2𝑔△𝜌

𝛾 (2.5)

where △ρ is the difference between the densities of the two liquids in contact. For

example, the density difference between air and liquid. While, g is the gravitational

constant, γ is the surface or interfacial tension, 𝑎 is the radius of curvature at the apex

of the drop, x, z and Φ are the defines coordinates and R1 is the radius of curvature at

the point with coordinates (x,z), as shown in Figure 2.11.

22

Figure 2.11: The pendant drop geometry [52].

2.6.4 Surface tension of liquid crystal

The research by [50] used bubble pressure method to measure the surface tension of

smectic liquid crystal (8CB) at room temperature, 25 °C. The surface tension

determined was 27.8 mN/m. While, from [53], surface tension measured for 5CB

liquid crystal using pendant drop method at temperature dependence is 16.22 mN/m.

Table 2.3 summarises the surface tensions value of 5CB and 8CB liquid crystals

using these two methods.

Table 2.3: Surface tension values for liquid crystal.

Technique Types of

liquid crystal

Temperature,

°C

Surface

tension, mN/m

References

Bubble

pressure 8CB 25 27.8 [50]

Pendant drop 5CB -15 to 10 16.22 [53]

23

2.7 Spectrophotometer

Spectrum was measured using a spectrometer. From the spectrophotometer, a graph

of intensity as a function of wavelength was shown. Optical Emission Spectroscopy

(OES) is one of the spectrometer that shows the intensity of light.

Figure 2.12: Light transmitted or reflected through a sample.

Figure 2.12 shows the block diagram of a spectrophotometer where incident

light beam can be absorbed, transmitted and reflected. Many compounds absorb

ultraviolet (UV) or visible (Vis) light. Based on Beer’s Law, the transmittance of any

sample at wave number is given by the ratio of the radiant power emerging from the

rear face of the sample at the wave number, I to the power of the radiation at the

front face of the sample Io [54].

Using Beer’s Law, the percentage of light transmitted can be measured using

equation (2.6):

%𝑇 =𝐼

𝐼𝑜× 100 (2.6)

Absorbance provides value of intensity of a light. Thus, if all the light passes through

a sample without any absorption, then the absorbance is zero and gives 100% of

transmittance.

Sam

ple

Incident light

beam

Transmission

Reflection

Absorption

24

2.8 Differential scanning calorimetry (DSC)

A Differential Scanning Calorimetry (DSC) measures the difference in heat flow rate

between a sample and inert reference as a function of time and temperature. DSC

provides information about the physical and chemical changes that involve

endothermic (absorption of heat) or exothermic (release of heat) activities from a

sample [55].

Figure 2.13: Schematic DSC heating curve of a semi-crystalline polymer.

The results of glass transition (Tg), melting and boiling points (Tm), and

evaporation temperature can be found from endothermic heat flows into the sample.

While results of exothermic heat flow out of the sample provides, crystallization time

and temperature, percent crystallinity, rate and degree of cure, and oxidation or

thermal stability. Figure 2.13 shows typical DSC heating curve of a semi-crystalline

polymer with glass transition, exothermic recrystallization, melting endothermic and

exothermic decomposition.

2.9 X-ray diffractometer (XRD)

The laboratory used in this project is equipped with a PANalytical X’Pert Powder X-

Ray Diffraction (XRD) machine (Figure 2.14). This machine operates with a power

of 3kW at 40 kV and 40 mA, with a cooper X-ray tube, which has a characteristic

92

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