THE STUDY OF FLUORESCENCE ENHANCING AND QUENCHING EFFECT OF
LUMINESCENT MATERIALS IN SOL GEL FOR THE DETECTION OF GASES
TEE SHIAU FOON
UNIVERSITI TEKNOLOGI MALAYSIA

UNIVERSITI TEKNOLOGI MALAYSIA
JUDUL : THE STUDY OF FLUORESCENCE ENHANCING AND QUENCHING
EFFECT OF LUMINESCENT MATERIALS IN SOL GEL
FOR THE DETECTION OF GASES.
SESI PENGAJIAN : 2005/2006
Saya
(HURUF BESAR) mengaku membenarkan tesis ini disimpan di Perpustakaan Universiti Teknologi Malayisa dengan syarat-syarat seperti berikut :
1.
Hakmilik tesis ini adalah dibawah nama penulis melainkan penulisan sebagai projek bersama dan dibiayai oleh UTM, hakmiliknya adalah kepunyaan UTM.
2.
Naskah salinan di dalam bentuk kertas atau mikro hanya boleh dibuat dengan kebenaran bertulis daripada penulis.
3.
Perpustakaan Universiti Teknologi Malaysia dibenarkan membuat salinan untuk tujuan pengajian mereka.
4.
Tesis hanya boleh diterbitkan dengan kebenaran penulis. Bayaran royalti adalah mengikut kadar yang dipersetujui kelak.
5.
*Saya membenarkan / tidak membenarkan Perpustakaan membuat salinan tesis ini sebagai bahan pertukaran di antara institusi pengajian tinggi.
6.
**Sila tandakan ( √ )
SULIT
(Mengandungi maklumat yang berdarjah keselamatan atau kepentingan Malaysia seperti yang termaktub di dalam
AKTA RAHSIA RASMI 1972)
TERHAD (Mengandungi maklumat TERHAD yang telah ditentukan oleh organisasi / badan di mana penyelidikan dijalankan)
√ TIDAK TERHAD
Disahkan oleh
( TANDATANGAN PENULIS) (TANDATANGAN PENYELIA)
Alamat Tetap : NO. 22, JLN. SG. PUTUS 42, PM. DR. MUSTAFFA NAWAWI
TAMAN HARAPAN, 42100, (NAMA PENYELIA)
KLANG, SELANGOR.
Tarikh : 03.01.2006 Tarikh : 03.01.2006
CATATAN : * Potong yang tidak berkenaan.
** Jika Tesis ini SULIT atau TERHAD, sila lampirkan surat daripada pihak
berkuasa / organisasi berkenaan dengan menyatakan sekali tempoh tesis
ini perlu dikelaskan sebagai SULIT atau TERHAD.

BAHAGIAN A – Pengesahan Kerjasama*
Adalah disahkan bahawa projek penyelidikan tesis ini telah dilaksanakan melalui kerjasama antara _____________________ dengan ________________________
Disahkan oleh:
Tandatangan : ………………………………………… Tarikh : ………………
Nama : ………………………………………….
(Cop rasmi)
* Jika penyediaan tesis/projek melibatakan kerjasama .
BAHAGIAN B – Untuk Kegunaan Pejabat Sekolah Pengajian Siswazah
Tesis ini telah diperiksa dan diakui oleh:
Nama dan Alamat : Prof. Madya Dr. Wan Saime Bin Wan Ngah
Pemeriksa Luar School of Chemical Science
Universiti Sains Malaysia
11800 MINDEN
Pulau Pinang
Nama dan Alamat : Prof. Madya Dr. Wan Aini Binti Wan Ibrahim
Pemeriksa Dalam Fakulti Sains
UTM, Skudai
Nama dan Alamat Pemeriksa Dalam II :
Nama Penyelia Lain
(jika ada) :
Disahkan oleh Timbalan Pendaftar di SPS:
Tandatangan : ___________________________ Tarikh : ________________
Nama : GANESAN A/L ANDIMUTHU

“I hereby declare that I have read this thesis and in my opinion this thesis is sufficient in terms of scope and quality for the award of the degree of Master of Science (Chemistry)”
Signature :
Name of Supervisor : Assoc. Prof. Dr. Mustaffa bin Nawawi
Date : 03.01.2006

THE STUDY OF FLUORESCENCE ENHANCING AND QUENCHING EFFECT
OF LUMINESCENT MATERIALS IN SOL GEL FOR THE DETECTION OF
GASES
TEE SHIAU FOON
A thesis submitted in fulfilment of the requirements for the award of the degree of
Master of Science (Chemistry)
Faculty of Science
Universiti Teknologi Malaysia
JANUARI 2006

ii
I declare that this thesis “ The Study of Fluorescence Enhancing and Quenching
Effect of Luminescent Materials In Sol Gel for the Detection of Gases ” is the result of my own research except as cited in the references. The thesis has not been accepted for any degree and is not concurrently submitted in candidature of any other degree.
Date : 03.01.2006

Here’s a token of love and gratitude.
Especially for my family and friends,
As my sources of strength, comfort and patience,
Thank you all for having faith in me,
I’m truly blessed and inspired! iii

iv
ACKNOWLEDGEMENTS
The author wish to express her heartfelt thanks and appreciation to Assoc.
Prof. Dr. Mustaffa bin Nawawi for his guidance, assistance, advice, valuable suggestions and sharing of knowledge as well as experience throughout this study.
The author would also like to acknowledge all the lecturers of Department of
Chemistry, Faculty Science, Universiti Teknologi Malaysia for their helps and supports. The author wish to show her gratitude to the UTM staff especially Mr.
Yasin, Miss Nurul and Mr. Hanan of the Analytical Laboratory for helping her.
The author especially dedicates this compilation to her dearest family and friends for their love, encouragement and support in the completion of her study.
Finally, the author acknowledges, with thanks, the financial support of UTM-
PTP for the scholarship.

v
ABSTRACT
The control of CO
2
, SO
2
and O
2
emission is important thus the determination of its concentration is important in many applications. This study is carried out to investigate and characterize the fluorescent properties of metal-chelate using fluorescent materials such as α -naphthoflavone (7, 8-benzoflavone), fluorescein and luminol. Initial investigation showed that α -naphthoflavone exhibit fluorescence at
λ em
= 426 nm ( λ ex
= 343 nm), fluorescein exhibit fluorescence at λ em
= 510 nm ( λ ex
=
483 nm) and luminol at λ em
= 421 nm ( λ ex
= 386 nm) at optimum pH 10.06, pH 8.90 and pH 8.62 respectively. The presence of CO
2
, SO
2
and O
2
were detected by α naphthoflavone, fluorescein and luminol. α -naphthoflavone, fluorescein and luminol giving a non-linear change in the emission intensity with increase volume of SO
2
and
O
2
gas. Therefore, α -naphthoflavone, fluorescein and luminol are not suitable for detecting SO
2
and O
2
gas. When dissolving CO
2
gas into the complex, the emission wavelength ( ∆λ em
) for fluorescein was shifted by 6 nm and luminol was 9 nm. The fluorescence properties of α -naphthoflavone, fluorescein and luminol entrapped in sol gel were also investigated. Results indicated that the emission intensity of the entrapped fluorescent material in sol gel decreased as compared to fluorescence in solvent. For the detection of CO
2
, the gas was bubbled onto the sol gel surface. It was observed that the emission intensity of α -naphthoflavone in sol gel increased linearly as the volume of CO
2
gases increased. Meanwhile, fluorescein and luminol shows a non linear change with the increased CO
2
amount in sol gel. Among the five metals ions that complex with α -naphthoflavone, fluorescein and luminol, it was found that the emission intensity of fluorescein-ferum complex decreases linearly after exposure to increasing amount of CO
2
in sol gel giving a linear relationship with an equation was y = -7.2252 x + 271.01 and a correlation of 0.9234.

vi
ABSTRAK
Pengawalan perlepasan gas karbon dioksida, sulfur dioksida dan oksigen adalah sangat penting dalam pengesanan kepekatannya dalam banyak aplikasi.
Dalam kajian ini, ciri-ciri bahan pendarfluor seperti α -naftoflavon (7,8-benzoflavon), fluoresin dan luminol dengan logam-kelat telah dikaji. Keputusan awal menunjukkan bahawa α -naftoflavon berpendarfluor pada puncak pemancaran λ em
426 nm ( λ ex
= 343 nm), fluoresin berpendarfluor pada puncak pemancaran λ em
483 nm ( λ ex
= 510 nm) manakala luminol pada λ em
386 nm ( λ ex
= 421 nm) masingmasing pada pH optimum 10.06, pH 8.90 dan pH 8.62. Kehadiran gas karbon dioksida telah diuji dengan menggunakan α -naftoflavon, fluoresin dan luminol dan hasil menunjukkan bahawa keamatan puncak pemancaran adalah berubah secara tidak linear dengan penambahan CO
2
. Didapati juga perubahan kedudukan puncak pemancaran ( ∆λ em
) bagi fluoresin adalah 6 nm dan luminol adalah 9 nm. Bahan berpendarfluor α -naftoflavon, fluoresin dan luminol terperangkap dalam sol gel juga dikaji. Keputusan menunjukkan keamatan bahan berpendarfluor yang terperangkap dalam sol gel berkurang. Gas CO
2
dialirkan di permukaan sol gel. Keamatan puncak pemancaran bagi α -naftoflavon dalam sol gel bertambah secara linear dengan kehadiran gas CO
2
. Sementara itu, fluoresin dan luminol menunjukkan perubahan yang tidak linear dengan penambahan CO
2
dalam sol gel. Dalam lima ion logam yang dipilih untuk membentuk kompleks dengan α -naftoflavon, fluoresin dan luminol, didapati bahawa keamatan puncak pemancaran bagi kompleks fluoresinferum berkurang secara linear setelah gas CO
2
dialirkan di permukaan sol gel menghasilkan satu persamaan linear dengan y = -7.2252 x + 271.01 dan dengan korelasi 0.9234.

vii
TABLE OF CONTENTS
TITLE
DECLARATION
DEDICATION
ACKNOWLEDGEMENTS
ABSTRACT
ABSTRAK
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF SYMBOLS
1 INTRODUCTION
1.1.1 Factors Affecting Fluorescence
Spectroscopy Quantitative Accuracy
1.1.1.1 Temperature Effect
1.1.1.2 pH Effect
1.1.1.3 Solvent Effect
1.1.1.4 Inner filter
3
3
4
4
5
Quenching i ii iii iv v vi vii xiii xiv xxi
Fluorescence Chemistry
1.3.2 Determination of gases in Fluorescence 7

1.3.3 Application Fluorescence in Polymer
1.3.4 Determination of Protein
1.3.5 Determination of Metals and Ions
1.4 α -naphthoflavone (7, 8-benzoflavone)
1.5.1 The Advantages for Using Fluorescein
1.7 Carbon Dioxide (CO
2
)
1.7.1 Preparation of Carbon Dioxide
1.7.2 Uses of Carbon Dioxide
1.8 The Determination of Carbon Dioxide with
Other Method
1.9 Sol Gel Glass and Sol Gel Process
Chemistry
1.9.1.1 Sol gel Encapsulated
Fluorescence Materials as
Gases Sensors
(Bioanalytical application)
1.9.1.3 The Others Uses of Sol gel
18
20
1.11 Statement of the Problem and the Needs of the Study
1.12 The Detection of Carbon Dioxide Gases
Using Fluorescent Materials
2 EXPERIMENTAL
2.1 Reagents and Materials
21
21
22
11
13
14
14
14
16
8
8
9
9 viii
2.3.1 Preparation of 1.0 mol L-1 Tris buffer
(tris hydroxymethyl amino methane)
24

2.3.2 Preparation of Hydrochloric Acid
(HCl) 0.01
2.3.3 Preparation of Sodium Hydroxide
(NaOH) 0.1 M
2.4 Preparation of Stock Solutions
2.4.1 Preparation of Solutions Fluorescent
Materials
25
25
24 ix
24
2.5.5 Standards Calibration Experiments
2.5.6 The Effect of Various Metals of Carbon Dioxide
2.6.1
Preparation of Sol Gel for Encapsulation of Sensing Material
2.6.2 Encapsulation of Fluorescent Materials
For the Detection of Carbon Dioxide
2.6.3 Encapsulation of Fluorescent Material
Complex For The Detection Of Carbon
Dioxide
3.
RESULTS AND DISCUSSIONS
3.1 The Fluorescence Study of α –naphthoflavone (ANF)
3.1.1 Effect
3.1.2 The Effect of Temperature
3.1.3 of
3.1.4 Standard Calibration Graph of
α –naphthoflavone
3.1.5 The Effect of Various Metals
28
28
27
27
28
29
37
38
31
34

3.1.6 The Detection of Carbon Dioxide,
Oxygen and Sulphur Dioxide Gaseous on the Fluorescence of α -naphthoflavone
3.1.6.1 The Effect of Carbon Dioxide (CO
2
)
Gases On The Emission Spectra of α -naphthoflavone i) α -naphthoflavone in Ethanol ii) α -naphthoflavone
3.1.6.2 The Effect of Oxygen (O
2
) gases on the Emission Spectra of
α -naphthoflavone in Ethanol
3.1.6.3 The Effect of Sulphur Dioxide (SO
2
)
Gases on the Emission Spectra of α -naphthoflavone in Ethanol
47
47
52
47
50
53 x as Fluorescent Carbon Dioxide Sensing
Material
3.2 The Fluorescence Study of Fluorescein
3.2.1 Effect
3.2.2 The Effect of Solvents
3.2.3 Standard Calibration Graph of Fluorescein
3.2.4 The Effect of Various Metals on the
Fluorescence of Fluorescein in Ethanol
3.2.5 The Detection of Gaseous Carbon Dioxide,
Oxygen and Sulphur Dioxide of Fluorescein
3.2.5.1 The Effect of Carbon Dioxide (CO
2
) on the Emission Spectra of α - naphthoflavone ii) Fluorescein in DMF 72
57
60
61
63
70
70

xi
3.2.5.2 The Effect of Oxygen (O
2
) Gases on the Emission Spectra of Fluorescein
74
3.2.5.3
The Effect of Sulphur Dioxide (SO
2
)
Gases on the Emission Spectra of
Fluorescein in Ethanol
3.2.6 Sol Gel Immobilized Fluorescein in Ethanol as Fluorescent Carbon Dioxide Sensing Material
3.2.7 Sol Gel Immobilized Complex Fluorescein- manganese and Fluorescein-ferum as
75
76
78
Fluorescent Carbon Dioxide Sensing Material
3.3 The Fluorescence Study of Luminol 80
3.3.1 Effect
3.3.2 Effect
3.3.3 The Effect of Solvents 84
Calibration 85 of luminol in Ethanol
3.3.5 The Effect of Various Metals
3.3.6 The Detection of Gases Carbon Dioxide,
Oxygen and Sulphur Dioxide on the
Fluorescence of Luminol
87
96
3.3.6.1
The Effect of Carbon Dioxide (CO
2
)
Gases on the Emission Spectra of Luminol
96
3.3.6.2 The Effect of Oxygen (O
2
) Gases on 97
3.3.6.3 The Effect of Sulphur Dioxide (SO
2
)
Gases on the Emission Spectra of
α -naphthoflavone in Ethanol
3.3.7 Property of Luminol which
Encapsulated Gel
98
100

3.3.8 Sol gel Immobilized luminol in Ethanol as
3.3.9
Sol Gel Immobilized Complex luminol
-cadmium and luminol-ferum in Ethanol as Fluorescent Carbon Dioxide Sensing
Material
4.
CONCLUSION
100
102
REFERRENCES 108 xii

LIST OF TABLES
3.1 Emission spectra ( λ em at different pH
) and intensity of fluorescein 59 xiii

LIST OF FIGURES
FIGURE NO. TITLE
1.1
1.2
1.3
1.4
The molecular structure of 7, 8-benzoflavone.
The molecular structure of fluorescein.
The reaction of luminol in basic condition.
1.5
2.1
The structural formula of carbon dioxide.
The Perkin Elmer LS50B luminescence spectrophotometer.
2.2 The process of bubbling carbon dioxide gas to the
3.1 surface of sol gel encapsulating fluorescent materials.
Excitation and emission spectra of α -naphthoflavone in ethanol ( λ ex
= 343.0 nm).
3.2
Emission spectra for α –naphthoflavone in ethanol at different pH ( λ ex
= 343.0 nm, λ em
= 426.0 nm).
3.3
Emission intensity for α -naphthoflavone at different pH.
3.4 Effect of temperature on α -naphthoflavone emission spectra.
3.5 Effect of temperature (
α -naphthoflavone. o C) on emission intensity of
3.6
The excitation peaks for α -naphthoflavone in N, N- dimethyformamide (DMF).
PAGE
9
10
12
13
24
30
32
33
33
34
35
36 xiv

3.7 The emission peaks for α -naphthoflavone in N,N-
dimethyformamide (DMF) ( λ em
= 430.83 nm).
3.8 Emission spectra of α –naphthoflavone in ethanol at different concentrations, a = 1 x 10 c = 1 x 10 -4 , d = 5 x 10 -5
343.0 nm (pH 10.06).
-3
, e = 1 x 10 -5
, b = 5 x 10 -4
M at λ ex at
,
3.9 Standard calibration graph for ethanol.
α –naphthoflavone in
3.10 Effect of lanthanum on the emission spectra of
α -naphthoflavone (a = 1 mL, b = 2 mL, c = 3 mL, d = 5 mL, e = 4 mL, f = 6 mL).
3.11 Effect of lanthanum volume on the emission spectra of α -naphthoflavone ( λ ex
343.0 nm, λ em
421.0 nm).
3.12 Effect of manganese on the emission spectra of
α -naphthoflavone in ethanol (a = 1 mL, b = 2 mL, c = 3 mL, d = 4 mL, e = 5 mL) ( λ ex
= 343.0 nm,
λ em
419.5 nm).
3.13 Effect of manganese volume on the emission spectra of α -naphthoflavone.
3.14 Effect of cadmium on the emission spectra of
α -naphthoflavone (a = 1 mL, b = 2 mL, c = 3 mL, d = 4 mL, e = 5 mL) ( λ ex
343.0 nm, λ em
421.6 nm).
3.15
3.19
Effect of cadmium volume on the emission intensity of α -naphthoflavone.
3.16 Effect of magnesium on the emission spectra of
α -naphthoflavone ( λ ex
343.0 nm, λ em
419.6 nm)
(a = 2 mL, b = 1 mL, c = 3 mL, d = 4 mL, e =
5 mL).
3.17 Effect of magnesium volume on the emission spectra of α -naphthoflavone.
3.18 Effect of ferum (II) on the emission spectra of
α -naphthoflavone ( λ ex
366.0 nm, λ em
421.46 nm)
(a = 1 mL, b = 2 mL, c = 3 mL, d = 5 mL, e = 4 mL, f = 6mL).
Effect of ferum (II) volume on the emission intensity of α -naphthoflavone ( λ ex
366.0 nm, λ em
421.5 nm).
47
36
37
38
39
40
41
42
43
43
44
45
46 xv

xvi
3.20
Effect of carbon dioxide gases on the fluorescence emission of α -naphthoflavone in ethanol.
3.21 Emission α -naphthoflavone (5 x 10 -3 M) and
200 uL NH
4
OH in DMF a) before and b) after response to CO
2
.
3.22 Emission spectra shows 1x10 in ethanol ( λ ex
= 343.0 nm, λ
-3 em
mol L -1
α -naphthoflavone dissolved with oxygen gases
48
49
51
= 423.21 nm).
3.23
Effect of sulphur dioxide gases on the emission intensity of α -naphthoflavone 1x10 -3 mol L ethanol ( λ ex
= 343.0 nm, λ em
= 421.74 nm).
-1 in
52
3.24
Effect of CO
2
exposured on the emission intensity of α -naphthoflavone in sol gel. ( λ
λ em
= 471.94 nm). ex
= 368.0 nm,
54
3.25 Effect of volume of CO
2 gases on the emission intensity of α -naphthoflavone encapsulated in sol gel.
54
3.26 Emission spectra of α -naphthoflavone-ferum complex exposure to carbon dioxide (a = 6 mL, b = 16 mL, c = 14 mL, d = 10 mL).
55
3.27
Effect of carbon dioxide volume on the emission intensity of α -naphthoflavone-ferum complex.
56
3.28 57
3.29
Effect of carbon dioxide volume on the emission intensity of α -naphthoflavone-lanthanum complex.
Fluorescence spectra of 1 x 10 -4 M of fluorescein showing excitation wavelength at 483.0 nm and emission at 510.2 nm.
58
3.30 Emission spectra showed the pH effect of fluorescein in ethanol ( λ ex
= 483.0 nm). (a = 7.38, b = 2.64, c = 9.61, d = 8.17, e = 8.90).
59
3.31 Emission spectra of fluorescein in DMF with λ em at 547.89 nm ( λ ex
at 475.0 nm).
61
3.32 The emission spectra of fluorescein at different concentration between 0.5 x 10 -4 M to 10.0 x 10 -4 M.
62
3.33 The standard curve showing that the intensity of emissions of fluorescein between 0.5 x 10
10.0 x 10 -4 M concentration.
-4 M to
62

3.34 Effect of manganese on the emission spectra of fluorescein (a = 1mL, b = 2 mL, c = 3 mL, d = 4 mL, e = 5 mL, f = 6 mL).
3.35 Effect of manganese volume on the emission spectra of fluorescein ( λ ex
475.0 nm, λ em
512.04 nm).
3.36 Effect of magnesium on the emission spectra of fluorescein in ethanol ( λ ex
475.0 nm, λ em
510.12 nm)
( a = 1 mL, b = 2 mL, c = 3 mL, d = 4 mL, e = 5 mL, f = 6 mL ).
3.37 Effect of magnesium volume on the emission spectra of fluorescein.
3.38 Effect of different volume of ferum (II) on the emission spectra of fluorescein.
3.39
Effect of lanthanum volume on the emission spectra of fluorescein ( λ ex
475.0 nm, λ em
502.94 nm).
3.40 Effect of cadmium volume on the emission spectra of fluorescein ( λ ex
= 475.0 nm, λ em
= 517.5 nm).
3.41 Emission spectra of 1 x 10 -4 M fluorescein + tris buffer (1ml) dissolved CO
2
gas (a = 2 mL, b = 6 mL, c = 4 mL, d = 20 mL, e = 12 mL, f = 16 mL).
3.42
Graph shows the effect carbon dioxide on the emission intensity of 1x10 -4 M fluorescein in ethanol ( λ ex
475.0 nm, λ em
516.0 nm).
3.43
Emission spectra of fluorescein after exposure to
CO
2 in DMF in alkaline condition (a = 1 mL, b = 2 mL, c = 3 mL, d = 4 mL, e = 5 mL, f = 6 mL, g = 7 mL, h = 12 mL, i = 13 mL).
3.44
3.45
Graph of 1 x 10 -4 M fluorescein in DMF dissolved with carbon dioxide gas ( λ ex
475.0 nm , λ em
546.1 nm).
Emission spectra of 1x10 -4 M fluorescein in ethanol bubbled with gaseous oxygen ( λ ex
475.0 nm,
λ em
516.0 nm).
3.46 Graph of 1x10 -4 M fluorescein in ethanol bubbled with gaseous oxygen ( λ ex
475.0 nm , λ em
516.1 nm).
64
64
66
66
67
69
69
71
71
73
73
74
75 xvii

3.47 Emission spectra shows fluorescein 1x10 -4 M dissolved with sulphur dioxide gas in ethanol
( λ ex
= 475.0 nm, λ em
= 510.53 nm) (a = before
dissolved SO
2 , b = 1 minute after dissolves SO
2 gases, c = 1 day after dissolves SO
2
gases, d = 2 day after dissolves SO
2
gases.
3.48 Effect of carbon dioxide gases in sol gel on the emission intensity of 1 x 10 -4 mol L -1 fluorescein ethanol λ em
= 518.56 nm).
3.49 Emission spectra show fluorescein-manganese complexes which dissolved carbon dioxide gases in sol gel ( λ ex
= 475.0 nm, λ em
= 482.52 nm)
(a = 2 mL, b = 4 mL, c = 6 mL, d = 16 mL, e = 10 mL).
3.50 Effect of carbon dioxide on the emission intensity of fluorescein-manganese complex encapsulated in sol gel.
3.51 Effect of carbon dioxide on the emission intensity of fluorescein-ferum complexes encapsulated in sol gel.
3.52 Fluorescence spectra of 1 x 10 -4 M luminol
( λ ex
= 386.0 nm and λ em
= 421.2 nm).
3.53 Emission spectra ( λ em
= 419.52 nm) showing the pH effect of luminol in ethanol ( λ ex
= 386.0 nm)
(a = pH 8.62, b = pH 4.71, c = pH 2.3, d = pH 9.00, e = pH 9.27, f = pH 10.07, g = pH 11.17).
3.54
3.55
Emission intensity of luminol at different pH.
The fluorescence emission spectrum of luminol at different temperatures.
3.56 Effect of temperature on luminol intensity.
3.57 Emission spectra of luminol in ethanol, DMF, sodium hydroxide and deionized water.
3.58 The emission spectra of luminol at different concentrations between 0.5 x 10
10.0 x 10 -4 mol L -1 .
-4 mol L -1 to
3.59 The standard calibration curve of luminol
(0.5 x 10 -4 mol L -1 to 10.0 x 10 -4 mol L -1 ).
76
77
78
79
80
81
82
82
83
84
85
86
86 xviii

3.60 Effect of lanthanum on the emission spectra of luminol (a = 5 mL, b = 3 mL, c = 5 mL, d = 1 mL).
88
3.61 Effect of lanthanum volume on the emission spectra of luminol ( λ ex
386.0 nm, λ em
419.12 nm).
89
3.62 Effect of ferum (II) on the emission spectra luminol
(a = 1 mL, b = 2 mL, c = 3 mL, d = 4 mL, e = 5 mL, f = 6 mL).
90
3.63 Effect of ferum (II) volume on the emission spectra of luminol ( λ ex
386.0 nm, λ em
412.12 nm).
91
3.64 Effect of cadmium on the emission spectra of luminol. 92
( λ ex
386.0 nm, λ em
419.12 nm) (a = 1 mL, b = 2 mL, c = 3 mL, d = 4 mL, e = 5 mL, f = 6 mL).
3.65 Effect of cadmium volume on the emission spectra 93 of luminol in ethanol.
3.66 Effect of magnesium on the emission spectra of luminol ( λ ex
386.0 nm, λ em
408.07 nm) (a = 1 mL,
94 b = 2 mL, c = 3 mL, d = 5 mL, e = 6 mL).
3.67 Effect of magnesium volume on the emission spectra of luminol.
3.68 Effect of manganese on the emission spectra of luminol ( λ ex
386.0 nm, λ em
406.83 nm) (a = 1 mL, b = 2 mL, c = 3 mL, d = 4 mL, e = 5 mL, f = 6 mL).
3.69 Effect of manganese volume on the emission spectra of luminol ( λ ex
386.0 nm, λ em
406.83 nm).
3.70 Graph of luminol emission intensity from 1x10 mol L -1 solution in ethanol bubbled with different volume of carbon dioxide gases ( λ ex
-3
= 386.0 nm,
λ em
= 412.0 nm).
3.71 Graph of luminol emission intensity from 1x10 -3 mol L -1 solution in ethanol bubbled with different volume of oxygen gases ( λ ex
λ em
= 412.7 nm).
= 386.0 nm,
3.72 Emission spectra shows the effect of sulphur dioxide on luminol in ethanol ( λ ex
= 346.0 nm,
λ em
= 409.74 nm) (a = 1 mL, b = 4 mL, c = 2 mL, d = 5 mL, e = 8 mL).
94
95
96
97
98
99 xix

3.73 Effect sulphur dioxide on the 1x10 -3 mol L -1 luminol in ethanol.
3.74 Effect of carbon dioxide on the emission spectrum of luminol in sol gel ( λ ex
= 366.0 nm, λ em
= 425.98 nm)
(a = 0 mL, b = 2 mL, c = 4 mL, d = 5 mL).
3.75 Effect of carbon dioxide on the emission intensity
of mol L -1 luminol in sol gel ( λ ex
386.0 nm,
λ em
421.6 nm).
3.76 Emission of luminol-ferum complex exposed to gaseous carbon dioxide (a = 20 mL, b = 18 mL, c = 14 mL, d = 6 mL).
3.77 Effect of carbon dioxide on the emission intensity of luminol-ferum complex.
3.78 Effect of carbon dioxide on the emission intensity of luminol-cadmium complex.
99
101
101
102
103
104 xx

LIST OF SYMBOLS
ANF
CO
2
- carbon
α –naphthoflavone
NH
4
λ em
- wavelength
λ ex wavelength
- nanometer
- [H + ] o Celsius
- millilitres
g - gram
R 2
Fe
- mole per litres
- lanthanum
- magnesium
- manganese
- ferum / iron xxi

CHAPTER 1
INTRODUCTION
1.1 Principle of Fluorescence
All chemical compounds absorb energy which causes excitation of electrons in the molecule, resulting in the transitions between discrete electronic energy states.
For a transition to occur, the absorbed energy must be equivalent to the difference between the initial electronic state and a high-energy state. This value is constant and is characteristic of the molecular structure. This is termed as the excitation wavelength. If conditions permit, an excited molecule will return to the ground state by emission of energy through heat and/or emission of energy quanta such as photons. The emission energy or wavelength of these quanta are also equivalent to the difference between the two discrete energy states and are characteristic of the molecular structure [1-3].
Fluorescence occurs when a molecule absorbs photons from the UV-visible light spectrum (200-900 nm), causing transition to a high-energy electronic state and then emits photons as it returns to its initial state, in less than 10 -9 sec. Some energy, within the molecule, is lost through heat or vibration so that emitted energy is less than the excited energy; i.e., the emission wavelength is always longer than the excitation wavelength. The difference between the excitation and emission wavelengths is called the Stokes shift [2, 5].

2
Fluorescent compounds or fluorophors can be identified and quantified on the basis of their excitation and emission properties. The excitation spectra is determined by measuring the emission intensity at a fixed wavelength, while varying the excitation wavelength. The emission spectra are determined by measuring the variation in emission intensity wavelength for a fixed excitation wavelength. The excitation and emission properties of a compound are fixed, for a given instrument and environmental condition, and can be used for identification and quantification
[1-3].
The principal advantage of fluorescence over radioactivity and absorption spectroscopy is the ability to separate compounds on the basis of either their excitation or emission spectra, as opposed to a single spectra. This advantage is further enhanced by commercial fluorescent dyes that have narrow and distinctly separated excitation and emission spectra.
Although maximum emission occurs only for specific excitation and emission wavelength pairs, the magnitude of fluorescent intensity is dependent on both intrinsic properties of the compound and on readily controlled experimental parameters, including intensity of the absorbed light and concentration of the fluorophor in solution. The intensity of emitted light, F, is described by the relationship
F =
I
0
(
1 − e ε bc
) where φ is the quantum efficiency, I
0
is the incident radiant power, ε is the molar absorptivity, b is the path length of the cell, and c is the molar concentration of the fluorescent dye [1, 5].
The quantum efficiency is the percentage of molecules in an excited electronic state that decay to ground state by fluorescent emission; i.e., rapid emission of a light photon in the range of 200-900 nm. This value is always less than or equal to unity and is characteristic of the molecular structure. A high efficiency is

3 desirable to produce a relative higher emission intensity. All non-fluorescent compounds have a quantum efficiency of zero.
The intensity of the excitation light, which impinges on the sample, depends on the source type, wavelength and other instrument factors. The light source, usually mercury or xenon, has a characteristic spectrum for emission intensity relative to wavelength.
At high dye concentrations or short path lengths, fluorescence intensity relative to dye concentration decreases as a result of "quenching". As the concentration of molecules in a solution increases, the probability that excited molecules will interact with each other and lose energy through processes other than fluorescent emission increases. Any process that reduces the probability of fluorescent emission is known as quenching. Other parameters that can cause quenching include presence of impurities, increased temperature, or reduced viscosity of the solution media [2-5].
1.1.1 Factors Affecting Quantitative Accuracy of Fluorescent Measurement
1.1.1.1 Temperature Effect
Temperature usually changes the fluorescence of a solution only a few percent per degree. Changes in temperature affect the viscosity of the medium and hence the number of collisions of the molecules of the fluorophors with solvent molecules. Fluorescence intensity is sensitive to such changes and the fluorescence of many certain fluorophors shows temperature dependence [1, 4, 5].
At low temperature the fluorescence reaches a limiting maximum, and at high temperatures tends to zero. Examples are the enhancement of fluorescence by low temperatures, by embedding molecules in rigid or highly viscosity glasses and

4 plastics by structural effects inhibiting free internal movements such as the oxygen and nitrogen bridges in xanthanene [5].
1.1.1.2 pH Effect
Relatively small changes in pH will sometimes radically affect the intensity and spectral characteristics of fluorescence. Accurate pH control is essential and when particular buffer solutions are recommended in an assay procedure, they should not be changed without investigation [1, 5]. Quinine or β -naphtol may be used as fluorescence indicator for titration purpose of coloured solutions because of their marked changes of fluorescence between acid and alkaline condition [1].
Most phenols are fluorescent in neutral or acidic media, but the presence of a base leads to the formation of non-fluorescent phenate ions [1]. The ionization of a weak acid or base produces a considerable change in the electronic structure of the molecule and this affect both the light-absorption curve and the power of fluorescence.
1.1.1.3 Solvent Effect
The intensity of fluorescence of a substance may vary considerably with change of solvent, but although the difference can be expressed formally as the influence of the solvent in facilitating non radiational inter- or intra-molecular deactivation processes. Gross interaction with the solvent, as the formation of oxonium ion compounds in strong sulphuric acid solutions or changes in the degree of ionization or of hydrogen bond effects are bound to affect the fluorescence intensity and colour through the changes of molecular structure involved.
In some instances, as for dimethyl-naptheurhodine, the fluorescence band moves to longer wavelength as the solvent changes from liquids of low to high

5 dielectric constant [1]. This could be due to the greater interaction of the solvent molecules with the excited than with the ground fluorescent molecules states, depending on the polarizabilities, and altering the positions of both the excitation and emission bands [1, 5].
1.1.1.4 Inner filter
The inner filter effect also occurs whenever there is a compound present in the sample with an absorption band and the fluorescence intensity will be reduced which overlaps either the excitation or emission band of the fluorescent analyte. It becomes a problem only when the absorption is high or when the concentration of the absorbing species varies from sample to sample. At high concentrations this is caused by absorption due to the fluorophor itself.
1.1.1.5 Quenching
Although the inner filter effect has the results of reducing the intensity of the radiation detection, it is not quenching. True quenching involves the removal of the energy from an excited molecule by another molecule, usually as the results of a collision. The decrease in the fluorescence intensity by the interaction of excited state of the fluorophor with the surroundings is known as quenching and is fortunately relatively rare. Quenching is not random [1, 2, 5]. Each example is indicative of a specific chemical interaction, and the common instances are well known.
Compounds containing unpaired electrons can also act as efficient quenching agents. The most important compound of this type is molecular oxygen. Moreover, quinine fluorescence is quenched by the presence of halide ion despite the fact that the absorption spectrum and extinction coefficient is identical in 0.5 M sulphuric acid
(H
2
SO
4
) and 0.5 M hydrochloric acid [5].

6
Synthesis and characterization of fluorescent particles is currently an important area of research. The synthesis of these fluorescent particles has attracted a great deal of attention for their interesting chemical and physical properties and potential technological applications [6-8].
In recent years, developments of high sensitive and selective sensory materials based on fluorescent materials have been carried out by several researchers.
Some recent developments have used the idea of trapping fluorescence materials in matrices [8]: fluorescence material trapped in matrices can be more stable [9]. Han and co-workers [10] have doped the fluorescent material Rhodamine B (Rh B) into organic-inorganic silica films and their pattering were fabricated by sol-gel process combined with a soft lithography. Besides that, the fluorescence materials were incorporated into porous glasses by diffusion [11]. A thin film dissolved oxygen sensor fabricated by trapping fluorescent material in sol-gel matrix was studied by
Bailey and co-workers [12].
Fluorescence detection offers several advantages over several other methods in terms of its sensitivity and specificity. The integration of fluorescence detection systems has received particular attention due to the large expense and size of current bio-fluorescence detection systems [13].
The possibility of obtaining new particles with enhanced fluorescent properties will be of great interest in the micro-analytical sciences. The new fluorophors will also be widely applicable in the gas sensing applications for industrial use as well as in bioassays. There has been much interest in the use of carbon dioxide and oxygen sensors based on luminescence quenching of organic fluorophors due to their fast response, high sensitivity and specificity [12-15].

7
1.3 Fluorescence Probe
Fluorescent and phosphorescent probes are widely used in applications for detecting biological events. Sensors based on luminescence detection usually result in higher sensitivity than those based on absorption or reflectance. The association with intensity-based systems such as drift in optoelectronic detection of luminescence lifetime, rather than intensity, can overcome many of the problem components.
New fluorescence optical sensing phases for pH measurements have been developed, based on the use of 2´, 7´-dibromo-5-(hydroxymercury) fluorescein
(mercurochrome) as fluorescent pH indicator. Fluorescence emission of mercurochrome changes reversibly with the pH in a relatively wide range of pH.
The pH sensing material has been prepared by trapping this fluorescent dye in a rigid inorganic matrix prepared following the sol–gel technology. The resulting sensing phase showed a strong pH dependence ( λ ex
= 528 nm, λ em
= 549 nm) over 6 pH units, with reversible fluorescent changes [16].
The fluorescence property of fluorescein isothiocynate (FITC) in acidalkaline medium was studied by spectrofluorimetry. A novel pH chemical sensor was prepared based on the relationship between the relative fluorescence intensity of
FITC and pH [17].
1.3.2 Fluorimetric Determination of Gases
In the past, a variety of O
2
gas sensor based on fluorescence quenching have been reported. The quenching of fluorescence of naphthalene in polymethyl methacrylate (PMMA) was studied by oxygen in thin films after displacement of

8 nitrogen atmosphere over the sample by oxygen [18]. A simple fluorescence technique was proposed for the measurement of the diffusion coefficient of oxygen into latex. These latex films were prepared by annealing pyrene (P) labelled polymethyl methacrylate (PMMA) particles above the glass transition temperature.
Diffusion coefficients of oxygen were determined using fluorescence quenching
[18].
Poly (1,4-phenylene diphenylvinylene), p-PDV, a photoluminescent conjugated polymer was synthesized by Mehamod et al . [19] for the detection of oxygen based on the occurrences of the quenching phenomena in the presence of oxygen.
1.3.3 Application of Fluorescence in Polymer
Sorption and drying processes were monitored in situ in polymer by fluorescence rotor probe, 4 tricyanovinyl-[N-(2-hydroxyethyl)-N-ethly]aniline (TCl), a solvatchromatic fluorescence probe, 4-(N,N-dimethylamino) 4’-nitrostibene
(DANS) and pyrene by Ellison and co workers [20]. The probes showed sensitivity to desorption or drying of both water and organic sorbates.
In addition, a method for encapsulation of a fluorescent molecule into silica
“nanobubbles” was reported [21]. Fluorescein isothiocyanate (FITC) dye molecules were coadsorbed onto the surface of gold nanoparticles with 3aminopropyltrimethoxysilane.
1.3.4 Determination of Proteins
Protein analysis continues to be an important area of investigation in the fields of chemical and biochemical analysis. Fukada and co-workers [22, 23] have established a series of new systems for protein determination using

9 chemiluminescence, which detect as little as nanogram amounts of protein.
Erythrosin B (EB) binding to proteins causes a decrease in the fluorescence maximum of EB at 550 nm [24]. Its measurable range was from 1.95 to 1000 ng/ml.
1.3.5 Determination of Metals and Ions
A highly selective and sensitive fluorometric method for the determination of bisphenol has been developed by Yoshida and co-workers [25]. This method is based on an intramolecular excimer-forming fluorescence derivatization with pyrene reagent. There were also many workers [26, 27] using the fluorescence quenching to determine the concentration of heavy metals such as copper (II), ferum, and zinc in industrial waste.
1.4
α -naphthoflavone (7, 8-benzoflavone)
α –naphthoflavone or 7,8-benzoflavone (Figure 1.1) is one of the fluorescent material that has found many uses in analytical chemistry. It is also a natural product although it has been used in a number of biological studies [28-31].
O
O
Figure 1.1: The molecular structure of 7, 8-benzoflavone.
For example, it is an activator of protein as investigated by Stermitz et al
[29]. Besides that, α –naphthoflavone also is an inhibitor of benzopyrene-caused
DNA damage [30] and Zangar and co-workers [31] found that α –naphthoflavone

10 binds to adenosine receptors. α –naphthoflavone has also been used to examine the mechanism of flavone action and further suggested as selective inhibitor to discriminate between human enzymes [32].
1.5 Fluorescein
Fluorescein was the first fluorescent dye used for water tracing work [33] and is still used for qualitative (visual) studies of underground contamination of wells. In recent years, Rhodamine WT has almost completely replaced fluorescein for flow measurements and circulation, dispersion, and plume studies [34]. Nonetheless, fluorescein has a role in such studies, and can be used for masking, hydraulic model studies, and underground studies. The molecular structure is as shown in Figure 1.2.
O O
COOH
Figure 1.2: The molecular structure of fluorescein.
OH
Fluorescein is also used widely in the fluorescence tracing of antibody and rapid diagnosis of some diseases in medical fields [35, 36]. The chemical sensor for pH measurement had been investigated based on the fluorescence property of FITC
[17]. A sensitive pH sensor using phospholipids coating the particles labelled with fluorescein was used for intra-cellular pH measurement in murine macrophages [17].
Fluorescein can be used quantitatively for underground tests, subject to limitations imposed by the higher background of naturally occurring fluorescent materials. An advantage of fluorescein in underground studies is its light sensitivity.
Should it reach an open receiving body of water, the colour will be less of a problem because it will disappear rapidly in the sunlight.

1.6
Luminol
11
Luminol (3-aminophthalhydrazide or 5-amino-2, 3-dihydro- 1,4phthalazinedione) is an organic compound which, when oxidized, emits light and this phenomenon is known as chemiluminescence . This is similar to the reactions that a firefly uses to emit light, and to those used in "glow-sticks" and some roadside emergency lights [37, 38]. The chemiluminescence reaction of luminol with oxidizing agents was first reported in 1928 by Albrecht [38]. Since then, the reaction has been mainly used to determine hydrogen peroxide, other oxidants and metal ions
[39 - 41].
Luminol is prepared by reduction of the nitro derivative (3) formed on thermal dehydration of a mixture of 3-nitrophthalic acid (1) and hydrazine (2). Highboiling triethylene glycol with a boiling point of 290 °C is added to an aqueous solution of the hydrazine salt. The excess water is distilled, and the temperature is raised to a point where dehydration to (3) is completed within a few minutes.
Nitrophthalhydrazide (3) is insoluble in dilute acid but soluble in alkali, by virtue of enolization. It is reduced to luminol by sodium hydrosulfite (sodium dithionite) in alkaline solution [1]. The synthesis scheme is shown Figure 1.3.
O
N
O
OH
OH
O
O
+
NH
2
NH
2
Heat
Triethylene glycol
(1) (2)
NH
2
O
NH
NH luminol
O
Figure 1.3: The synthesis scheme of luminol.
O
N sodium hydrosulfite
O
O
O
(3)
NH
NH

12
The luminol is converted by the basic solution into the resonance-stabilized dianion (1), (Figure 1.4) which is oxidized by hydrogen peroxide into the dicarboxylate ion (2), accompanied by the loss of molecular nitrogen, N
2
. When the molecule (2) is formed, it is in an excited (higher energy) electronic state, and sheds its "extra" energy by emitting a photon of light (h λ ), allowing the molecule to go to its ground state to form (3).
NH
2
O
NH
NH base
O
NH
2
O
N
N
NH
2
O
O
N
N
(1)
O
H
2
O
2
NH
2
O
N
N
O
(2)
*
+ N
2
-h ν
NH
2
O
O
(3)
O
O
Figure 1.4: The reaction of luminol in basic condition.
In aqueous solutions, the luminol oxidation is catalyzed by the presence of a metal ion, such as iron (II) or copper (II). For this reason, luminol can be used in the detection of blood, since it can be activated by the iron in hemoglobin.
Most recently, Yuan and Shiller [42] report a subnanomolar detection limit for H
2
O
2 using luminol chemiluminescence. This method was used to determine hydrogen peroxide content in sea water, based on the cobalt (II) catalytic oxidation of luminol. While cobalt is the most sensitive luminol metal catalyst; it is also present in sea water at very low concentrations. Moreover, luminol was also

13 observed to determine bromide ion in seawater with chemiluminescence reaction
[40].
1.7 Carbon Dioxide (CO
2
)
Carbon dioxide is an essential constituent of tissue fluids and as such should be maintained at an optimum level in the blood. The gas therefore is needed to supplement various anaesthetic and oxygenation mixtures under special circumstances such as cardiac pulmonary by-pass surgery and the management of renal dialysis. It also has a limited place as a respiratory stimulant and is used in the investigation and assessment of chronic respiratory disease.
The carbon dioxide present in the atmosphere is produced by respiration and by combustion. However, it has a short residence time in this phase as it is both consumed by plants during photosynthesis.
Carbon dioxide is a colourless odourless gas and is soluble in water, ethanol and acetone. It has a melting point of -55.6 o C, boiling point at -78.5 o C and density is 1.977 g cm -3 [43].
The CO
2
molecule has a linear shape. This means that the atoms in carbon dioxide are arranged as in Figure 1.5. The green circle represents one atom of carbon and the two grey circles represent oxygen atoms.
Figure 1.5: The structural formula of carbon dioxide.

14
Carbon dioxide is an acidic oxide and reacts with water to give carbonic acid as equation 1.1.
CO
2
(g) + H
2
O(aq) H
2
CO
3
(aq) (1.1)
1.7.1 Preparation of Carbon Dioxide
Carbon dioxide is prepared by treating any metallic carbonate with dilute mineral acids as in equation 1.2
CaCO
3
(s) + 2 HCl(aq) CaCl
2
(s) + H
2
O(aq) + CO
2
(g) (1.2) or by heating carbonates of metals other than alkali metals as in equation 1.3.
MgCO
3
Heat
(s) MgO(s) + CO
2
(g) (1.3)
1.7.2
Uses of Carbon Dioxide
Large quantities of solid carbon dioxide (i.e. in the form of dry ice) are used in processes requiring large scale refrigeration. Carbon dioxide is also used in fire extinguishers as a desirable alternative to water for most fires. It is a constituent of medical gases as it promotes exhalation. It is also used in carbonated drinks [43].

15
1.8 The Determination of Carbon Dioxide
Recently, carbon dioxide level in air has increased considerably due to continuous damage to the environment and the increased use of fossil fuels.
Accordingly, it has become an important task to monitor and control the carbon dioxide which causes both green house effect and possibility of respiratory organ disease. Until now, many works have been carried out to develop solid-state carbon dioxide gas sensors [44, 45].
An optical sensor for the measurement of high levels of carbon dioxide in gas phase has been developed [46]. It is based on fluorescence resonance energy transfer
(FRET) between a long-lifetime ruthenium polypyridyl complex and the pH-active disazo dye Sudan III. The donor luminophore and the acceptor dye are both immobilized in a hydrophobic silica sol–gel/ethyl cellulose hybrid matrix material.
Pt/Na + ion conductive ceramic thin film/Pt/carbonate (Na
2
CO
3
:BaCO
3
=
1:1.7 mol) system CO
2
micro gas sensor was fabricated and the sensing properties were investigated [44].
Fiber-optic carbon dioxide sensors with a dip-coated sol–gel film containing indicator dye of thymol blue were prepared and characterized. The sensitive film has both organic and inorganic parts with good gas permeability. The difference between attenuations in N
2
gas and in CO
2
/N
2
mixture gas increases with the increase in the CO
2
concentration in the 0.55 – 0.7 M range [45].
A thin film sensor for detection of carbon dioxide dissolved in liquids with attention focused on its use for clinical blood gas analysis. Carbon dioxide from the analyte penetrated into a hydrogel electrolyte through a gas permeable membrane and is chemisorbed on a rhodium working electrode. The concentration of CO
2 collected by this way and determined by an amperometric measurement technique based on the inverse voltammetry [47, 48].
The detection of CO
2
is usually based on infrared detection [49] and a
Severinghaus electrode with bulky and expensive devices used. There have been

16 many publications on single optical fibre O
2
sensor based on fluorescent quenching of a dye by molecular O
2
and CO
2
sensors based on the pH modulation accomplishing with the colour change of a dye [50].
1.9
Sol Gel Glass and Sol Gel Process
The sol–gel technique is a low-temperature route widely employed to prepare thin films for use in the different fields, because it can offer homogeneous thin films at molecular scale and control of chemical purity. In recent years, great interest was devoted to the preparation of thin films for optical applications.
Avnir and co-workers [51] demonstrated the possibility and applications of doping a gel with an organic dye. Recent work with silica gel has attracted a great deal of attention because of its potential utility indicated higher stability and better lasting properties than those based on polymer [52-54]. The sol-gel reaction occurs at room temperature, therefore, organic molecules can be incorporated in the gel network with no risk of thermal degradation.
In the sol-gel process, hydrolysis and condensation reaction are highly affected by water content and solution pH [52]. Under acidic conditions, most commonly used for tetraalkyloxysilane (TAOS) sol-gel, hydrolysis is fast relative to condensation and the polymers formed are more open, three-dimensional structures.
Under basic condition, hydrolysis is the rate-limiting step [52-56]. Repulsion between negatively charged particles prevents chain-like linkages and promotes denser, more colloidal gels [52, 57].
Moreover, sol gel technology provides a relatively straightforward way to fabricate glasslike or ceramic material via the hydrolysis and condensation of suitable metal alkoxides. The most popular starting precursors for the fabrication of silica based materials are tetramethoxysilane (TMOS) and tetraethoxysilane (TEOS).
These reagents can be hydrolyzed (equation 1.4) and condensed (equation 1.5 and/or

17
1.6) under relatively mild conditions (Room temperature and pressure) as illustrated in the following simplified reaction sequence for TMOS (1.5).
Si(OCH
3
)
4
+ n H
2
O Si(OCH
3
)
42n
(OH) n
+ nCH
3
OH (1.5)
Si-OH +HO-Si Si-O-Si + H
2
O (1.6)
Si-OCH
3
+ HO-Si Si-O-Si + CH
3
OH (1.7)
In a typical procedure, TMOS is mixed with water in a mutual solvent
(methanol) and catalysts [acid (HCl), base (NH
3
) or nucleophile (F )] is added.
During sol-gel formation, the viscosity of the solution gradually increases when the sol (colloidal suspension of small particles) becomes interconnected to form rigid, porous structure gel [57]. Gelation can take place on a timescale ranging from the seconds to months depending on the proceeding conditions. (Si:H
2
O ratio, type and concentration of catalyst, alkoxide precursors, etc).
During drying, alcohol and water evaporate from the pores, causing the matrix to shrink. Xerogels or fully dried gels are significantly less porous than their hydrated counterparts. To maintain porosity and pore structure, the gel can be supercritically dried to form aerogel. The surface area of these materials often exceed 1000 m 2 /g. The physical properties of the resultants structure, such as average, pore size distribution, pore shape and surface area, strongly depends on the sol-gel process parameters and the method at which the material is prepared and dried [57-60].
The sol-gel process provides a relatively simple way to encapsulate reagents in a stable host matrix. Moreover, sol-gel derived glasses used as host materials provide better optical transparency, stability and permeability than many organic polymers. Protein and enzymes entrapped in silica gel have been used in numerous biological-sensing applications, and sol-gel materials doped with organic and organometallic compounds have been utilized as sensors for gases, metals, ions and pH [52].

18
Today the use of sol-gel to prepare inorganic and organic-inorganic composite materials is blossoming. Compared with commonly used organic polymers, the sol-gel process affords enormous flexibility in terms of the types of materials that can be prepared, the surface can be formed on, and their ion-exchange properties.
1.9.1 Application of Sol Gel in Analytical Chemistry
Nowadays, there are many researchers using the sol gel as a probe for the detection of gases such as carbon dioxide, oxygen and others. Tetraethylorthosilicate
(TEOS) and tetramethylorthosilicate (TMOS) are the common chemicals that are used in the preparation of sol gel [60-65].
1.9.1.1 Sol Gel Encapsulated Fluorescent Materials as Gas Sensors
The sensing of molecular oxygen based on luminescence quenching is regarded as one of the most typical and widespread optosensing application.
As for the matrix, materials such as polymer films, sol gel phases, zeolites and siloxanes have been tested. A good matrix for inclusion of the sensing molecules is chemically inert and optically transparent, possesses photochemical and thermal stability and shows negligible intrinsic fluorescence. Many polymer and sol gel phases largely fulfil these requirements [57, 63]. The porosity of the sensing phase is also important since the quencher should be able to interact with the immobilized luminophore [63, 64].
Oxygen sensors based on this principle have also been extensively studied
[59, 61]. The most common sensor elements studied are those based on an organic or inorganic compound suspended in a thin silicone membrane. Advantages of using an aerogel-based sensor element over other systems include a rapid response time (due

19 to rapid diffusion of gases through the aerogel pore network), and improved resistance to photo-bleaching (as the photoluminescence is caused by stable defect centers in SiO
2
).
The sol-gel coating processes of the Rhodamine B-doped SiO
2
-TiO
2
system, and the influence of the compositions of the SiO
2
-TiO
2
system on the fluorescence properties of Rhodamine B–doped films have been investigated by Hao and coworkers [60].
1.9.1.2 Detection of Proteins
The entrapment of biomolecules in a silica sol gel matrix and their use in chemical sensing applications have blossomed during the past decade. It has been that proteins and enzymes can be entrapped in a random orientation in sol gel derived glasses while maintaining their native properties and relativities [65, 66].
Work by Jordan and co-workers [67] has demonstrated the potential for solgel entrapment of active proteins in an array format. They have optimized alkoxysilane-based sol-gel formulations for protein (keratinocyte growth factor and glucose axidase) stability and antibody (anti-fluorescein) activity, and has recently demonstrated fluorescence based glucose biosensing using a glucose oxidase-based microarray deposited onto a tris(diphenylphenanthroline) ruthenium (II) chloride doped sol-gel film.
Silica xerogel membranes are well suited for encapsulating biomolecules and biosensors have been described using encapsulated enzymes and proteins [16-18, 68,
69]. The glass polymer provides a rigid structure that prevents protein movement and intramolecular interaction, while allowing the biomolecules to retain their activity [16].
Jordan and co-workers [67] used sol gel glass for encapsulated of antifluorescein antibodies, an artificial receptor element, and ribonuclease inhibitor.

20
Many workers [67, 70] used absorption and fluorescence spectroscopies to characterize the properties of bovine serum albumin (BSA) and horse heart myoglobin (Mb) entrapped in sol gel. They observed that a large fraction of BSA entrapped in the sol gel glass was in a native conformation but the reversible conformational transitions were sterically restricted.
1.9.1.3 The Other Uses of Sol gel
Great interest was devoted to the preparation of thin films for optical applications especially elaboration of planar waveguides doped with the active elements such as rare-earth ions. Among the rare-earth ion-doped planar waveguides, Er 3+ doping in different matrix materials prepared by the sol–gel method has attracted much attention [71].
Metal adsorption using porous films can be of great interest for the development of fuel cells or microbatteries. The cluster formation necessary for fuel cells requests the presence of small structures, easy to manipulate, such as microchannels. It has already been shown in the literature that copper can be adsorbed by SiO
2
films obtained by sol–gel process [1].
Conventional methods of enzyme immobilization include physical or chemical adsorption at a solid surface; covalent binding or cross-linking to a matrix, and entrapment within a membrane, surfactant matrix, polymer or microcapsule [1].
Previous work [8, 9, 15] has demonstrated that sol–gel method could be a promising alternative method for the enzyme immobilization. With the combination of the unique features of sol–gel process including high purity and uniformity, low process temperature and easy control on the reaction degree, the sol–gel encapsulation method is supposed to offer several advantages over conventional entrapment method.

21
1.10 Statement of the Problem and the Needs of the Study
The industrial and automobile exhausts are identified as one of the major sources of air pollution [29]. Due to incomplete combustion of fuel, automobiles emit toxic gases such as carbon monoxide (CO), carbon dioxide (CO
2
), and sulphur dioxides (SO
2
) to the environment. Therefore, a study on the method of detection of toxic gases released to the air is needed.
Since many workers used luminescence materials to detect the toxic gases, therefore this study will be carried out to investigate the possible use of fluorescent metal-chelate based luminol, fluorescein and α -naphthoflavone in sol gel matrices for the detection of carbon dioxide, sulphur dioxide and oxygen gases.
α -naphthoflavone was chosen due to lack of previous study on the detection gases. Luminol has been mainly used to determine hydrogen peroxide and metal ions [40, 41] and fluorescein has been used for the detection of carbon dioxide and oxygen [49]. However, the study of fluorescein complexes and luminol complexes for the detection of carbon dioxide, oxygen and sulphur dioxide have not been previously reported.
1.11
Objectives of Research
The research focuses on the study of the effect of carbon dioxide, oxygen and sulphur dioxide on the fluorescence intensity of α -naphthoflavone, fluorescein and luminol.
In this study, the fluorescent properties of metal-chelate based on fluorescent particles in matrices such as sol gel will be investigated using fluorescence spectrophotometer. The physical and chemical properties of these fluorophors also will be observed, at the same time the excitation and emission characteristic and the reaction of gases on the fluorescents materials would be carried out.

22
1.12 The Detection of Gases Using Fluorescent Materials
Initial works were carried out to study the optimum conditions for the fluorescent materials α -naphthoflavone, fluorescein and luminol. Five parameters pH, temperature, effect of solvent, metal-chelate and concentration were firstly optimised. The detection of CO
2
, O
2
and SO
2
were carried out by observing the effect of these gases on the changes in the fluorescent intensity of the fluorescent materials.

CHAPTER 2
2.1 Reagents and Materials
EXPERIMENTAL
All chemicals used were of analytical-reagent grade. The selected fluorescent reagent fluorescein was obtained from BDH Chemical (England); luminol and α naphthoflavone (7,8-benzoflavone) were purchased from Sigma Chemicals (U.S.A).
The metals used in this study include iron (II) chloride from Unilab (Australia) (MW:
198.8 g mol -1 ), magnesium hexahydrate from Fluka Chemical (Switzerland) (MW:
256.41 g mol -1 ), manganese (II) chloride-2-hydrate from Merck (Germany) (MW:
161.88 g mol -1 ), lanthanum nitrate from BDH (England) (MW: 433.02 g mol -1 ) and pure cadmium chloride from Merck (Germany) (MW: 201.32 g mol -1 ). Sol gel silicate precursors tetraethoxysilane (TEOS) was from Fluka Chemika (Switzerland).
Water purified in a Nano Pure Ultrapure water system (Barnstead /Thermolye) was used for all dilution and sample preparation. All chemicals were used without further purification or treatment. All glassware used in this work was washed in 10 percent nitric acid and sterilized before use.

24
2.2 Apparatus
2.2.1
Luminescence Spectrophotometer
Spectrofluorimetric measurements were made using a Perkin-Elmer Model
LS-50B Luminescence Spectrometer (Figure 2.1). All measurements were performed at room temperature using 1 cm x 1 cm quartz cell with software Version
FL Winlab. A Cyber-scan pH meter model equipped with a glass electrode combined with an Ag / AgCl reference electrode, was employed for the pH measurements.
Figure 2.1: The Perkin Elmer LS50B luminescence spectrophotometer.
2.2.1.1 Fluorimetric Analysis
Emission and excitation spectra of all the solutions were obtained using luminescence spectrophotometer. Fluorescence instrument parameters were set up prior to analysis. All the spectra were measured using 1cm x 1cm quartz cell and scanned with a fixed scan speed (250 nm / min). The slit width for emission and excitation spectra was adjusted accordingly in order to obtain good spectrum. The fluorescence excitation spectra were recorded in the range from 200 nm to 800 nm and the fluorescence emission spectrum was scanned with a fixed excitation.

25
During the signal processing for each analyte, the wavelength programming mode is operated. The instrument automatically sets the excitation and emission wavelengths for each analyte and dwell time for specified integration time. The instrument then averages the appropriate number of lamp pulse cycles for the specified integration time. Longer integration time reduces the signal-to-noise ratio for the sample fluorescence intensity.
2.3 Preparation of Standard Solutions
2.3.1 Preparation of 1.0 mol L -1 Tris buffer (tris hydroxymethyl amino methane)
Tris buffer (tris hydroxymethyl amino methane) (6.057 g) was dissolved into a 50 mL volumetric flask using distilled water and the solution made up to the mark.
2.3.2 Preparation of 0.01 M Hydrochloric Acid (HCl)
For the preparation of 0.01 M hydrochloric acid (HCl), 0.041 mL of 12.23 M hydrochloric acid was added by means of micropipette into a 50 mL volumetric flask. The solution was diluted with distilled water, made up to the mark and shaken well.
2.3.3 Preparation of 0.1 M Sodium Hydroxide (NaOH)
Sodium hydroxide (4.00 g) was dissolved in a beaker and transfered to a 1000 mL volumetric flask of distilled water and the solution made up with distilled water to the mark and shaken well.

26
2.3.4 Preparation of Metal Solutions
The metal solutions were prepared by dissolving iron (II) chloride (0.1990 g), magnesium hexahydrate (0.2560 g), manganese (II) chloride-2-hydrate (0.1620 g), lanthanum nitrate (0.4330 g) and pure cadmium chloride (0.2010 g) with 95% ethanol in a 100 mL volumetric flask to produce an individual stock solution of approximately 0.1 mol L -1 . The solution was diluted with ethanol to the mark and shaken well.
2.4 Preparation of Stock Solutions
2.4.1 Preparation of Fluorescent Material Solutions
α -naphthoflavone (7, 8-benzoflavone, 0.0270 g), luminol (0.0177 g) and fluorescein (0.0332 g) each prepared by adding the powder in 100 mL 95% ethanol in a volumetric flask to produce stock solutions of approximately 1 x 10 -3 mol L -1 .
Each stock solution was freshly prepared for experimental work to avoid oxidation and degradation. Appropriate dilution was made for the study on the effect of concentration, temperature, pH and solvent.
2.5 Optimization Studies
The optimization experiment was conducted for the effect of concentration, temperature, pH and solvents were studied.

27
2.5.1 Effect of pHs
The effect of pH for fluorescent materials was optimized. Solution of fluorescent materials in the pH 2-11 range were prepared with 0.1mol L -1 tris buffer
(tris (hydroxymethy) amino methane) and 0.01 mol L -1 hydrochloride acid. A pH meter (Cyber-scan model) was used for pH measurement. After that, the excitation and emission intensity of solution was measured with LS 50B luminescence spectrophotometer. The optimum pH was used in 2.54.
2.5.2 Effect of Temperature
Temperature effect was observed by controlling the temperature of the fluorescence solutions in water bath and ice bath. The intensity of the fluorescence solutions were measured in the temperature range 5 - 70 o C.
2.5.3 Effect of Solvents
Fluorescent materials α -naphthoflavone (7, 8-benzoflavone, 0.027 g), luminol
(0.0177 g) and fluorescein (0.0332 g) were dissolved in 100 mL of N, Ndimethyformamide (DMF) in volumetric flask to produce stock solution of approximately 1 x 10 -3 mol L -1 and its intensity measured with luminescence spectroscopy. This procedure was repeated by dissolving the same mass of fluorescent materials but in different solvent such as deionized water, 0.1 mol L -1 sodium hydroxide and 95% ethanol.

28
2.5.4 Effect of Concentration
The effect of functionalized α -naphthoflavone (7, 8-benzoflavone) concentration on the fluorescence intensity was performed by increasing the concentration from 1 x 10 -5 mol L -1 to 1.0 x 10 -3 mol L -1 at the selected optimum pH.
The emission spectra at λ em
= 368 nm wavelength were measured until the concentration of α -naphthoflavone (7, 8-benzoflavone) solution reached the maximum value. These procedures were repeated for the measurement of fluorescein in the range 1 x 10 -4 mol L -1 until 1 x 10 -3 mol L -1 and luminol in the range 1 x 10 -4 mol L -1 until 1 x 10 -3 mol L -1 at optimum pH.
2.5.5 Standards Calibration Experiments
The suitable solvent for dissolving the fluorescent materials in this study is ethanol at optimum pH. Therefore, a set of calibration solution of α -naphthoflavone
(range 1 x 10 -3 to 1 x 10 -5 mol L -1 ), fluorescein (range 0.5 x 10 -4 to 5.0 x 10 -4 mol L -1 ) and luminol (range 0.5 x 10 -4 to 10.0 x 10 -4 mol L -1 ) was prepared by diluting the appropriate volume into 100 mL of volumetric flask. Dilutions were repeated at optimum pH. The fluorescence intensity of emission wavelengths were measured for each solution
2.5.6 The Effect of Various Metals
The effects of various metals on the fluorescence of α -naphthoflavone were examined in these investigations. The metals used were lanthanum (La), magnesium
(Mg), manganese (Mn), cadmium (Cd) and Ferum (II) (Fe). Lanthanum solution was prepared by adding its powder in a 100 mL volumetric flask to prepare 0.1 M concentration in ethanol. After that, the solution of lanthanum was added in the range 1 mL to 5 mL to the solution of α -naphthoflavone (7, 8-benzoflavone) (5 mL).

29
The light intensity-time decay data were measured immediately after mixing the metals. The other metals were also observed by repeating the same method with fluorescein and luminol.
2.6 Application of Fluorescence Materials for Detection of Carbon Dioxide
2.6.1 Preparation of Sol Gel for Encapsulation of Sensing Material
The basic sol gel was prepared following a method described by Deshpande and Kumar [56]. The doped silica gel matrices were prepared by hydrolysis and polycondensation of tetraethyorthosilicate (TEOS) in water and ethanol solution under acidic condition and stirring for 17 hours until the solution became homogeneous. After that, the solution was cooled at room temperature for 30 minutes. Nitric acid (1 mL) was used as a catalyst. The organic dyes previously dissolved in ethanol will be coated on the glass slides.
2.6.2 Encapsulation of Fluorescent Materials for the Detection of Carbon
Dioxide
Glass slides were cleaned by deionized water followed by immersion in a solution of 10% nitric acid for at least 24 hours. This was followed by thorough washing with ethanol at room temperature. A layer of sol gel was coated on the glass slide. 20 µ L of fluorescent material was dropped on the surface of sol gel and air dried. After that, the carbon dioxide gas was bubbled on the surface of sol gel at volumes ranging from 2 mL to 20 mL. Figure 2.2 shows the process of dissolution of gaseous carbon dioxide on the surface of sol gel encapsulated with fluorescent materials. In this experiment, the gas flow rate was maintained at 1.00 mL / s.
Silica gel was used to ensure the gaseous CO
2
was in dry condition. The fluorescence excitation and emission spectra of the fluorescent materials coated on

30 glass slide was then measured. The glass slide was put on the specific location for the detection of fluorescence materials in solid form.
2.6.3 Encapsulation of Fluorescent Material Complex for The Detection Of
Carbon Dioxide
The same procedures as in 2.6.2 were done for the glass slides followed by a layer of sol gel coated on the glass slide. The fluorescent material-metal complex chosen was dissolved in volumetric flask. Fluorescent material-metal complex (20
µ L) was dropped on the surface of sol gel and allowed to air dried. The gaseous carbon dioxide (2 mL to 20 mL) was dissolved on the surface of sol gel encapsulated fluorescent material-metal and the fluorescence excitation and emission spectra were recorded.
Flow meter
Silica gel
Burette
CO
2
Gas tank slide pump connector
Figure 2.2: The process of bubbling carbon dioxide gas to the surface of sol gel
encapsulating fluorescent materials.

CHAPTER 3
RESULTS AND DISCUSSIONS
3.1 The Fluorescence Study of α –naphthoflavone (ANF)
α -naphthoflavone is a yellow powdery substance with a molecular weight of
272.30 g mol -1 . The IUPAC name for α -naphthoflavone is 7, 8-benzoflavone and the empirical formula is C
19
H
12
O
2
. It has been used to prevent protein inactivation since its first successful application as an aid to solubilization during purification. α naphthoflavone seems to bind to proteins and to block the enzymes inactivation [25,
27]. The fluorescent properties of α –naphthoflavone (ANF) were studied by varying various factors that affects its emission intensity.
α –naphthoflavone was dissolved in different solvents to obtain a suitable solution for further investigating. Early result indicates ethanol to be the most suitable solvent for α –naphthoflavone and giving a strong peak in the visible region.
There were three excitation peaks of α –naphthoflavone in ethanol. But, the excitation wavelength showed the strongest excitation peak at 343.0 nm and the emission peak at 426.0 nm (Figure 3.1).

32
λ ex at 343nm
301.5
250
200
INT
150
λ em at 426nm
100
50
0.0
200.0
300 400 500 600 700 800.5
Figure 3.1: Excitation and emission spectra of α -naphthoflavone in ethanol ( λ ex
343.0 nm).
=
3.1.1 The Effect of pH
The pH of the solution will generally influence the fluorescence intensity.
Figure 3.2 showed that pH does not affect the position of emission spectrum but affects the fluorescence intensity. Optimum pH for α -naphthoflavone is at pH 10.06
(Figure 3.3). This could be due to the formation of different ionizable chemical species by α -naphthoflavone in solution [1, 72].

33
476.4
450 pH 10.06
400
350
300 pH 3.06 pH 2.14 pH 2.03 pH 8.14
250
INT
200
150 pH 6.80
Normal
100
50
0.0
370.0
400 420 440 460 480 500 520 540 560 580 600.5
Figure 3.2: Emission spectra for α –naphthoflavone in ethanol at different pH ( λ ex
343.0 nm, λ em
= 426.0 nm).
=
600
500
400
300
200
100
0
1 3 5 7 pH
9 11
Figure 3.3: Emission intensity for α -naphthoflavone at different pH.
13

34
3.1.2 The Effect of Temperature
Experimental works were carried out at different temperatures to observe the effect of temperature on the fluorescence properties of α –naphthoflavone (Figure
3.4). Results have shown that in some cases fluorescence intensity increases at low temperature (Figure 3.5). It can be seen that the emission peak does not shift as the temperature increases from 5 o C to 70 o C meanwhile the fluorescence intensity of α naphthoflavone decreases gradually with the increasing heat treatment temperature.
However, the shape of the emission spectra of α -naphthoflavone remains unchanged.
The intensity was found to increase by as much as 15 percent per degree
Celsius as the solution temperature decrease from 25 o C to 5 o C. This is probably due to collision quenching. The intensity of the emission decreases as the viscosity of α naphthoflavone increases. Since the viscosity of a liquid increases with decreasing temperature, collision quenching of fluorescence in liquid media becomes less as the temperature is lowered [5].
342.6
300
300
250
200
200 emission
INT
150
100
100
50
5.
o C
10 o C
15 o C
25 o C
55 o C
70 o C
-0.1
370.0
400 450 500 550 600.5
nm
Figure 3.4: Effect of temperature on α -naphthoflavone emission spectra.

35
400
350
300
250
200
150
100
50
0
0 20 40 temperature ( o C)
60 80
Figure 3.5: Effect of temperature ( o C) on emission intensity of α -naphthoflavone.
3.1.3 The Effect of Solvents
α -naphthoflavone does not dissolve in potassium hydroxide and water but dissolves in ethanol and DMF. Figure 3.6 and Figure 3.7 show the fluorescence spectral recording for α -naphthoflavone in DMF. The fluorescence spectra show that the excitation peak was observed at 385.5 nm and the emission peak is at 430.8 nm. The excitation peak was different compared to α -naphthoflavone in ethanol
(Figure 3.1). The location of peak excitation of α -naphthoflavone-ethanol and α naphthoflavone-DMF was about ∆λ ex
= 30 nm. It can also be seen that the emission intensity of α -naphthoflavone in DMF is lower than the emission intensity in ethanol.
The presence of hydrogen bonding in ethanol causes red shifts in the emission wavelength of α -naphthoflavone in DMF. Hydrogen bonding of a

36 fluorescent solute to the solvent can cause significant changes in the emission frequency of the solute [1, 4, 5]. The fluorescence of α -naphthoflavone is quenched by DMF (Figure 3.7). It appears that hydrogen bonding involves the π electron system of the proton donor.
310.0
Non aqueous
250 alfa+N,N-Dimethyforamide ex at 385.05nm
200
INT
150
100
50
0.0
300.0
350 400 450 500 550 600.5
nm
Figure 3.6: The excitation peaks for α -naphthoflavone in N, N-dimethyformamide
(DMF).
Wavelength (nm)
110.0
105
100
95
λ non aqueous em
= 430.83nm alfa + N,N-Dimethyforamide
INT
90
85 em 430.83nm
80
75
70.0
408.0
420 440 460 480 500.5
Figure 3.7: The emission peaks for α -naphthoflavone in N,N-dimethyformamide
(DMF) ( λ em
= 430.83 nm).

37
3.1.4 Standard Calibration Graph of α –naphthoflavone
Figure 3.8 shows the emission spectrum of α -naphthoflavone in the visible region. It was found that the emission intensity of the α -naphthoflavone in ethanol increases with the increase in the concentration from 1 x 10 -3 to 1 x 10 -5 mol L -1 at pH
10.06. No concentration quenching effect was observed in the studied α – naphthoflavone concentration range. The calibration graph showed that the emissions of α –naphthoflavone is a curvilinear curve between 1x10 -3 mol L -1 to
1x10 -5 mol L -1 . (Figure 3.9) and regression equation y = 2.3148 x + 106.96.
Additionally a red shift for the emission band can be observed from low α – naphthoflavone concentration to high concentration. This may be caused by two factors: First it may be due to the changes in the molecules polarity or polarizability.
Secondly: a self-absorption process could also be responsible for the observed small red shift in the emission band [5, 73].
314.7
300 a
250 b
200
INT 150 c
100
50 d e
0.0
370.0
400 420 440 460 480 nm
Wavelength (nm)
500 520 540 560 580.5
Figure 3.8: Emission spectra of α –naphthoflavone in ethanol at different
concentrations, a = 1 x 10
e = 1 x 10 -5 M at λ ex
-3 , b = 5 x 10 -3 , c = 1 x 10 at 343.0 nm (pH 10.06).
-4 , d = 5 x 10 -4 ,

38
400
350
300
250
200
150
100
50
0
0 20 40 60 80 concentration ( x10
-3
) (mol/L)
100 120
Figure 3.9: Standard calibration graph for α –naphthoflavone in ethanol.
3.1.5
The Effect of Various Metals
The formation of highly fluorescent metal chelates by the combination of a metal ion with an organic ligand has provided one of the most specific and highly sensitive methods for determination of many elements. Conversely, the particular effective group in the organic molecule can be identified by chelate formation.
The effects of various metals on the fluorescence of α -naphthoflavone were examined. The metals used were lanthanum (La), magnesium (Mg), manganese
(Mn), cadmium (Cd) and iron (Fe). Iron compounds are essential to all life. Iron atom in hemoglobin is responsible for carrying oxygen around the blood stream. The hydroxide of magnesium (milk of magnesia), chloride, sulphate (Epsom salts) and the citrate are use in medicine. Manganese is important in the utilisation of vitamin
B
1
. Cadmium is a heavy metal that causes diseases and lanthanum is a rare-earth metal that is seldom investigated in previous studies. We choose these metals and

39 hope that there are some fluorescent materials that can complex with these metals and become a gas sensor and can be used in our body in future.
Lanthanum exhibits an enhancing effect on the emission intensity of α naphthoflavone-lanthanum complexes as shown in Figure 3.10. A red shift for the emission band can be observed which could be due to the reaction between the lanthanum and the α -naphthoflavone and formed complexes. Meanwhile, manganese, magnesium and cadmium produce the same emission peaks at 421.0 nm.
As can be seen in Figure 3.11, the emission intensity of α -naphthoflavonelanthanum complex increased with increasing amount of lanthanum. The emission intensity increased by about 70% as the increasing amount of lanthanum was added to α -naphthoflavone. Meanwhile, Arbelov et al.
[73] observed that aggregate formation at high concentration had a negligible effect on the observed emission peak shift which as shown in Figure 3.10.
846.2
800
750
700
650
600 f d e
550
500
INT
450
400
350
300
250
200
150 c b a
100
50
0.0
375.0
400 420 440 460 480 500 520 540 560 580 600 620 640 665.7
Figure 3.10: Effect of lanthanum on the emission spectra of α -naphthoflavone ( a =
1 mL, b = 2 mL, c = 3 mL, d = 5 mL, e = 4 mL, f = 6 mL).

40
In this case, quenching phenomenon does not occur. It could be due to α naphthoflavone-lanthanum complex was in aqueous form. The fluorescence of the lanthanide complex increased through the use of certain ions such as La 3+ . In the presence of these ions, the fluorescence of their respective complexes can be enhanced. The graph of α -naphthoflavone-lanthanum complex is shown in Figure
3.11.
In addition, works on lanthanum (III)-metal complex have been reported by several workers [74]. Rapid formation of phases of variable composition
La
2
(CO
3
) x
(OH)
2(3-x)
has been reported when lanthanum species are in contact with
CO
2
in a humid atmosphere.
800
750
700
650
600
550
500
0 1 2 3 4 volume of lanthanum (mL)
5 6
Figure 3.11: Effect of lanthanum volumne on the emission spectra of α -
naphthoflavone ( λ ex
343.0 nm, λ em
421.0 nm) ii) manganese (Mn)
7
Manganese is one of the cations that formed fluorescent metal chelates, hence complexes of manganese will be used. Figure 3.12 shows the influence of

41 manganese on α -naphthoflavone fluorescence intensity. It was found that the fluorescent intensity of α -naphthoflavone-manganese complex decreases linearly with the increase in the amount of manganese. But, the emission peak was still at λ em
419.5 nm. However, it was found that the emission intensity of manganeseα naphthoflavone complex was (Figure 3.13) different from α -naphthoflavonelanthanum complex (Figure 3.11) which changed non-linearly with the increase in the amount of metals.
As shown in Figure 3.13, emission intensity versus volume of manganese
(mL) changes linearly and giving an equation with y = -32.988 x + 648.71 and R 2 =
0.9413. The emission intensity decreased with the increased of amount of manganese. However, the decrease in emission intensity was less as compared to α naphthoflavone-ferum complex. In previous study, manganese is one of the least effective quenchers. This fact immediately suggests that metal-ion quenching proceeds by formation of an excited-state charge-transfer complex between the latter acting as electron donor [1]. This result was supported by Parker et al.
who found that the interaction of manganese complex also exhibit fluorescence quenching [75].
663.6
600
550 a b
500
450
400
INT
350
300
250
200
150
100 c d e
50
0.0
378.4
400 420 440 460 480 500 520 540 560 580
Figure 3.12: Effect of manganese on the emission spectra of α -naphthoflavone in
ethanol (a = 1 mL, b = 2 mL, c = 3 mL, d = 4 mL, e = 5 mL) ( λ ex
=
343.0 nm, λ em
419.5 nm).
604.3

42
650
600
550
500
450 y = -32.988x + 648.71
R 2 = 0.9413
400
0 1 2 3 volume of manganese (mL)
4 5
Figure 3.13: Effect of manganese volume on the emission spectra of α -
naphthoflavone. iii) cadmium (Cd)
6
It was found that the intensity of cadmiumα -naphthoflavone complex shows the emission peak remains unchanged (Figure 3.14) but a non-linear change as the volume of cadmium increased (Figure 3.15). It was also noticed that some white spots occur when the mixture was left for one day in α -naphthoflavone-cadmium.
This suggests that α -naphthoflavone aggregation occur to a significant extent in the solution. As mention by Gunnlaugsson et al.
[76], the fluorescence response of Cd
(II) anthracene was affected by the concentration of cadmium.

43
759.6
700
650
600
550
500
450 a b e c d
INT
400
350
300
250
200
150
100
50
0.0
378.4
400 420 440 460 480 500 520 540 560 580 605.9
nm
Wavelength (nm)
Figure 3.14: Effect of cadmium on the emission spectra of α -naphthoflavone (a = 1
mL, b = 2 mL, c = 3 mL, d= 4 mL, e = 5 mL) ( λ em
421.6 nm). ex
343.0 nm, λ
280
260
240
220
340
320
300
200
0 1 2 3 volume of cadmium (mL)
4 5
Figure 3.15: Effect of cadmium volume on the emission intensity of α -
naphthoflavone.
6

44 iv) magnesium (Mg)
Magnesiumα -naphthoflavone complex had no significant effect on the fluorescence intensity. The intensity of the emission increased as the first addition of magnesium as shown in Figure 3.16. However, the emission intensity decreased with the increasing amount of magnesium and shows a non-linear change in the emission intensity versus concentration. The emission spectrum was at the same λ em
419.6 nm. But, this was different with the original emission spectrum λ em
426.0 nm.
It could be due to the occurrences of reaction and aggregation between α naphthoflavone and magnesium. As a result, quenching phenomenon occurred.
As suggested by Roshal and co-workers [77] that in the excited state chelating magnesium complexes are more stable than in the ground state. In addition,
Armas et al.
[78] proposed that the complex formation between Mg (II) and 8hydroxyquinoline-5-sulfonic acid (HQS) and the intensity of magnesium complexes was influenced by the concentration of magnesium. Feigl and Heisig [79] showed that the emission intensity of magnesium complexes gave a different change with concentration.
759.6
700
650
600
550
500
450
400
INT
350
300
250
200
150
100 a b e c d
50
0.0
378.4
400 420 440 460 480 500 nm
Wavelength (nm)
520 540 560 580 604.3
Figure 3.16: Effect of magnesium on the emission spectra of α -naphthoflavone ( λ ex
343.0 nm, λ em
5 mL).
419.6 nm) (a = 2 mL, b = 1 mL, c = 3 mL, d = 4 mL, e =

45
750
700
650
600
550
500
450
0 1 2 3 4 volume of magnesium (mL)
5
Figure 3.17: Effect of magnesium volume on the emission spectra of α -
naphthoflavone.
6 v) Iron (II) (Fe)
Iron is the fourth most abundant element in the Earth’s crust, but occurs at extremely low concentrations in the ocean due to the low solubility of the ferum (III) hydroxides.
In this experiment, α -naphthoflavone-ferum (II) complex quenches the emission intensity as the amount of the ferum (II) increased from 1 mL to 6 mL
(Figure 3.18). However, the emission peak shows a small red shift as compared to
α -naphthoflavone in ethanol spectrum.
Among the metal ions selected, ferum (II) quenched the emission intensity of
α -naphthoflavone. Comparing to the other metals, the emission intensity of α naphthoflavone-ferum complex was quenched gradually. However, result showed that a non-constant change in the emission intensity of α -naphthoflavone-magnesium and α -naphthoflavone-cadmium were observed (Figure 3.15 and 3.17).

46
The graph of emission intensity versus concentration of ferum (II) gives a linear equation with y = -82.693 x + 477.32 and R 2 = 0.9676 (Figure 3.19). As the volume of ferum (II) increases, the emission intensity decreases. The results indicate that the emission intensity decreased linearly with increasing amount of ferum (II).
It could be due to the self absorption and concentration quenching. On the other hand, this phenomenon may be due to the formation of aggregates [1, 5, 80] and formation of ferrous.
451.9
400
350 a
300 b
250
INT
200
150 c
100 d e
50 f
0.0
372.5
400 420 440 460 480 500 520 540 560 568.4
nm
Wavelength (nm)
Figure 3.18: Effect of ferum (II) on the emission spectra of α -naphthoflavone ( λ ex
366.0 nm, λ em
421.46 nm) (a = 1 mL, b = 2 mL, c = 3 mL, d = 5 mL,
e = 4 mL, f = 6 mL).
Aodeng and co-workers [81] demonstrated a new spectrofluorimetric method for the determination of trace amounts of iron (II) based on the fluorescence quenching in the formation of Fe (II)-phen complexes. This result was similar with the observation by Cabantchik et al.
[82].

47
300
250
200
150
450
400
350
100
50 y = -82.693x + 477.32
R
2
= 0.9676
0
0 1 2 3 volume of ferum (mL)
4 5
Figure 3.19: Effect of ferum (II) volume on the emission intensity of α -
naphthoflavone ( λ ex
366.0 nm, λ em
421.5 nm)
3.1.6
The Detection of Carbon Dioxide, Oxygen and Sulphur Dioxide
Gaseous by the Fluorescence of α -naphthoflavone
6
3.1.6.1
The Effect of Carbon Dioxide (CO
2
) On The Emission Spectra of α naphthoflavone i) α -naphthoflavone in Ethanol
There have been many publications on single optical fibre sensors based on the fluorescent quenching of a dye by molecular O
2
and CO
2
sensors based on the pH modulation accomplishing with the colour change of a dye [19, 49, 53]. Beside that, fluorescence optical sensing phases for pH measurements have been developed [16,
17].
In this study, carbon dioxide gas was bubbled through into α -naphthoflavoneethanol solution. It was observed that the emission intensity of α -naphthoflavone was affected by CO
2
. The emission intensity showed a non-linear change (Figure

48
3.20) as the amount of CO
2
increased in α -naphthoflavone-ethanol solution.
However, the emission spectrum of α -naphthoflavone-ethanol remains unchanged with λ em
= 421.2 nm. It could be due to the dissolution of CO
2
in α -naphthoflavoneethanol and the reaction of CO
2
and α -naphthoflavone resulting in only about 40% increase in the emission intensity. This can be clearly seen in Figure 3.20. The plot of emission intensity versus carbon dioxide volume (mL) shows a non-linear change.
In previous study, Chang et al.
[83] demonstrated a sensing film for monitoring dissolved CO
2
using fluorescence quenching phenomenon.
800
750
700
650
600
550
500
450
400
0 5 10 15 volume of carbon dioxide (mL)
20
Figure 3.20: Effect of carbon dioxide gases on the fluorescence emission of α -
naphthoflavone in ethanol.
It was also observed that some pH-sensitive dyes change colour upon exposure to gaseous CO
2
. The presence of water is crucial for response to CO
2
at pH
10.06 is based on the hydrolysis of the dyes as shown in the following equations [2]:
CO
2
(g) + H
2
O(aq) H
2
CO
3
(aq) (3.1)
H
2
CO
3
(g) + H
2
O(g) HCO
3
(aq) + H
3
O + (aq) (3.2)

49
HCO
3
- (aq) + H
2
O(aq) CO
3
2(aq) + H
3
O + (aq) (3.3)
DH(aq) + H
2
O(aq) D (aq) + H
3
O + (aq) (3.4)
(colour A) (colour B)
Where DH and D are the protonated and deprotonated forms of the dye, respectively. The change of the colour in dye is sensitive to pH change of its environment. ii) α -naphthoflavone in DMF
A solution of 5x10 -3 M α -naphthoflavone and 200 µL NH
4
OH in DMF was tested for its response to CO
2
. Figure 3.21 shows the emission spectra before and after exposure to gaseous carbon dioxide.
39.0
36
34
32
30
INT
28
26
24 b4 b
DMF+ alfa+ CO2 slit 5
21.0
415.0 420 430 440 450 460 470.5
Figure 3.21: Emission spectra α -naphthoflavone (5 x 10
DMF a) before and b) after response to CO
2
-3 M) and 200 uL NH
4
OH in
.

50
Figure 3.21 showed one emission peak is observed before exposure to CO
2 and two emission peaks were observed in DMF after reaction with CO
2
. The change in the emission spectrum arises from the change in colour of the solution from colourless to yellow. These changes were brought about by the change in the pH of the solution with the dissolution of CO
2
in DMF and can be envisaged in the following reaction [43, 49]:
CO
2
(g) + OH (aq) HCO
3
(g)
H + (aq) + OH (aq) H
2
O(aq)
(3.5)
(3.6)
3.1.6.2 The Effect of Oxygen (O
2 in Ethanol
) on the Emission Spectra of α - naphthoflavone
Molecular oxygen is one of the best-known collisional quenchers. It quenches the fluorescence of a great number of fluorophors. Among these are polycyclic aromatic hydrocarbons, metal-organic complexes of ruthenium, osmium and variety of surface-adsorbed heterocyclic compounds including acridine yellow, rhodamine B, tetraphenylporphyrin and perylene tetracarboxylic acid N-alkylimide [1].
As shown in Figure 3.22, it is noticed that the emission intensity of α naphthoflavone decreases as the volume of oxygen increased. It could be that the α naphthoflavone aggregated as oxide and the aggregation occurred in ethanol.
Meanwhile, there were few studies showing that oxygen causing fluorescence quenching [14, 19]. It was known that the fluorescence of most organic molecules in a polar solvent is strongly quenched by oxygen although the effect of water is usually negligible.

51
160.0
140
120
100
INT
80
60
0.079g
after O2
Before reaction with oxygen
After reaction with oxygen
40 alfa+fe(II)+b4 O2
0.158g
After reaction with more oxygen
20
0.0
378.4
400 420 440 460 480 500 nm
Wavelength (nm)
520 540 560 580 598.4
Figure 3.22: Emission spectra shows 1 x 10 -3 mol L -1
with oxygen gases in ethanol ( λ ex
α -naphthoflavone dissolved
= 343.0 nm, λ em
= 423.21 nm).
Many of these sensors operate on the basis of fluorescence quenching where the target analyte decreases the luminescence of an immobilized indicator dye.
Dynamic quenching is measured and related to analyte concentration through the well-known Stern-Volmer relationship [18]. Molecular oxygen quenches many of these dyes and, as such, represents a serious interference that must be removed before the analytical measurement [17, 18, 59].
Fujiwara and co-workers [84-86] observed the optical sensor for oxygen with pyrene derivatives with carboxyl group. They also found that the oxygen quenched the emission intensity. In addition, there were a lot of researches showing that the intensity of emission was quenched by oxygen [14, 19, 85, 86].

52
3.1.6.3 The Effect of Sulphur Dioxide (SO
2 naphthoflavone in Ethanol
) on the Emission Spectra of α -
There were many analytical methods reported for measuring sulphur dioxide either continuously or discreetly. As we know that sulphur dioxide is a primary air pollutant.
Many chemical sensors are under development for sulphur dioxide with the goal of generating devices capable of real-time, remote monitoring. Examples of chemical sensors include: high-temperature solid electrolyte sensors that are capable of process gas control; sensors based on changes in the dielectric properties of silicone membranes, and gas-sensing potentiometric electrodes with anion-selective internal sensing elements [87-90].
In this experiment, gaseous sulphur dioxide was passed through into 1x10 -3
M α -naphthoflavone in ethanol. The intensity of emission increased dramatically with the addition of sulphur dioxide gases as shown in Figure 3.23. This result indicates that due to the presence of two simultaneous quenching process, dynamic and static occurred in nature with or without a heterogeneous environment for the α naphthoflavone. This result was supported by Razek and co-workers [87].
175
170
165
160
155
150
145
140
1 2 3 4 5 volume of SO
2
(mL)
6 7 8
Figure 3.23: Effect of sulphur dioxide gases on the emission intensity of 1x10 -3 mol
L -1 α -naphthoflavone in ethanol ( λ ex
= 343.0 nm, λ em
= 421.74 nm).

53
3.1.7 Sol gel Immobilized α -naphthoflavone as Fluorescent Carbon Dioxide
Inorganic sol-gel based structures have gained a great importance in the last decade in the field of optical sensors developments due to the advantages they offer.
Sol gel technology provides flexibility in shaping sensors configuration and a simple way of immobilizing organic reagents in porous supports. Moreover, the sol gel resulting matrices exhibit special favourable characteristics for optical sensors, development, including inertness, optical transparency, chemical, photochemical and thermal stability, low temperature preparation and high rigidly, which enhances the luminescence emission of the entrapped dye [52].
Avnir and co-workers [51] demonstrated the possibility and applications of doping a gel with an organic dye. The recent research work with silica gel has attracted a great deal of attention because of its potential utility indicated higher stability and better lasting properties than those based on polymer. The sol-gel reaction occurs at room temperature; therefore, organic molecules can be incorporated in the gel network with no risk of thermal degradation.
The emission of α -naphthoflavone in sol gel was observed and is shown in
Figure 3.24. The emission intensity of α -naphthoflavone coated on the surface of sol gel in this experiment was found to increase linearly after exposure to increasing amount of CO
2
. The linear relationship is shown in Figure 3.25 giving an equation of y = 0.1668 x + 5.9878 and the correlation R 2 = 0.9549. The intensity of α naphthoflavone in sol gel was lower compared to α -naphthoflavone in solution.
Preliminary experiments have shown that fluorescent materials entrapped in sol gel had given a lower intensity [51].
In the sol gel glass, the α -naphthoflavone is entrapped inside the pores of the matrices where it may move freely inside the pores or it may have some interactions with silanol groups on the inner surfaces of pores [13, 16].

54
10.3
9
8
7
6
20 mL CO
2
2 mL CO
2
INT 5
4
3
2
Fluorescein in sol gel
1
0.0
401.0
420 440 460 480 500 520 540 560 580 600 620 640 650.5
nm
Wavelength (nm)
Figure 3.24: Effect of CO
2
exposure on the emission intensity of α -naphthoflavone
in sol gel ( λ ex
= 368.0 nm, λ em
= 471.94 nm).
8
6
4
2
12
10 y = 0.1668x + 5.9878
R 2 = 0.9549
0
0 5 10 15 volume of carbon dioxide (mL)
20
Figure 3.25
: Effect of volume of CO
2 gases on the emission intensity of α -
naphthoflavone encapsulated in sol gel.
25

55
For comparison, the fluorescence emission intensity of α -naphthoflavone in ethanol showed a non linear changed which can be seen in Figure 3.20 meanwhile α naphthoflavone encapsulated in sol gel showed a good linear relationship changes in emission intensity (sol gel). It could be due to α -naphthoflavone forming a stable complex in the presence of carbon dioxide in sol-gel and the entrapped gas in pores surfaces. It can be also seen that the excimer emission of α -naphthoflavone coating has good sensitivity.
3.1.8 Sol Gel Immobilized α -naphthoflavone-ferum and α -naphthoflavonelanthanum as Fluorescent Carbon Dioxide Sensing Material
The α -naphthoflavone-lanthanum complexes were chosen for further study of the fluorescent properties when react with gaseous carbon dioxide. An appropriate amount of carbon dioxide was dissolved into α naphthoflavone-ferum and α -naphthoflavone-lanthanum solution and the emission spectrum measured with a LS-50B luminescence spectrophotometer.
232.9
220
200
180
160 a
INT
140
120
100
80 b
60 c
40
20 d
0.0
359.7
380 400 420 440 460 480 nm
Wavelength (nm)
500 520 540 560 585.1
Figure 3.26: Emission spectra of α -naphthoflavone-ferum complex exposure to
carbon dioxide (a = 6 mL, b = 16 mL, c = 14 mL, d = 10 mL).

56
Results showed that the intensity of emission increased when 2-6 mL carbon dioxide was bubbled into the solution. But, as 8 mL CO
2
was bubbled, the intensity decreased suddenly from 200 units to 50 units, corresponding to around 75% decrease in the amount of intensity. This can be seen in Figure 3.27.
The peak emission of α -naphthoflavone-ferum complex is broad compared to other complexes and the shape of emission spectra was still similar. However, there was a small shift from long wavelength to short wavelength when 6 mL – 16 mL
CO
2
gaseous was added. It is most probably due to the formation of the α naphthoflavone-ferum-CO
2
complex. As previously reported [1, 76, 91], the shift of the wavelength could be due to the interaction between carbon dioxide with α naphthoflavone-ferum complex.
250
200
150
100
50
0
0 2 4 6 8 10 12 volume of carbon dioxide (mL)
14 16 18 20
Figure 3.27: Effect of carbon dioxide volume on the emission intensity of α -
naphthoflavone-ferum complex.
The effect of carbon dioxide on the fluorescent properties of α naphthoflavone-lanthanum complex was also observed. As shown in Figure 3.28, the emission intensity of α -naphthoflavone-lanthanum-CO
2
shows a non linear change. However, the peak emission was still at λ em
421.6 nm. This phenomenon was slightly different from α -naphthoflavone-ferum, which show a slight shift. This trend is observed for all the studied samples. The emission intensity increased

57 suddenly when 10 mL carbon dioxide gaseous was bubbled into the solution meanwhile the emission intensity decreased around 30 % as 14 mL gaseous CO
2 dissolved. This is a non consistent change. Therefore, it is not suitable for the detection of CO
2
.
1100
900
Alfa lanthanum CO2
700
500
0 5 10 15 20 25 volume of carbon dioxide (mL)
Figure 3.28: Effect of carbon dioxide volume on the emission intensity of α -
naphthoflavone-lanthanum complex.
3.2 The Fluorescence Study of Fluorescein
Fluorescein was the first fluorescent dye used for water tracing work [32] and is still used for qualitative (visual) studies of underground contamination of wells. It has a role in such studies, and can be used for masking, hydraulic model studies, and underground studies. It emits a brilliant green fluorescence, which gives an excellent visual. Therefore it is easy to visualize the progress of an experiment.
The fluorescence response for fluorescein in ethanol was recorded with the excitation and emission wavelengths at 483.0 nm and 510.2 nm (Figure 3.29).

58
804.4
INT
750
700
650
600
550
500
450
400
350
300
250
200
150
100
λ ex
= 483.0 nm
λ em
= 510.2 nm
50
0.0
200.0
250 300 350 400 450 500 550 nm
Wavelength (nm)
600 650 700 750 800.0
Figure 3.29: Fluorescence spectra of 1 x 10 -4 M of fluorescein showing excitation
wavelength at 483.0 nm and emission at 510.2 nm.
3.2.1 The Effect of pHs
The effect of ethanol as a solvent on the fluorescent behaviour and the effect of pH on the fluorescent property of fluorescein was also investigated.
As shown in Figure 3.30, it was observed that fluorescent emission of fluorescein in solution is strongly dependent on pH. A blue shift for the emission band can be observed from acidic condition to basic condition. Moreover, the intensity of the emission peak was different for each other (Table 3.1). The lowest intensity was achieved at pH 7.38. However, the optimum pH for fluorescein occurred at pH 8.90. This could be due to the different ionized chemical species formed by fluorescein. According to literature, the existence of fluorescence materials in a definite molecular form depends mainly on solvent polarity and

59 proticity (pH) [1, 5, 17, 49]. In these media hydrogen bonding with the solvent molecules may play an important role in the stabilization of the ionic forms.
However, according to Carvell et al.
[92], Sanchez-Barragan and co-workers [16], the emission intensity of fluorescein is increased from pH 3 to pH 13.
1000.0
900
800
700
600
INT
500
400
300 e d c b
200
100
0.0
a
400.0
420 440 460 480 500 520 540 560 580 600 620 640 660 680 700.0
Figure 3.30: Emission spectra showed the pH effect of fluorescein in ethanol ( λ ex
=
483.0 nm) (a = 7.38, b = 2.64, c = 9.61, d = 8.17, e = 8.90).
Table 3.1 : Emission spectra ( λ pH em
) and intensity of fluorescein at different pH
λ em
Intensity
2.64 511.06 172.07
7.38 522.12 155.31
8.17 525.53 549.36
8.90 529.36 756.05
9.61 531.06 330.19

60
3.2.2 The Effect of Solvents
Initial study was undertaken to determine the best solvent for fluorescein optimum linear range and sensitivity. The effects of various solvents such as deionized water, ethanol, DMF and sodium hydroxide (NaOH) were investigated.
The result of the experiments for DMF are shown in Figure 3.31. The intensity of fluorescein in DMF is lower compared with fluorescein in ethanol
(Figure 3.29). It was noticed that fluorescein change colour from orange to pink in
DMF. It was assumed that the intermolecular interaction between the solvent and solute often produces a tangible effect on the absorption-emission properties of luminophors. This effect may be caused by both usual and special interactions between the solvent and solute [1, 2, 5]. In additional, the dipole moments and permittivities of solvents, the size of the solute molecules, and the difference between the dipole moments in the ground and excited states also can affect the emission intensity.
In deionized water and sodium hydroxide, the emission intensity was higher than other solvents. It could be due to the present of some metals in deionized water and sodium hydroxide. Fluorescein is very sensitive to the metals. Therefore, ethanol was found to be the most suitable solvent or diluent for this purpose. Thus, it was used exclusively throughout this work.

61
1000.0
INT
800
600
400
200
λ ex= 475.0nm
0.0
λ em= 547.89 nm
200.0
300 400 500 600 700
Figure 3.31: Emission spectra of fluorescein in DMF with λ em
at 547.89 nm
( λ ex
at 475.0 nm).
800.5
3.2.3 Standard Calibration Graph of Fluorescein
Figure 3.32 shows the emission spectra of fluorescein in the visible region when dissolved in ethanol. It was found that the emission intensity of fluorescein increased with the increase in the concentration from 0.5 x 10 -4 to 5.0 x 10 -4 mol L -1 with regression equation y = 152.36 x – 64.579 and R 2 value 0.9425. The absorption spectra do not show any significant change in the spectra shape, but the intensity decreased at certain concentrations (10.0 x 10 -4 M) in Figure 3.33. This indicated a quenching phenomenon [1, 2, 5]. Fluorescence quenching is a process, which decreases the intensity of fluorescence emission. Quenching may occur by several mechanisms such as collisional or dynamic quenching, static quenching, quenching by energy transfer and change transfer reactions.
As suggested by Bowen and Wokes [5], the self-quenching of dye molecules in aqueous solution is much more complicated. In addition, to collisional deactivation, changes in molecular complexity occur. At high concentrations,

62 dimeric and sometimes polymeric molecules are formed in the dark whose presence can be recognized by changes in the absorption spectrum.
480.0
450
400
10.0 x 10 -4 M
5.0 x 10 -4 M
350
300
2.0 x 10 -4 M
INT
250
200
1.0 x 10 -4 M
150
100
50
0.5 x 10 -4 M
0.0
400.0
420 440 460 480 500 520 540 560 580 600 620 640 660
Figure 3.32: The emission spectra of fluorescein at different concentration
between 0.5 x 10 -4 M to 10.0 x 10 -4 M.
680 700.0
800
700
600
500
400
300
200
100
0 y = 152.36x - 64.579
R
2
= 0.9425
0.5
1 2 concentration (x10
-4
) (mol L
-1
)
5 10
Figure 3.33: The standard curve showing the intensity of emissions of fluorescein
between 0.5 x 10 -4 M to 10.0 x 10 -4 M concentration.

63
The dimer of fluorescein is either non-fluorescent or fluorescent with low efficiency. The cause of the change of molecular complexity is not normal electrical dipoles associations, since it occurs far more readily in aqueous than in alcoholic solutions. It must either be due to hydrogen bonding by intercalated water molecules or to strong polarization forces [5].
3.2.4 The Effect of Various Metals
Metal xanthate complexes and their reaction products have been extensively studied. The soluble alkali metal xanthanes are widely used in extraction and separation of Hg, Ag, Cd etc [1-3].
The effects of various metals such as lanthanum (La), magnesium (Mg), manganese (Mn), cadmium (Cd) and iron (Fe) on the fluorescence of fluorescein observed in these investigations were similar to α -naphthoflavone. It was noticed that manganese, ferum and magnesium exhibit enhances emission intensity of fluorescein-metal complex. Meanwhile, cadmium and lanthanum shows a non-linear change in the emission intensity. The results are different compared with α naphthoflavone.
Figure 3.34 shows the emission spectra of fluorescein-manganese complex.
The increasing amount of manganese decreased the intensity of fluorescein with increasing the volume of metal in ethanol. This indicated a quenching phenomenon
[1, 5, 80]. The emission spectra do not show any significant change in the shape of the spectra.

64
70.8
65 a
60
55
50 b
45
40 c
INT
35 d
Mn
30 e
25
20
15
10
5
0.0
491.3500
520 540 560 580 600 nm
Wavelength (nm)
620 640 660 680 702.9
Figure 3.34
: Effect of manganese on the emission spectra of fluorescein (a = 1mL,
b = 2 mL, c = 3 mL, d = 4 mL, e = 5 mL, f = 6 mL).
70
65
60
55
50
45
40
35
30
25
20 y = -7.861x + 69.227
R
2
= 0.9613
0 1 2 3 4 volume of manganese (mL)
5
Figure 3.35: Effect of manganese volume on the emission spectra of fluorescein
( λ ex
475.0 nm, λ em
512.04 nm).
6

65
According to Lopez and et al.
[5], the fluorescence quenching of both molecular forms were separately studied in different solvents. In addition, it appears that the hydroxyl group of ethanol molecules cause disaggregation of the dye [94].
The graph of intensity of emission versus volume of manganese (mL) (Figure 3.35) shows a negative straight line with additional amount of fluorescein. The equation of the line is given by y = -7.861 x + 69.227. A good linear correlation still between the emission intensity and the quantities of manganese added with R 2 = 0.9613. ii) magnesium (Mg)
Strong fluorescence quenching is observed when the fluorescein complex with magnesium excited at 483.0 nm, with emission maximum of 510.12 nm. The results in Figure 3.36 show that magnesium gives a surprisingly strong quenching effect from blue to red wavelengths. The reduction in emission intensity in the presence of magnesium could be due to some collision formed by addition of the amount of magnesium. In addition, the molecules have the tendency to conjugate or coagulate [5, 77, 80].
It was noticed that the emission intensity of fluorescein-magnesium complex decreased with increasing the amount of magnesium and giving a straight line with y
= - 4.654 x + 53.678 and R 2 = 0.9509 (Figure 3.37). The current result is in agreement with results from Bos and co-workers [95] who have demonstrated that the fluorescence of the fluorescent complex decreased in the presence of magnesium.

66
51.3
45
40
35
30
INT
25 a b c d mg e
20
15
10
5
0.0
492.0 500 520 540 560 580 600 620 640 652.9
Figure 3.36: Effect of magnesium on the emission spectra of fluorescein in ethanol
( λ ex
475.0 nm, λ em
510.12 nm) (a = 1 mL, b = 2 mL, c = 3 mL, d = 4
mL, e = 5 mL, f = 6 mL).
55
50
45
40
35
30
25 y = -4.654x + 53.678
R
2
= 0.9509
20
0 1 2 3 volume of magnesium (mL)
4 5 6
Figure 3.37: Effect of magnesium volume on the emission spectra of fluorescein.

67 iii) iron (II) (Fe)
The effect of addition of ferum (II) or iron on the intensity of fluorescein was investigated by recording the emission intensity. Ferum leads to a surprisingly strong quenching effect from blue to red wavelengths. It was noticed that the intensity decrease with the increase in the amount of ferum. A curvilinear line was obtained as shown in Figure 3.38.
The reduction in emission intensity in the presence of ferum is due to fluorescence quenching. This process however proceeds via the formation of aggregation which in the presence of large excess of ferum probably forms ferric, thus reducing the emission intensity in ethanol medium [1, 5].
Moreover, the quenching molecule does not merely stimulate a change in the energy levels of the fluorescent molecule (fluorescein) but take part in a chemical reaction like methylene blue and ferrous ion [1, 80]. An oxidation-reduction reaction may occur by transfer of an electron from one molecule to the other.
55
50
45
40
35
30
25
0 1 2 3 volume of ferum (mL)
4 5
Figure 3.38: Effect of different volume of ferum (II) on the emission spectra of
fluorescein.
6

68
The light absorption in equation (3.7) implies the jump of an electron from D, fluorescein molecule, from one orbital to another higher energy. The reverse process in equation (3.8) gives fluorescence. Ferrous ion removes excited D molecules given by equation (3.9) in which an electron from the Fe 2+ fills the vacated orbital in D* and block the fluorescence process [1].
D + hv D* (3.7)
D* D + hv’ (3.8)
D* + Fe 2+ D + Fe 3+ (3.9) iv) cadmium (Cd) and lanthanum (La)
Cadmium and lanthanum effect on the fluorescence of fluorescein complexes also have been studied. It was found that the intensity of fluorescein-lanthanum complexes shows a non-linear change in the intensity (Figure 3.39). This is assumed that fluorescein aggregation occur to a significant extent in the solution. In addition, the complexes molecules have the tendency to conjugate or coagulate at certain solution [5].
The absorption spectra of fluorescein-cadmium complexes do not show any significant change in the shape of spectra, but the intensity decreased slightly with increasing volume of cadmium in fluorescein-cadmium complex as shown in Figure
3.40. Cadmium (II) atom is an octahedral environment surrounded by two chelating xanthate anions and two fluorescein ligands. Two nitrogen atoms occupy the apical site. Cadmium xanthate metal was demonstrated to have a similar nonlinear optical property [96].

69
125
120
115
110
105
100
95
90
1 2 3 volume of lanthanum (mL)
4 5
Figure 3.39
: Effect of lanthanum volume on the emission spectra of fluorescein ( λ ex
= 475.0 nm, λ em
= 502.94 nm).
50
45
40
35
30
25
20
0 1 2 3 volume of cadmium (mL)
4 5 6
Figure 3.40
: Effect of cadmium volume on the emission spectra of fluorescein ( λ ex
= 475.0 nm, λ em
= 517.5 nm).

70
3.2.5
The Detection of Gaseous Carbon Dioxide, Oxygen and Sulphur Dioxide of Fluorescein
3.2.5.1
The Effect of Carbon Dioxide (CO
2
) on the Emission Spectra of
Fluorescein i) Fluorescein in Ethanol
The control of carbon dioxide concentration is important in many applications. Non-expensive and robust detection systems are required for air quality, food control and for early fire detection. Medical diagnosis and treatment of critically ill patients in intensive care units and operating theatres often requires monitoring of CO
2
and O
2
partial pressures of arterial blood. To date, optical and electrochemical sensors have been used, but the high cost of the former and the unreliability of the latter present serious disadvantages.
In these studies, a solution of 1 x 10 -4 M fluorescein and tris buffer (1 mL) was tested for its response to CO
2
(Figure 3.42). It is assumed that fluorescein (FL) exists only as FL 2 ions in alkaline solvent. Neutral form of fluorescein can occurs in three different tautomers, i.e. zwitterions, quinoid and lactone. However, it has also been reported colourless lactonic form is usually the dominant tautomer present in organic solvents [93].
A plot of fluorescence intensity ( λ em
= 516.0 nm) against concentration of
CO
2
is shown in Figure 3.42. We noticed that the intensity of fluorescein decreased with the increase in dissolved CO
2
in ethanol. This is due to the presence of water in ethanol which is crucial for response to CO
2
and is based on the hydrolysis of the dyes as shown in the following equations and phenomenon quenching happened [49]:
CO
2
(g) + H
2
O(aq) H
2
CO
3
(aq) (3.10)
H
2
CO
3
(aq) + H
2
O(aq) HCO
3
(aq) + H
3
O + (aq) (3.11)
HCO
3
(aq) + H
2
O(aq) CO
3
2(aq) + H
3
O + (aq) (3.12)

71
DH(aq) + H
2
O(aq) D (aq) + H
3
O + (aq) (3.13)
(colour A) (colour B)
423.1
400 a b
350 d c e
300 f
INT
250
200
150
100
50
0.0
491.3
500 510 520 530 540 550 560 570 580 590 600 610 620 630 640.0
nm
Wavelength (nm)
Figure 3.41
: Emission spectra of 1 x 10 -4 M fluorescein + tris buffer (1ml) dissolved
CO
2
gas. (a = 2 mL, b = 6 mL, c = 4 mL, d = 20 mL, e = 12 mL, f = 16
mL).
410
390
370
350
330
310
290
270
0 5 10 volume of carbon dioxide (mL)
15 20
Figure 3.42
: Graph shows the effect carbon dioxide on the emission intensity of
1 x 10 -4 M fluorescein in ethanol ( λ ex
475.0 nm, λ em
516.0 nm).

72 ii) Fluorescein in N, N-dimethyformamide (DMF)
DMF is a hydrophilic compound with propensity to absorb water vapour. As can be seen in Figure 3.43, it was noticed that the emission intensity of fluorescein in
DMF decreased with increasing volume of carbon dioxide. A nice curve was produced when emission intensity of fluorescein versus volume of carbon dioxide
(mL) (Figure 3.44) was plotted. These changes were brought about from the change of the pH (pH 8.90) of solution with the dissolved of CO
2
in DMF and can be envisaged in the following reactions [49]:
CO
2
(g) + OH (aq) HCO
3
(aq) (3.14)
H + (aq) + OH (aq) H
2
O(aq) (3.15)
2H + (aq) + FL 2(aq) H
2
FL(aq) (3.16)
Dissolved CO
2
removed the OH ions the ethanol. As the results, the H + ion concentration is increased which shifts the equilibrium of equation (3.16) from left to right.
Chattopadhyay and co-workers [97] observed that the flow rate of the carbon dioxide gas affect the reaction between fluorescent materials and carbon dioxide gases. They also mentioned that for a given analyte concentration, the slow flow rate offers the advantage of increased sensitivity whereas the higher flow rate offers larger dynamic ranges. In other work, Ahmad and Narayanaswamy [59] reported that higher flow rate offer higher reaction rate, shortened response time but lower maximum reflectance difference, whereas lower flow rate gave the opposite effect.
In this experiment, the gas flow rate was not optimized and maintained at 1.00 mL / s.

73
432.4
400
350
300 a b
INT
250 c d
200 h e
150 g f
100 i
50
0.0
485.4
500 520 540 560 580 600 620 640 660 680 699.4
nm
Wavelength (nm)
Figure 3.43
: Emission spectra of fluorescein in DMF in alkaline condition after
exposure to CO
2
(a = 1 mL, b = 2 mL, c = 3 mL, d = 4 mL, e = 5 mL,
f = 6 mL, g = 7 mL, h = 12 mL, i = 13 mL).
450
400
350
300
250
200
150
100
50
0
0 2 4 6 8 volume of CO
2
(mL)
10 12 14
Figure 3.44
: Graph of 1 x 10
gas ( λ ex
-4 M fluorescein in DMF dissolved with carbon dioxide
475.0 nm , λ em
546.1 nm).

74
3.2.5.2 The Effect of Oxygen (O
2
) Gases on the Emission Spectra of Fluorescein
The fluorescence intensity of fluorescein in ethanol decreased as the
576.6
550
500
450
400
350
INT
300
250
200
150
100
50 dissolved volume of oxygen increases (Figure 3.45) at λ em
515.01 nm. The quenching phenomenon occurred as oxygen dissolved in ethanol from 1 – 4 mL
(Figure 3.46). It can be attributed to several reasons. Firstly, it could be due to aggregation of fluorescein-oxide at higher oxygen concentration. The addition of oxygen into the solvent fluorescein-ethanol formed non-fluorescent compound. Basu et al.
[98] also found that emission intensity of fluorescent material effectively quenched by oxygen. As can be seen in the result, fluorescein is not suitable as an oxygen sensor. d = 4 mL c = 1 mL a = 2 mL b = 3 mL
0.0
479.4
490 500 510 520 530 540 550 560 nm
Wavelength (nm)
Figure 3.45: Emission spectra of 1x10 -4
gaseous oxygen ( λ ex
570 580 590 600 610 620
M fluorescein in ethanol bubbled with
475.0 nm, λ em
516.0 nm).
634.0

75
600
500
400
300
200
100
0
1 2 3 volume of oxygen (mL)
4
Figure 3.46
: Graph of 1x10 -4 M fluorescein in ethanol bubbled with gaseous oxygen
( λ ex
475.0 nm , λ em
516.1 nm).
3.2.5.3 The Effect of Sulphur Dioxide (SO
2
) on the Emission Spectra of
Fluorescein
Sulphur dioxide was bubbled in 1x10 -4 M fluorescein in ethanol. It was found that the emission intensity of fluorescein-ethanol decrease as SO
2
gases increase. However, the emission spectrum remains unchanged at 510.53 nm (Figure
3.47). But, the intensity increased 10 % after 1 day. The emission intensity of α naphthoflavone-ethanol increased when reacted with SO
2
compared with fluoresceinethanol. This is most probably due to the reaction between 1 M hydrochloric acid and ferum sulphate was slow to form SO
2
gas and the increased of sulphur dioxide in solution formed a non fluorescent compound. Aggregation could occur in fluorescein-ethanol solution.

76
360.0
300 a b
INT
250
200 d c
150
100
50
0.0
485.4
500 510 520 530 540 550 nm
560 570 580 590 600 610.2
Wavelength (nm)
Figure 3.47: Emission spectra shows fluorescein 1x10 -4 M dissolved with sulphur
dioxide gas in ethanol (
dissolved SO
2 ,
λ ex
= 475.0 nm, λ em
= 510.53 nm) (a = before b = 1 minute after dissolves SO
2
gases, c = 1 day
after dissolves SO
2
gases, d = 2 day after dissolves SO
2
gases.
3.2.6
Sol Gel Immobilized Fluorescein as Fluorescent Carbon Dioxide Sensing
Material
The sol gel process is attractive for making porous membranes for optical sensors because of the relative simple chemistry and because of the low polymerisation temperature which allows encapsulated or attachment of organic probe molecules that are unstable at higher temperature [57]. A silica gel is made by hydrolysis of an alkoxide precursor followed by condensation of silanol in the sol-gel process. Evaporation of the solvent and the alcohol liberated during hydrolysis forms a dried gel or xerogel. On the other hand, the sol gel glass has large porosity
(about 30 %) and very large specific surface area (>300 cm 2 /g) [57]. Therefore, a substantial fraction of the entrapped reagents (fluorescein) may be exposed to a neighbouring phase and intrapore volume and they can react with the analytes there in.

77
Carbon dioxide was dissolved into fluorescein in ethanol to observe the effect of the fluorescence property of fluorescein encapsulated in sol gel. As shown in
Figure 3.48, the emission intensity of fluorescein decreased dramatically as it is exposed to 2 mL of dissolved CO
2
. However, the emission intensity of fluorescein remained at 1.55 units with the continuous dissolution of CO
2
gas. Quenching phenomenon occurred due to fluorescein forming carbonate on the surface of sol gel.
Published reports [56, 57] related to photo physical properties of fluorescein in SiO
2
sol gel matrices show the existence of dimers. The reason for fluorescein dimer formation has been associated to water content in SiO
2
sol gel matrices. On the other hand, decreases in polarity from sol to xerogel increases in acid during fluorescein doped SiO
2
sol gel preparation.
4
3.5
3
2.5
2
1.5
1
0.5
0
0 2 4 6 8 volume of CO
2
(mL)
10 12 14
Figure 3.48: Effect of carbon dioxide gases in sol gel on the emission intensity of
1 x 10 -4 = 388.0 nm, λ em
= 518.56
nm).
mol L -1 fluorescein in ethanol ( λ ex

78
3.2.7 Sol Gel Immobilized Complex Fluorescein-manganese and Fluoresceinferum as Fluorescent Carbon Dioxide Sensing Material
The fluorescein-manganese and fluorescein-ferum complexes solutions were encapsulated in sol gel and the properties of fluorescence were observed in the following experiments. The different amount gases carbon dioxide (2 - 20 mL) was dissolved into the complex solution and the emission intensity was measured.
Figure 3.49 shows the emission spectra of fluorescein-manganese complex encapsulated in sol gel after reacted with carbon dioxide. It was found that the emission wavelength of fluorescein-manganese-CO
2
was found to be at 482.58 nm while excitation wavelength was at 475.0 nm with a slit width of 10 nm. The intensity of the emission decreased with the increase of the amount of carbon dioxide.
However, the emission peaks remain unchanged.
666.9
600
550
500
450
400
350
INT
300
250
200
150
100
50
0.0
450.0
a b c d e
460 470 480 490 500 510 520 531.5
Figure 3.49: Emission spectra show fluorescein-manganese complexes which
dissolved carbon dioxide gases in sol gel ( λ ex
= 475.0 nm, λ em
=
482.52 nm) (a = 2 mL, b = 4 mL, c = 6 mL, d = 16 mL, e = 10 mL).

79
The plot of emission intensity versus the volume of carbon dioxide (mL) is shown in Figure 3.50. The intensity of emission decreased as the carbon dioxide gases dissolved in the first 2-10 mL. This could be due to the addition of carbon dioxide in fluorescein-manganese complex solution caused aggregation in sol gel and the phenomenon of quenching occurred. However, this phenomenon changes and the emission intensity increased again when 12 mL of gases carbon dioxide was dissolved. This could be most probably due to the diffusion of carbon dioxide through the aerogel pore network and the entrapped carbon dioxide gases achieved an optimum condition and exhibit fluorescence in fluorescein-manganese-CO
2
form
[56, 57].
600
500
400
300
200
100
0
0 5 10 15 volume of carbon dioxide (mL)
20 25
Figure 3.50: Effect of carbon dioxide on the emission intensity of fluorescein-
manganese complex encapsulated in sol gel.
As shown in Figure 3.51, it was found that the bubbling of carbon dioxide in the fluorescein-ferum complex produce a linear relationship in the emission intensity as the amount of carbon dioxide is increased. The equation was y = -7.2252 x +
271.01 with a correlation of 0.9234. It could be due to fluorescein-ferum complex reacting with carbon dioxide and fluorescein-ferum-CO
2
formed. The amount of
CO
2
will influence the formation of carbonate and affects the fluorescence probably of fluorescein-ferum complexes.

80
300
250
200
150
100
50 y = -7.2252x + 271.01
R 2 = 0.9234
0
0 5 10 15 volume of carbon dioxide (mL)
20
Figure 3.51: Effect of carbon dioxide on the emission intensity of fluorescein-
ferum complex encapsulated in sol gel.
3.3 The Fluorescence Study of Luminol
Luminol is also widely used as a chemiluminescent reagent [37, 39, 40]. It is soluble in ethanol, sodium hydroxide but is absolutely insoluble in water. The fluorescence spectrum of luminol in ethanol is shown in Figure 3.52. The maximum emission was observed at wavelength of 421.2 nm when the luminol solution is excited at wavelength of 386.0 nm. Luminol exhibits strong fluorescence band in the visible spectral region.

81
1000.1
INT
900
800
700
600
500
400
λ ex
= 386.0 nm
300
200
λ em
= 421.2nm
100
0.0
200.0
250 300 350 400 450 500 nm
550 600 650 700 750 802.9
Wavelength (nm)
Figure 3.52: Fluorescence spectra of 1x10 -4
421.2 nm).
M luminol ( λ ex
= 386.0 nm and λ em
=
3.3.1 The Effect of pHs
The effect of pH on the fluorescence intensity was studied using tris buffer and sodium hydroxide solution. As shown in Figure 3.53, the maximum relative fluorescence intensity could be reached at pH 8.62. But, it was noticed that the fluorescence intensity decreased by about 75 % in the pH range 8.62 to 11.93. The emission intensity of luminol in basic conditions showed a non linear change.
However, the location of the emission peak was still at 412.0 nm and the shape of emission peak remains unchanged. This was different with the pH effect of fluorescein as shown in Figure 3.54. It could be due to the deionization of luminol and aggregation occurred in the basic condition. Thus, pH 8.62 was considered to be optimum. According to Zhou and co-workers [41], the fluorescent properties of luminol can only be produced in alkaline media and this corroborated with the results.
From the experiment, the luminescence of luminol was achieved and stabilized in the

82 pH range of 7.62 to 8.62. Therefore, the pH of the test samples should be adjusted in this pH range.
449.2
400 a
INT
350
300
250
200
150
100
50 c d b e f g
0.0
396.0
420 440 460 480 500 520 540 560 580 600 620 634.0
Figure 3.53: Emission spectra ( λ em
= 419.52 nm) showing the pH effect of luminol in ethanol ( λ ex
= 386.0 nm) (a = pH 8.62, b = pH 4.71, c = pH 2.3, d
= pH 9.00, e = pH 9.27, f = pH 10.07, g = pH 11.17).
450
400
350
300
250
200
150
100
50
0
2.3
4.71
5.87
6.06
7.61
8.62
9 9.27
10.07 11.17 11.93
pH value
Figure 3.54: Emission intensity of luminol at different pH.

83
3.3.1.2 The Effect of Temperature
Luminol is a kind of fluorescent material, whose optical properties depends on many factors, such as solvents (polarity and aprotic character), concentration, pH value, etc. [1, 5, 39, 40]. With the increase of the densification temperature, luminol will decompose at even higher temperature. Thus, it is understanding that the emission intensity of luminol decreased with the increased of temperature.
The effect of temperature on the determination of luminol intensity was investigated from 5 – 45 o C (Figure 3.55). Increase of temperature within the range of 20 o C – 45 o C markedly decrease the peak intensity of luminol (Figure 3.56). At higher temperature, evaporation of ethanol may cause error to the analysis.
Therefore, a temperature of 20 o C – 25 o C (room temperature) is suitable as it gives sufficient sensitivity as well as keeping evaporation of ethanol to a minimum. The shape of the emission spectra remains unchanged. The emission intensity decreases with the increase of temperature.
632.8
INT
600
550
500
450
400
350
300
250
200
150
10 o C
20 o C
25 o C
30 o C
45 o C
100
50
0.0
394.3
420 440 460 480 500 520 540 560
Figure 3.55: The fluorescence emission spectrum of luminol at different
temperatures.
580 600.5

600
550
500
450
400
84
350
5 10 15 20 25 temperature ( o C)
30 35 40 45
Figure 3.56: Effect of temperature on luminol intensity.
3.3.3 The Effect of Solvents
Luminol dissolved into ethanol, DMF, sodium hydroxide and deionized water was used to observe the effect of different solvents on emission intensity. It was found that the emission intensities and the emission wavelengths were different for each solvent. Luminol dissolved in ethanol give the highest emission intensity as shown in Figure 3.57. However, the lowest emission intensity was achieved in sodium hydroxide (NaOH) solution. There was a small wavelength shift between luminol-ethanol and luminol-DMF. It could be due to the specific nature of solutesolvent interaction and hydrogen bonding between luminol and ethanol. Therefore, ethanol was chosen for further experiment.

85
1044.5
1000
900
800 Ethanol
700
600
INT 500
400
300
200
DMF
Water
100
0.0
300.0
350 400
NaOH
450 500 550 600 660.4
Figure 3.57: Emission spectra of luminol in ethanol, DMF, sodium hydroxide and
deionized water.
3.3.4 Standard Calibration Graph of luminol in Ethanol
A concentration dependent change in fluorescence spectra was observed. The fluorescence spectra of the highest concentration differed strongly from that recorded at the lowest concentration. The effect of the concentration of luminol solution was evaluated from 0.5 x 10 -4 mol L -1 to 10.0 x 10 -4 mol L -1 and the results are shown in
Figure 3.58. The fluorescence intensity of luminol in ethanol increased with the increased of concentration. There is no quenching phenomenon effect observed in the studied luminol concentration between 0.5 x 10 -4 mol L -1 to 10.0 x 10 -4 mol L -1 range. As can be seen in Figure 3.59, the calibration graph of emission intensity versus concentration of luminol (mol L -1 ) showed a curvilinear relationship with increasing the concentration.

528.9
500
450
400
350
300
INT 250
200
150
100
50
0.0
394.0
10.0 x 10 -4
5.0 x 10 -4
2.5 x 10 -4
1.0 x 10 -4
0.5 x 10 -4
420 440 460 480 500 520 540 560 580 600 616.2
Figure 3.58: The emission spectra of luminol at different concentrations
between 0.5 x 10 -4 mol L -1 to 10.0 x 10 -4 mol L -1 .
600
500
86
400
300
200
100
0
0 2 4 6 8 10 12
10 -4 mol L -1 concentration (x10 -4 ) (mol L -1 )
Figure 3.59: The standard calibration curve of luminol (0.5 x 10 -4
.
mol L -1 to 10.0 x

87
3.3.5 The Effect of Various Metal Solutions
The effects of various metals lanthanum (La), magnesium (Mg), manganese
(Mn), cadmium (Cd) and iron (Fe) on the fluorescence of luminol to form complexes were observed in these investigations same as in α -naphthoflavone and fluorescein.
The most effective type of complexing reagent for the formation of a fluorescent complex is one with which the metal form a bonds in two position to produce a chelate ring. This normally requires the presence of two functional groups in the reagents, one containing an acidic (replaceable) hydrogen atom and the other an atom carrying a lone pair. The acidic hydrogen is usually supplied by an –OH group (phenolic or carboxylic) and the lone pair comes from an oxygen, nitrogen or sulphur atom [1].
From the results obtained, it was noticed that lanthanum, manganese and cadmium exhibit an enhancing effect on the emission intensity of luminol-metal complexes. It was found that the intensity emission of ferum-luminol complex decreased with increasing volume of ferum and quenching phenomenon happened.
Meanwhile, magnesium-luminol complex had no significant effect on the fluorescence intensity and shows a non-linear change. Detection is selective since only a few metals give stable complex. Nevertheless, metal ions like Fe (II) can interfere because they are able to quench at room temperature. i) lanthanum (La)
The effect of lanthanum on the fluorescent properties of luminol observed was similar to fluorescein and α -naphthoflavone. The same amount of lanthanum (1 mL - 6 mL) was added to the luminol solution (5 mL) to investigate the emission intensity effect.

88
Figure 3.60 showed that the emission spectra of luminol at different amount of lanthanum. The emission peak was at λ em
419.12 nm and the excitation peak was at λ ex
386.0 nm. Figure 3.61 showed that the emission intensity increased when the volume of lanthanum increased from 1 mL to 6 mL. It could be most probably due to the increased volume of the lanthanum from 3 mL to 6 mL forming a complex that exhibit an enhancing effect in the intensity of fluorescence with a linear equation y =
70.779 x + 390.73 and R 2 = 0.9176.
817.0
a
INT
750
700
650
600
550
500
450
400
350
300
250
200
150
100 b c e d f
50
0.0
330.8
360 380 400 420 440 460 480 500 520 540 560 580 600 622.1
Figure 3.60: Effect of lanthanum on the emission spectra of luminol (a = 5 mL, b =
3 mL, c = 5 mL, d = 1 mL).

89
300
200
100
600
500
400
900
800
700 y = 70.779x + 390.73
R 2 = 0.9176
0
1 2 3 4 volume of lanthanum (mL)
5 6
Figure 3.61: Effect of lanthanum volume on the emission spectra of luminol ( λ ex
386.0 nm, λ em
419.12 nm). ii) iron (II) (Fe)
Ferum or iron is always found in biological samples. Its effect on the fluorescent emission peak of luminol in ethanol was further investigated by observing its effect when present in relatively small quantities.
Figure 3.62 indicated that the fluorescence spectrum of the ferum-luminol complexes was shifted to smaller energies when the volume of the metal was increased. Ferum suppressed the luminol emission peak height down to 10%. Such a suppressive effect is probably due to the formation of insoluble ferric. A linear plot of emission intensity versus concentration of ferum was obtained (Figure 3.63) with the equation y = -21.682 x + 211.95 and R 2 = 0.9543.

90
222.0
200
180
160
140 a b c
INT
120
100 d e
80
60
40
20
0.0
330.8
f
360 380 400 420 440 460 480 500 520 540 560 580 600 622.1
Figure 3.62: Effect of ferum (II) on the luminol emission spectra (a = 1 mL, b = 2
mL, c = 3 mL, d = 4 mL, e = 5 mL, f = 6 mL).
The excitation and emission spectra as well as the quantum yields of the fluorescent molecules are influenced by metal solution. In order to have any effect of aggregates on the emission spectrum, Arbelov and co-workers [73] reacted the fluorescent material while varying optical path-length of the sample and found that the fluorescence spectrum of concentrated solutions recorded using very short optical path had the same shape as that observed in dilute solution. Therefore, aggregate formation at high concentration had a negligible effect on the observed emission peak shift.
Furthermore, the observed peak shift in the fluorescence spectra is a consequence of re-absorption and re-emission [73]. Since the emitted wavelength was longer than the absorbed one, re-absorption of the fluorescence generated fluorescence emission at redder side. Absorption saturation would leads to similar red shift, depending on the medium. If the molecules have closely lying levels of

91 different absorption cross-section, absorption saturation to each level would lead to a wavelength shift. In general, the red shift in dyes is not due to saturation. Reagent
3-hydroxyflavone was specified for the detection of ferum [1].
250
200
150
100
50 y = -21.682x + 211.95
R 2 = 0.9543
0
0 1 2 3 4 volume of ferum(II) (mL)
5 6 7
Figure 3.63: Effect of ferum (II) volume on the emission spectra of luminol ( λ ex
386.0 nm, λ em
412.12 nm). iii) cadmium (Cd)
Cadmium (Cd) is a best-known toxic heavy metal. Cadmium occupies an interesting position in the periodic table, being placed in group 12 between zinc and mercury, both of which play essential roles in several biological processes. There were many workers who worked on the formation of fluorescent cadmiumcomplexes, especially CdS. Therefore, it is interesting to study the interaction of cadmium (II) ion with luminol.
The intensity of luminol-cadmium complexes increases with the increase in the volume of cadmium (Figure 3.64). A good linearity was observed between the

92 emission intensity and the volume (mL) of cadmium (Figure 3.65) as compared with other metals that had been investigated in this experiment.
1000.0
900
800
700
600
500
INT f e d c b
400 a
300
200
100
0.0
330.8
360 380 400 420 440 460 480 nm
Wavelength (nm)
500 520 540 560 580
Figure 3.64
: Effect of cadmium on the emission spectra of luminol ( λ ex
600 622.1
386.0 nm,
λ em
419.12 nm) (a = 1 mL, b = 2 mL, c = 3 mL, d = 4 mL, e = 5 mL, f
= 6 mL).
The linear regression equation is given by y = 110.66 x + 343.07 and R 2 =
0.9696 (Figure 3.65). In another study on the same complex, metal cadmium gives a non-linear relation to the fluorescein and α –naphthoflavone complexes. Increasing the volume of cadmium causes the ternary complex to become less stable, which resulted in the non-linearity of fluorescence intensity.

93
1200
1000
800
600
400
200 y = 110.66x + 343.07
R 2 = 0.9696
0
0 1 2 3 4 volume of cadmium (mL)
5 6 7
Figure 3.65: Effect of cadmium volume on the emission spectra of luminol in
ethanol. iv) magnesium (Mg)
Figure 3.66 shows the effect of different volumes (mL) of magnesium on the emission spectra of luminol at λ ex
386.0 nm and λ em
408.07 nm. The shapes of emission spectra remain unchanged. As shown in Figure 3.67, it was found that the intensity of magnesium-luminol complex shows a non-linear change. This result was similar with cadmiumα -naphthoflavone complex as shows in Figure 3.15. This suggests that luminol aggregation occur to a significant extent in the solution. It was found that there were a lot of studies on the complexes of magnesium. For example,
Odom et al.
[100] and Chandrasekhar at el.
[101] synthesized and characterized the magnesium and zinc complexes with other ligands.

94
908.6
INT
800
700
600
500
400
300
200
100
0.0
330.0 350 400 450 e d b c a nm
Wavelength (nm)
500 550 600 622.5
Figure 3.66
: Effect of magnesium on the emission spectra of luminol ( λ ex
386.0 nm,
λ em
408.07 nm) (a = 1 mL, b = 2 mL, c = 3 mL, d = 5 mL, e = 6 mL).
1200
1000
800
600
400
200
0
1 2 3 4 volume of magnesium (mL)
5 6
Figure 3.67
: Effect of magnesium volume on the emission spectra of luminol.

95 v) manganese (Mn)
Manganese and heavy metal cation chosen in the study are able to form complex compounds. As shown in Figure 3.68, the emission intensity of the luminol-manganese complex increased with the addition of manganese volume. The calibration graph of emission intensity versus volume of manganese showed a linear relationship with equation y = 117.0 x + 287.04 and correlation coefficient of R 2 =
0.9344 (Figure 3.69). These results were not similar with fluorescein and α naphthoflavone-manganese complexes which can be found in Figure 3.34 and Figure
3.13. The position of emission peak remains unchanged.
795.6
750 d
700 e
650
600
550
500 c
450
INT
400
350
300
250
200
150 b a
100
50
0.0
330.8
360 380 400 420 440 460 480 500 520 540 560 580 600 622.1
Figure 3.68
: Effect of manganese on the emission spectra of luminol ( λ ex
386.0 nm,
λ em
406.83 nm) (a = 1 mL, b = 2 mL, c = 3 mL, d = 4 mL, e = 5 mL).

96
1000
900
800
700
600
500
400
300
200
100
0
0 y = 106.08x + 287.04
R 2 = 0.9344
1 2 3 4 volume of manganese (mL)
5 6 7
Figure 3.69: Effect of manganese volume on the emission spectra of luminol ( λ ex
386.0 nm, λ em
406.83 nm).
3.3.6 The Detection of Gaseous Carbon Dioxide, Oxygen and Sulphur Dioxide on the Fluorescence of Luminol
3.3.6.1
The Effect of Carbon Dioxide (CO
2
) on the Emission Spectra of
Luminol i) Luminol in Ethanol
The analytical technique for CO
2
(Figure 2.2) as developed in the present experiment was applied to the analysis of luminol in ethanol. The carbon dioxide gas was bubbled into the luminol solution at different concentration of CO
2
.

97
Figure 3.70 shows a non-linear change as the amount of CO
2
gaseous increases. This can be attributed to several reasons. Additionally, the bubbling of gaseous CO
2
decreases the pH of the solution to become acidic. It could be due to the carbonic acid formation. Previous study showed that luminol exhibit fluorescence in basic condition [38]. Similar result was obtained for CO
2
detection with fluorescein (Figure 3.42).
630
620
610
600
590
580
570
560
550
540
0 2 4 6 volume of carbon dioxide (mL)
8 10
Figure 3.70: Graph of luminol emission intensity from 1x10 -3 mol L -1 solution in
ethanol bubbled with different volume of carbon dioxide gases ( λ ex
=
386.0 nm, λ em
= 412.0 nm)
3.3.6.2 The Effect of Oxygen (O
2
) Gases on the Emission Spectra of Luminol in
Ethanol
The sensing of molecular oxygen based on luminescence quenching is regarded as one of the most widespread optosensing applications. In all luminescence quenching detection schemes, a preferably strong and low noise luminescence background signal generated, which is dynamically quenched in the presence of molecular oxygen or other quencher.

98
As shown in Figure 3.71, the emission intensity increased when 2 mL of oxygen was dissolved. Then, the intensity decreases as more 4 - 6 mL of oxygen gas dissolved. Quenching phenomenon occurred in the presence of oxygen. It could be due to oxidation of luminol occurring during the dissolving of the oxygen. When samples containing fluorescent group, are exposed to the air or their solutions are saturated with oxygen, the fluorescence intensities of the samples decrease and the rates of the fluorescence decay increase.
480
460
440
420
400
380
360
0 2 4 volume of oxygen (mL)
6
Figure 3.71
: Graph of luminol emission intensity from 1x10 -3 mol L -1 solution in
ethanol bubbled with different volume of oxygen gases ( λ ex
= 386.0 nm,
λ em
= 412.7 nm).
3.3.6.3 The Effect of Sulphur Dioxide (SO
2
) on the Emission Spectra of Luminol
As shown in Figure 3.72 and Figure 3.73, the emission intensity of luminol decreased as the volume of sulphur dioxide increased. There was only a minor change in the emission wavelength. This is possible because the sulfonate groups on the luminol do not interact with sulphur dioxide added externally. Result shows that luminol is not suitable as a sulphur dioxide sensor.

99
360.0
INT
300
250
200
150
100
50 a b c d e
0.0
380.0
400 420 440 460 480 500 520 540.5
Figure 3.72: Emission spectra shows the effect of sulphur dioxide on luminol in
ethanol ( λ ex
= 346.0 nm, λ em
= 409.74 nm) (a = 1 mL, b = 4 mL, c = 2
mL, d = 5 mL, e = 8 mL).
450
400
350
300
250
200
150
0 2 4 volume of SO
2
6 8 10
Figure 3.73: Effect of sulphur dioxide on the 1x10 -3 mol L -1 luminol in ethanol.

100
3.3.7 The Fluorescent Property of Luminol Encapsulated in Sol Gel
Lately, the doping of the sol–gel glass with photoactive substances has received considerable interest. The sol-gel process provides a relatively simple way to encapsulate reagents in a stable host matrix. Moreover, sol-gel derived glasses used as host materials provide better optical transparency, stability and permeability than many organic polymers.
Luminol was encapsulated in sol gel and it was found that the measured intensity of luminol in sol gel was lower compared with luminol in ethanol (Figure
3.74). This could be due to the trapping of luminol in the pores of sol gel and make the molecules cannot move freely and they lowering the emission intensity [57].
3.3.8 Sol gel Immobilized Luminol as Fluorescent Carbon Dioxide Sensing
Material
Figure 3.75 shows the emission spectra of luminol-ethanol encapsulated in sol gel. 1 mL – 8 mL of CO
2 gases was bubbled into luminol-ethanol solution. It can be seen that the emission intensity produce a non-linear change as the carbon dioxide gases are exposed to luminol in ethanol. It could be due to the reaction between carbon dioxide and luminol forming an unstable compound. Moreover, the difference between the emission intensity before and after dissolved CO
2
gases changed by a small value. The intensity of emission is lower compared to luminol in ethanol.

101
5.00
4.5
4.0
3.5
3.0
INT
2.5
2.0
c d a b
1.5
1.0
0.5
0.00
380.0
400 420 440 460 480 500 520 540 560 580 600.5
Figure 3.74: Effect of carbon dioxide on the emission spectrum of luminol in sol gel
( λ ex
= 366.0 nm, λ
5 mL). em
= 425.98 nm) (a = 0 mL, b = 2 mL, c = 4 mL, d =
6
5
2
1
4
3
0
0 1 2 3 4 5 volume of carbon dioxide (mL)
6 7 8
Figure 3.75: Effect of carbon dioxide on the emission intensity of 1x10 -3 mol L -1
luminol in sol gel ( λ ex
386.0 nm , λ em
421.6 nm).

102
3.3.9
Sol Gel Immobilized Luminol-cadmium and Luminol-ferum Complexes as Fluorescence Carbon Dioxide Sensing Material
Luminol was complexed with cadmium and ferum (II) because cadmium gives the best linearity among the five metals and ferum (II) shows a linear decrease in the emission intensity of luminol. The intensity of emission spectra were found to increase when the amount of CO
2
increased. However, the shapes of the emission peaks remain unchanged (Figure 3.76). As can be seen in Figure 3.77, over a wide range of CO
2
concentration, the sensor produces a typical curve. The fluorescence intensity of the luminol-ferum complex increases with the CO
2
concentration and this was expected due to more complexes being formed when more CO
2
became available in the solution. A levelling off at high CO
2
concentration is due to saturation of the immobilised CO
2
with luminol-ferum complex. On the other hand, low concentration of CO
2
will limit the amount of luminol-ferum-CO
2
complex being formed.
1044.5
1000
900
800
700
600
INT 500
400
300 b c a d
200
100
0.0
387.4
400 420 440 460 480 nm
Wavelength (nm)
500 520 540
Figure 3.76: Emission spectra of luminol-ferum complex exposed to gaseous
carbon dioxide (a = 20 mL, b = 18 mL, c = 14 mL, d = 6 mL).
560.4

103
1200
800
400
0
0 5 10 15 20 volume of carbon dioxide (mL)
Figure 3.77: Effect of carbon dioxide on the emission intensity of luminol-ferum
complex.
Ferum or iron can act as a catalyst when it reacts with luminol due to chemiluminescent reaction of the luminol reagent with the iron or ferum (II) in hemoglobin.
Figure 3.78 showed the emission spectra of luminol-cadmium complexes exposed to carbon dioxide. The addition of carbon dioxide to luminol-cadmium complex, produce a single peak with larger emission intensity with no shifting of emission spectra could be observed. The increase in the amount of gaseous carbon dioxide in the luminol-cadmium complexes solution results in a non linear change in the emission intensity plot. It could most probably be due to the formation of cadmium carbonate. However, the increase in the amount of CO
2
increases the acidity of the solution. The cadmium carbonate may have dissolved in the solution.
As a result, the emission intensity fluctuates.

104
660
640
620
600
580
560
540
520
2 4 6 8 10 12 14 16 volume of carbon dioxide (mL)
18 20
Figure 3.78: Effect of carbon dioxide on the emission intensity of luminol-cadmium
complex.

CHAPTER 4
CONCLUSION
4.1 Conclusion
This study involved a detection of carbon dioxide, sulphur dioxide and oxygen using fluorescent materials such as α -naphthoflavone (7, 8-benzoflavone), fluorescein and luminol.
The fluorescence properties of α -naphthoflavone observed were found to exhibit excitation wavelength ( λ ex
) at 343.0 nm and emission wavelength ( λ em
) at 426.0 nm.
The optimization procedures were carried out in order to obtain a reliable measurement.
The optimum pH 10.06 and tris buffer was adopted for the analysis and the maximum emission intensity occurred at α -naphthoflavone concentration of 1 x 10 -3 mol L -1 . The influence of the temperature on the α -naphthoflavone showed that the fluorescence intensity reached maximum when the solution is at 5 o C.
The standard calibration for quantitative analysis of α -naphthoflavone gives a curvilinear curve. A comparison of the effect of five metals on the α -naphthoflavone emission intensity was also investigated. It was found that lanthanum was the only metal that caused an increase in emission intensity. Ferum (II) gives the best correlation coefficient as compared to the other metals. Lanthanum and ferum (II) were chosen for further investigation for the detection of CO
2
in sol gel.

106
The emission spectrum of α -naphthoflavone-ferum complex in sol gel is found to be broad compared to other complexes and the shape of emission spectrum was found to show no Stoke shift. However, there was a small shift from long wavelength to short wavelength when CO
2
was added. The shift of the wavelength could be due to the interaction between carbon dioxide with α -naphthoflavone-ferum complex. The emission intensity of α -naphthoflavone-lanthanum-CO
2
complex shows a non linear change.
The use of fluorescein for the detection of carbon dioxide was also investigated.
The maximum emission fluorescence peak for fluorescein is located at 510.2 nm ( λ ex
=
483.0 nm) with optimum pH 8.90. The regression plot gives an equation of y = 152.36 x
– 64.579 and R 2 0.9425. Maximum emission intensity occurred at fluorescein concentration of 1 x 10 -3 mol L -1 .
The emission intensity was found to decrease with an increased in the amount of manganese. The graph of emission intensity versus volume of manganese (mL) gives a linear regression line with equation y = -7.861 + 69.227 and R 2 = 0.9613 meanwhile the emission intensity of fluorescein-ferum complex decreased with an increased in the amount of CO
2
in the complex solution. Manganese and ferum (II) was quenched the emission intensity of fluorescein and this complex was chosen for further study for the detection of CO
2
in sol gel. The emission intensity of fluorescein-ferum complex decreases linearly after exposure to increasing amount of CO
2
in sol gel. The relationship gives a regression equation was y = -7.2252 x + 271.01 and a correlation of
0.9234. However, the emission intensity of fluorescein-manganese complex decreased with an increase in the amount of carbon dioxide
The maximum emission of luminol was observed at wavelength of 421.2 nm when excited at wavelength of 386.0 nm in ethanol. The optimum pH was reached at pH 8.62. It was found that the emission intensity decreases with as increase in the temperature.

107
Under the optimum condition, calibration graphs of intensity against concentration of luminol gives a curvilinear relationship for concentration of 0.5 x 10 -4 mol L -1 to 10.0 x 10 -4 mol L -1 . Cadmium was found to decrease the emission intensity of luminol and gives a linear equation y = 110.66 x + 343.07 and R 2 = 0.9696. However, ferum (II) was found to quench the emission intensity of luminol with an equation y = -
21.682 x + 211.95 and R 2 = 0.9543.
The luminol-cadmium complex and lumimol-ferum complex in sol gel were chosen to detect CO
2
. Results showed that the emission intensity of luminol-cadmium complex increased with a increase in the amount of CO
2
in the complex solution giving a non linear change in the emission intensity. The fluorescence intensity of the luminolferum complex increasing with an increase in the CO
2
concentration and this was expected due to more complexes being formed when more CO
2
became available in the solution.
In conclusion, the development of carbon dioxide sensor based on the fluorescence enhancement technique in sol gel has achieved its objectives in this work with successful and promising results.

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