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DEVELOPMENT OF CONDUCTING POLYMER-BASED ANTIOXIDANT PACKAGING MATERIALS
CHYONG FANG HSU
DEVELOPMENT OF CONDUCTING POLYMER-BASED ANTIOXIDANT PACKAGING MATERIALS
CHYONG FANG HSU
This thesis is submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy in Food Science, The University of Auckland, 2009
CONTENTS
CONTENTS
i
ABSTRACT
viii
ACKNOWLEDGEMENT
x
LIST OF SCHEMES
xi
LIST OF TABLES
xiii
LIST OF FIGURE
xv
LIST OF SYMBOLS AND ABBREVIATIONS
xxiii
Chapter 1 Introduction 1.1 Antioxidants
1
1.2 Determination of antioxidant capacity
3
1.3 Conducting polymers
11
1.4 Conducting polymer composites
18
1.5 Packaging
20
1.6 Objectives of this thesis
21
i
Chapter 2 Experimental Methods 2.1 Introduction
27
2.2 Materials
28
2.3 Polymerization of polypyrrole, polyaniline and
30
poly(3,4-ethylenedioxythiophene) powders 2.4 Reduction of PPy, PANI and PEDOT powders and
30
preparation of dedoped PANI powder 2.5 Elemental analysis
31
2.6 Preparation of PANI EC film
31
2.6.1 Solution blending of EC and previously prepared PANI powders
31
2.6.2 Reduction of PANI EC film
32
2.6.3 Preparation of an in situ oxidatively polymerized PANI(OX)EC film
32
2.7 Preparation of PANI PE film
33
2.8 Preparation of PANI and PPy films electrochemically
33
2.9 Evaluation of antioxidant capacity of CPs powders and films
34
2.9.1 DPPH assay
34
2.9.2 ABTS assay
34
2.9.3 ORAC-FH assay
36
2.10 Characterization of polypyrrole, polyaniline and PEDOT
36
2.10.1 SEM images
36
2.10.2 Spectroscopy
37
2.10.3 Conductivity
37
ii
2.10.4 Cyclic Voltammograms of PANI powders
38
2.11 Mechanical properties of PANI films
38
2.12 Measurement of the peroxide value (PV) of oil samples
39
2.13 Determination of fatty acids in fish oil
39
2.13.1 Preparation of the GC internal standard
40
2.13.2 Preparation of saponifying agent
40
2.13.3 Preparation of methylating agent
40
Chapter 3 DPPH scavenging activity of conducting polymers 3.1 Introduction
41
3.2 Experimental
41
3.3 Development of a DPPH assay for conducting polymer powders
42
3.4 Antioxidant activity of PPy
45
3.4.1 Polymerization of PPy
45
3.4.2 Spectroscopic Characterisation
50
3.4.3 DPPH free radical scavenging activity of PPy powders
56
3.4.4 Properties of fully reduced PPy powders
58
3.4.5 Comparison of DPPH• scavenging activity of PPy and PNMPy
62
3.5 DPPH scavenging activity of PANI
64
3.5.1 Polymerization of PANI
64
3.5.2 SEM morphology of PANI
67
3.5.3 Cyclic Voltammetric investigation
70
iii
3.5.4 FTIR spectroscopy
73
3.5.5 Raman spectroscopy
74
3.5.6 ESR spectroscopy
75
3.5.7 DPPH• scavenging activity of PANI powders
77
3.5.8 Structural changes in PANI upon reaction with DPPH
79
3.6 DPPH• scavenging activity of poly (3,4-ethylenedioxythiophene)
85
3.6.1 Polymerization of PEDOT
85
3.6.2 SEM images
87
3.6.3 FTIR spectroscopy
88
3.6.4 DPPH• scavenging activity
90
3.6.5 Conductivity of the PEDOT samples
91
3.7 Conclusions
92
Chapter 4 ABTS•+ scavenging activity of polypyrrole, polyaniline and poly(3,4-ethylenedioxythiophene) 4.1 Introduction
96
4.2 Experimental
97
4.2.1 ABTS• radical scavenging activity of PPy 0.5, PANI 1.5 and PEDOT 1.0
97
4.2.2 Spectroscopic characterization of the conducting polymer powders
98
+
4.2.3 Cyclic voltammetry (CV) and UV- visible characterisation of ABTS• and +
DPPH• in an ethanolic solution
iv
98
4.3 Results and discussion
100
4.3.1 Development of an ABTS assay for conducting polymer powders
100
4.3.2 ABTS• scavenging activity of conducting polymer powders
103
4.3.3 Comparison of DPPH• and ABTS• scavenging capacity of the
111
+
+
conducting polymers 4.3.4 Spectroscopic characterization of PANI before and after reaction with
113
ABTS• and DPPH• +
4.3.5 Comparison of ABTS• and DPPH• oxidant strength using cyclic +
117
voltammetry and UV-visible spectrometry 4.4 Conclusions
123
Chapter 5 Antioxidant capacity of a conducting polymer – ethyl cellulose film 5.1 Introduction
124
5.2 Experimental
124
5.3 Results and Discussion
125
5.3.1 Development of the PANI EC films
125
5.3.2 Morphological study
126
5.3.3 Development of an ORAC assay for PANI-containing films
128
5.3.4 Peroxyl free radical scavenging activity of PANI EC films
130
v
5.3.5 Characterization of PANI powders by XPS and IR
138
5.3.6 Peroxyl free radical scavenging capacity of PANI EC films with different
141
PANI contents 5.3.7 Mechanical properties of the PANI EC films
142
5.3.8 Peroxyl radical scavenging activity of reduced PANI in PANI EC films
144
5.3.9 Peroxyl radical scavenging activity of the in situ oxidatively polymerized
146
PANI(OX)EC film 5.3.10 Peroxyl free radical scavenging of pure PANI and of PPy films prepared
148
electrochemically on a stainless steel working electrode 5.3.11 Peroxyl free radical scavenging activity of PANI PE film 5.4 Conclusions
151 153
Chapter 6 The application of the conducting polymer films in food packaging 6.1 Introduction
155
6.2 Experimental
155
6.2.1 Measurement of peroxide value (PV)
155
6.2.2 Determination of DHA and EPA in fish oil using gas chromatography
156
6.2.3 Statistical Analysis
156
6.3 Results and discussion
156
6.3.1 Oxidation of lipid
156
6.3.2 The influence of PANI EC film on oxidation of Ropufa oil
158
vi
6.3.3 Influence of α-tocopherol on the oxidation of Ropufa oil
162
6.3.4 The influence of PANI EC film on oxidation of avocado oil
163
6.3.5 Fatty acid composition of Ropufa oil
164
6.4 Conclusions
167
Chapter 7 Conclusions and future work 7.1 General conclusions
168
7.2 Future research
174
References
176
vii
Abstract
Antioxidants are commonly used as preservatives for protecting foodstuffs from oxidation. Packaging is believed not only to increase the economic value of products but also improve their qualities. Conducting polymers (CPs), such as soluble polyaniline and soluble polypyrrole, have been reported to exhibit antioxidant activity. Development of conducting polymer containing packaging materials, with antioxidant properties, is therefore the focus of this project. Polypyrrole (PPy), polyaniline (PANI) and poly (3,4ethylenedioxythiophene) (PEDOT) powders were prepared using various amounts of the oxidant ammonium persulfate (APS). Spectroscopic methods, including IR and Raman, were used to identify the structures of the CP powders synthesized at various APS levels. It was found that a high level of APS led to overoxidation of the CP powders. The conductivity and doping level of the CP powders formed using higher concentrations of the APS oxidant were found to be lower than those prepared using low concentrations of APS, which might be due to overoxidation, damaging the CP structures during the preparation processes.
The antioxidant activity of PPy, PANI and PEDOT powders were evaluated using the DPPH and ABTS assays. The results showed that CP powders synthesized at a high concentration of APS presented lower free radical quenching effects, likely also due to overoxidation. The optimum initial ratio of APS to monomer for the synthesis of CP powders with a superior DPPH free radical scavenging was found to be around 0.5 for PPy, 1.5 for PANI and 1.0 for PEDOT, and the antioxidant ranking of the CP powders
viii
was as follows: PANI1.5 > PPy0.5 > PEDOT1.0. The reduced forms of PPy, PANI and PEDOT exhibited better radical scavenging abilities than the as-prepared powders. PANI nanotubes, synthesized at high pH, was also found to exhibit stronger free radical scavenging activity than the regular granular PANI formed under acidic conditions. Structural changes of PANI powders after reaction with free radicals were observed in IR and XPS spectra, showing an increase in the ratio of imine (C=N) to amine (C-N) units.
PANI EC films were prepared using a solution method and the antioxidant capacity of the PANI EC films are examined using the ORAC assay. The results showed efficient peroxyl free radical scavenging activity of the PANI EC films. A very good correlation between the ORAC response and the area of tested film was also observed, indicating a homogenous dispersion of active PANI powder across the film. Similar to the results obtained from the DPPH and ABTS assays, reduced PANI presented greater peroxyl radical scavenging activity than the as-prepared powders. The influence of the PANI EC films on the oxidation of Ropufa oil was determined by peroxide value (PV) measurement. After incubation at 60oC for several days, the oil stored in the presence of the PANI EC film was found to exhibit a lower PV than in the absence of a CP film, indicating that the conducting polymer is effective in inhibiting oxidation of fish oil.
ix
Acknowledgements
I would like to thank my supervisors A/Prof. Paul A. Kilmartin and A/Prof. Jadranka Travas-Sejdic for their patient guidance throughout the work and their assistance and comments in writing this thesis. I have to thank A/Prof. Paul A. Kilmartin especially for giving me his valuable time in discussion and direction in research.
I would like to express my appreciation to Dr. Hui. Peng for patiently helping me with many of my experiments and ideas, to Dr. William Chiu, Dr. Lijuan Zhang, Dr. Sally Xiong, Dr. Sudip Ray and Dr. Kwong-chi Li for giving me research ideas and sharing with me their experiences and to Sreeni Pathirana for her technical assistance and help.
Thanks also go to my friends, colleagues of my research group and fellow Food Science students for their help and encouragement, and to funding support from the University of Auckland Research Committee grant (No. 3606261) and the New Zealand Foundation for Science and Technology (contract No. UOAX0408).
I would like to give my special thanks to my husband Kuo-Ming and my son Mike for their endless love and support.
x
LIST OF SCHEMES
Scheme 1.1 Formation of the ABTS• radical cation in the presence and absence +
5
of an antioxidant. Ferrylmyoglobin is produced by oxidation of metmyoglobin. Scheme 1.2 The production of ABTS• from the ABTS diammonium salt and +
7
reaction of ABTS• with an antioxidant. +
Scheme 1.3 Illustration of the principle of the ORAC assay.
11
Scheme 1.4 Structures of polypyrrole, polyaniline and PEDOT.
16
Scheme 1.5 Oxidation of PEDOT.
17
Scheme 2.1 Chemical structures of some of the key chemicals used in this research.
29
Scheme 3.1 Chemical polymerisation of pyrrole.
46
Scheme 3.2 A: Overoxidation processes for PPy. B: Initial reaction phase for
52
a Wolff-Kishner reduction of the carbonyl group of overoxidised polypyrrole to a hydrazone. Scheme 3.3 Polaron and bipolaron forms of oxidised PPy.
54
Scheme 3.4 Reaction scheme for polypyrrole with DPPH•.
63
Scheme 3.5 Chemical polymerisation of aniline.
65
Scheme 3.6 Oxidation of polyaniline.
73
Scheme 3.7 Polymerization of PEDOT.
85
Scheme 4.1 Schematic representation of the cell used for spectroelectrochemistry.
99
experiments
xi
Scheme 5.1 Formation of PANI radical cations and resonance stabilisation of
132
PANI radical cations. Scheme 5.2 The main chemical forms of polyaniline showing different proportions of (reduced) benzenoid and (oxidized) quinoid units.
xii
146
LIST OF TABLES
Table 2.1 Formula and molecular weight of the chemicals.
28
Table 3.1 pH value of the PPy reaction mixture after 2 hours.
47
Table 3.2 Elemental analysis results for PPy samples prepared with
47
different APS/pyrrole ratios. Table 3.3 Elemental analysis results (mass %) for PPy samples prepared with
60
an APS/pyrrole ratio of 0.5 or 2.0, before (PPy0.5 and PPy2) and after reduction with hydrazine (RedPPy0.5 and RedPPy2), with molar ratios given in brackets, normalised to a value of 4 for carbon in each case. Table 3.4 Elemental analysis results for PANI samples prepared with different
65
APS/aniline ratios. Table 3.5 Conductivity and pH values of the PANI reaction mixture after 24 hours.
66
Table 3.6 Elemental analysis of PANI before (A) and after (B) reaction with
82
DPPH free radicals, according to XPS wide spectra. Table 3.7 Elemental analysis results for PEDOT samples prepared with
86
different APS/EDOT ratios. Table 3.8 Calculation of molar ratio of HSO4-/SO42- to EDOT units for
86
PEDOT1.0 based on the elemental analysis results. Table 3.9 pH values of the PEDOT reaction mixture after 24 hours.
87
Table 3.10 Conductivity values and DPPH scavenging activity for PEDOT powders.
92
xiii
Table 4.1 Solution absorbance values at 753 nm, to indicate the degree of ABTS•
+
105
scavenging for PPy, PANI and PEDOT at different times with and without shaking. Table 4.2 Comparison of ABTS• (%) scavenging capacity for as-prepared +
108
and reduced PPy0.5, PANI1.5 and PEDOT1.0 after 3 hours of reaction. Table 4.3 Comparison of DPPH• and ABTS• scavenging activity of the +
113
conducting polymers. Table 5.1 Molar percentage of O, N and C in PANI cluster and EC matrix.
128
Table 5.2 Regression Coefficients for fitting the Weibull distribution function
137
curve to the ORAC response curves for PANI EC films (9 % PANI). Table 5.3 Net AUC of PANI EC films.
142
Table 5.4 Net AUC of PANI EC and PPy EC cast films, and of PANI and
149
PPy film prepared electrochemically on a stainless steel working electrode. Table 6.1 Fatty acid composition in wt % of Ropufa oil determined by GC (n = 2).
xiv
166
LIST OF FIGURES
Figure 1.1 Fluorescence decay curve of (a) blank (b) sample. The area between
10
curve a and curve b provides the net AUC of the tested sample. Figure 1.2 (A) Formation of a polaron and a bipolaron, and (B) charge-carrier
13
delocalization, for all-trans polyacetylene. Figure 1.3 Representative conducing polymer spectra: (A) FTIR spectrum of
23
overoxidised PPy; (B) Raman spectra of (a) regular and (b) overoxidised PPy; (C) ESR spectra of PPy; (D) XPS (N1s) spectra of PANI. Figure 2.1 Preparation of PANI powder.
30
Figure 2.2 Preparation of PANI EC film.
32
+
Figure 2.3 UV-VIS spectra of ABTS• .
35
Figure 3.1 Decline in the absorbance at 516 nm of a 255 µM methanolic
44
DPPH• solution with (a) nil, (b) 1.0, (c) 2.0 and (d) 5.0 mg of PPy powder added (formed using an APS/pyrrole ratio of 1.5). Figure 3.2 Doping level (■) and conductivity (▲) of PPy powders prepared
49
with various ratios of APS/pyrrole. Figure 3.3 SEM images of PPy prepared with APS / Py ratio of (A) 0.25
50
(B) 0.5 (C) 1.0 (D) 1.5 (E) 2.0. Figure 3.4 Comparison of the FTIR spectra of PPy powders prepared with different APS/pyrrole ratios, from top to bottom: (a) 0.25, (b) 0.5, (c) 1.0, (d) 1.5 , (e) 2.0 and (f) reduced PPy 2.0.
xv
51
Figure 3.5 Raman spectra of PPy powders formed with different APS/pyrrole
53
ratios, from top to bottom: (a) 0.25, (b) 0.5, (c) 1.0, (d) 1.5 and (e) 2.0; (A) in the range of 200-2000 cm-1, and (B) highlighting the peak at around 1600 cm-1. Figure 3.6 ESR spectra of the PPy powders formed using different
55
APS/pyrrole ratios, from top to bottom: (a) 1.5, (b) 2.0, (c) 1.0, (d) 0.5 and (e) 0.25. Figure 3.7 The change in the EPR signal for 30 mg of a PPy powder in
56
10 mL of Milli-Q water upon reduction with various amounts of hydrazine. Figure 3.8 DPPH scavenging capacity of PPy synthesized with different
57
APS/pyrrole ratios: (a) DPPH only, (b) 2.0, (c) 1.5, (d) 1.0, (e) 0.25, (f) 0.5. Figure 3.9 DPPH• scavenging capacity of PPy synthesized with two
61
APS/pyrrole ratios, before and after reduction of 30 mg samples in 10 mL Milli-Q water with 2 mL of hydrazine for 48 hours: (a) DPPH only, (b) PPy2, (c) RedPPy2, (d) PPy0.5, (e) RedPPy0.5. Figure 3.10 Decline in the absorbance at 516 nm of a 255 µM methanolic
64
DPPH• solution with (a) nil, and 1 mg of (b) PNMPy, (c) PPya and (d) PPy powders. Figure 3.11 SEM images of PANI prepared with various ratios of
68
APS/aniline ratio (A) 1.0 (B) 1.5 (C) 2.0 (D) 2.5. Figure 3.12 TEM image of PANI1.5.
69
xvi
Figure 3.13 SEM image of PANI prepared with the APS/aniline/H2SO4 of
70
(A) 1:1:0.5 (B) 1:1:1 (C) 1:1:1.5 (D) 1:1:2. Figure 3.14 Cyclic voltammograms of PANI powders, prepared with various
72
APS/ANI ratios: (a) 1.5 (b) 1.0 (c) 1.5 (d) 2.0, cast on a glassy carbon working electrode and cycled in 0.1 M HCl at a scan rate of 20 mV s-1. Figure 3.15 FT-IR spectra of polyaniline prepared with various ratios of
74
APS/aniline: (a) 1.0 (b) 1.5 (c) 2.0 and (d) 2.5. Figure 3.16 Raman spectra of polyaniline prepared with various ratios of
75
APS to aniline : (a) 1.0 (b) 1.5 (c) 2.0 and (d) 2.5. Figure 3.17 ESR spectra of polyaniline with various ratios of APS to aniline,
76
from a to d, the ratios are: 1:1.5, 1:1, 1:2 and 1:2.5. Figure 3.18 (A) DPPH• scavenging activity of PANI powders prepared with
78
various APS/aniline ratios: (a) DPPH• only, (b) 2.5, (c) 2.0, (d) 1.0 and (e) 1.5. (B) Decline in the absorbance at 516 nm of a 255 µM methanolic DPPH• solution with (a) nil and PANI prepared with molar ratio of APS: aniline: H2SO4 of (b) 1:1:1, (c) 1:1:1.5, (d) 1:1:0.5 and (e) 1:1:0. Figure 3.19 DPPH• scavenging capacity of PANI powders synthesized with
80
an APS/ANI ratio of 1.5: (a) reduced PANI, (b) as-prepared PANI, (c) dedoped PANI. Figure 3.20 IR spectra of PANI (a) after (b) before reaction with DPPH•.
81
Figure 3.21 ESR spectra of PANI (a) before (b) after reaction with DPPH•.
83
xvii
Figure 3.22 Cyclic voltammograms of PANI powders (a) before (b) after
84
reaction with DPPH•, cast on a glassy carbon working electrode and cycled in 0.1 M HCl at a scan rate of 20 mV s-1. Figure 3.23 Morphology of PEDOT prepared with various ratios of APS/EDOT
88
(1) 1.0 (2) 1.5 (3) 2.0 (4) 2.5. Figure 3.24 IR spectra of PEDOT prepared with the ratio of APS to EDOT:
89
from top to bottom are 1, 1.5, 2 and 2.5. Figure 3.25 DPPH• scavenging capacity of PPy synthesized with different
91
APS/EDOT ratios: (a) DPPH• only, (b) 2.5, (c) 2.0, (d) 1.5, (e) 1.0. Figure 3.26 Amount of DPPH scavenged over a 24 hour period for powders
95
formed with different polymerization ratios of (A) APS to Py, (B) APS to ANI and (B) APS to EDOT (n = 2). +
Figure 4.1 Inhibition of production of ABTS• by addition of (a) nil (b) 5.0,
101
(c) 3.0, (d) 1.0 and (e) 2.0 (Δ) mg of PANI powder (prepared using an APS/aniline ratio of 1.5). +
Figure 4.2 ABTS• scavenging activity of as-prepared conducting
104
polymer powders: (A) ABTS•+ only, (b) PEDOT1.0, (c) PPy0.5 and (d) PANI1.5. +
Figure 4.3 ABTS• scavenging activity of the reduced conducting polymer
106
+
powders: (a) ABTS• only, (b) PEDOT1.0 (c) PPy0.5 (d) PANI1.5. Figure 4.4 SEM images of (a) PANI15H0.5 (b) PANI15H1.
xviii
109
+
Figure 4.5 ABTS• scavenging activity of conducting polymer powders:
110
+
(a) ABTS• only, (b) PANI15H1 (c) PANI15H0.5 (d) PANI1.5. Figure 4.6 IR spectra of (A) PPy0.5 (B) PANI1.5 (C) PEDOT1.0 :
114
+
(a) before (b) after reaction with ABTS• . Figure 4.7 XPS spectra of PANI1.5 (A) before, and after reaction with
116
+
(B) ABTS• and (C) DPPH•. Figure 4.8 Cyclic voltammogram of 0.1 mM ABTS in 0.1 M LiClO4
118
in ethanol using an Indium tin oxide (ITO) electrode as the working electrode, platinum wire as a counter electrode and silver covered with AgCl as the reference electrode at a scan rate of 50 mV/s. +
Figure 4.9 Generation of ABTS• monitored using cyclic voltammetry and
120
UV-visible spectrometry: (A) CV voltammograms from -0.1 to 0.8 V; (B) associated absorbance changes at 753 nm with time; (C) cyclic representation of absorbance change at 753 nm as a +
function of applied potential; and (D) generation of ABTS• at 0.5 V after 0, 10, 120, 240, 360, 480, 600, 720 and 840 seconds. Figure 4.10 Oxidation and reduction of DPPH• monitored using cyclic voltammetry and UV- visible spectrometry: (A) CV voltammogram from -0.1 to 0.9 V, (B) absorbance changes at 753 nm for various times, (C) first cycle of the CV trace and absorbance at 753 nm from -0.1 to 0.9 V, (D) second cycle of CV tracing and absorbance at 753 nm from -0.1 to 0.9 V.
xix
121
Figure 5.1 SEM images of (A) Pure EC, (B) a PANI EC film prepared with
127
9 % PANI viewed from above, and (C) a cross section of the PANI EC film, where ○ is an area high in EC (part І) and □ is an area high in PANI (part П). Figure 5.2 The decline of fluorescence of a fluorescein solution with (a) nil
129
(b) 18.2 mg of EC film and (b) 20.0 mg of PANI EC film, in presence of AAPH at 37 oC. Figure 5.3 Correlation between ORAC area and weight of multiple PANI EC
130
film pieces Figure 5.4 ORAC results of PANI EC films (9 % PANI): (A) fluorescence decay
133
curve of fluorescein induced by peroxyl radicals in presence of various PANI EC films; (a) no film present, (b) 16 mm2 (c) 36 mm2 (d) 64 mm2 and (e) 100 mm2 of PANI EC film; (B) correlation between net AUC and film area (deducted 91% ORAC area of EC, with net AUC of 1.6, 3.6, 6.7 and 8.4 units for film areas of 16 mm2, 36 mm2, 64 mm2 and 100 mm2 respectively). Figure 5.5 Plot of net AUC versus Trolox concentration (µM).
135
Figure 5.6 Relative ORAC value (Trolox equivalents, µ moles) as a function
135
of film area (mm2) for the PANI EC films (9 % PANI). Figure 5.7 Fluorescence decay curve of AAPH /FL system in presence of (a) nil (b) 16 mm2 (c) 36 mm2 (d) 64 mm2 and (e) 100 mm2 of PANI EC film. The dots represent experimental data points and the lines are the fitting results.
xx
138
Figure 5.8 Correlation between the tested PANI EC film area and (a) parameter α
138
and (b) parameter β. (a) y = 0.355x + 22.0, R2 = 0.9807 (b) y = -0.008x + 2.33, R2 = 0.9617 Figure 5.9 XPS spectra of PANI (A) before and (B) after reaction with
140
peroxyl free radicals. Figure 5.10 IR spectra of PANI (A) before and (B) after reaction with
141
peroxyl free radicals. Figure 5.11 Mechanical properties of PANI EC films with varying amounts
144
of PANI added: (Δ) modulus and (●) ultimate tensile strength. Figure 5.12 Comparison of peroxyl free radical scavenging ability of
145
(■) PANI EC film, (●) reduced PANI + EC and (△) reduced PANI EC film. Figure 5.13 SEM image of PANI(OX)EC film (cross section) prepared by
147
synthesizing PANI in the matrix of EC and casting the mixture onto a Teflon sheet. Figure 5.14 Net AUC of (a) PANI(OX)EC film prepared in matrix of EC
148
(17 % PANI), and (b) PANI EC as-prepared films (17 % PANI). a) y = 0.859x-1.05, R2 = 0.9850 b) y = 0.446x-2.39, R2 = 0.9968 Figure 5.15 Cyclic voltammograms for the polymerization of (A) PANI in
150
0.1 M H2SO4, and (B) PPy in 0.1M NaHSO4. Inserts: the first three cycles of PPy and PANI. Figure 5.16 ORAC results of PANI PE film (10% PANI): (A) relative fluorescence
152
versus time; and (B) ORAC area versus PANI PE film area. Figure 6.1 PV of Ropufa oil under accelerated storage conditions (60oC).
xxi
158
Figure 6.2 Peroxide value (PV) of Ropufa oil at 60oC with (a) no film,
160
(b) PANIEC film (9 %), (c) PANIEC film (17 %) (n = 3). Figure 6.3 Peroxide value of Ropufa oil in the presence of various amounts of
161
the antioxidant BHT after 8 days of storage at 60oC. y = 243 – 7.21x, R2 = 0.9567 Figure 6.4 Peroxide value (PV) of Ropufa oil at 40oC with (▲) no film,
162
(•) PANIEC film (9 %), (c) PANIEC film (17 %) (n = 3). Figure 6.5 Peroxide value (PV) of 3.0 g of Ropufa oil with (•) nil and
163
(▲) 4.5 mg of α-tocopherol added (n = 3). Figure 6.6 Peroxide value (PV) of avocado oil at 60oC in presence of (▲) nil and (•) PANI EC film (9 %) (n = 3).
xxii
164
LIST OF SYMBOLS AND ABBREVIATIONS
A
amps
A•
antioxidant radical
AAPH
2-2’-azobis(2-amidinopropane) dihydrochloride
ABTS
2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
ABTS•
+
ABTS radical cation
ABTSH
reduced ABTS•
AH
antioxidant (with proton attached)
ANI
aniline
APS
ammonium persulfate
AUC
under the ORAC fluorescence decay curve
B
benzenoid
BHT
butylated hydroxytoluene
CP
conducting polymer
CPS
counts per second
CV
cyclic voltammetry
DBSA
dodecylbenzenesulfonic acid
DHA
docosahexaenoic acid
DMSO
dimethyl sulfoxide
DPPH
2,2’-diphenyl-1-picrylhydrazyl
DPPH•
DPPH free radical
+
+
xxiii
e-
electron
EC
ethyl cellulose
EDOT
3,4-ethylenedioxythiophene
EDXS
energy-dispersive X-ray spectrometry
EPA
eicosapentaenoic acid
ESR
electron spin resonance spectroscopy
ET
electron transfer
eV
electron volt
FH
fluorescein (with proton attached)
FTIR
fourier transformation infrared spectroscopy
GC
gas chromatography
H+
proton
HAT
hydrogen atom transfer
HIPEF
high-intensity pulsed electric fields
HX-Fe+3
metmyoglobin
ITO
Indium tin oxide electrode
LH
lipid (with proton attached)
mS
milli-Siemens
mT
milli-Tesla
ORAC
oxygen radical absorbance capacity
β-PE
β-phycoerythrin
PANI
polyaniline
PANI1.5
PANI prepared with a ratio of APS to ANI of 1.5 to 1
xxiv
PANI(OX)EC
PANI EC composite prepared by polymerizing aniline in a EC matrix with presence of APS oxidant
PE
polyethylene
PEDOT
poly (3,4-ethylenedioxythiophene)
PEDOT1.0
PEDOT prepared with a ratio of APS to EDOT of 1.0 to 1
PNMPy
polyN-methylpyrrole
PPy
polypyrrole
PPy0.5
PPy prepared with a ratio of APS to Py of 0.5 to 1
PPya
PPy made with addition of acid
PV
peroxide value
Py
pyrrole
Q
quinoid
RedPEDOT1.0
PEDOT1.0 reduced with hydrazine
RedPANI1.5
PANI1.5 reduced with hydrazine
RedPPy0.5
PPy0.5 reduced with hydrazine
R2N2
AAPH
RH
organic substrate (with proton attached)
ROO•
peroxyl radicals
SEM
scanning electron microscopy
SHE
standard hydrogen electrode
V
volt
X•
oxidizing agent
•
X-[Fe+4 = O]
ferrylmyoglobin
xxv
XPS
X-Ray photoelectron spectroscopy
xxvi
Chapter 1 Introduction
1.1 Antioxidants An antioxidant is defined as “any substance that, when present at low concentrations compared to those of an oxidisable substrate, significantly delays or prevents oxidation of that substrate.”1. An oxidation reaction is often caused by the presence of oxygen and free radicals and can be catalyzed by metals. An antioxidant can interfere with the oxidation process by scavenging active oxygen species, chelating catalytic metals or by terminating the free radical chain reaction. In a chemical system, the strength of an antioxidant is commonly evaluated by examination of its ability to scavenge free radicals and thereby terminate the chain reaction2, 3. The termination of a chain reaction by an antioxidant is shown as follows (X• = oxidizing agent; RH = substrate; AH = antioxidant)4:
X• + RH → R• + XH
(eq. 1.1)
R• + O2 → ROO•
(eq. 1.2)
ROO• + RH → ROOH + R•
(eq. 1.3)
R• + AH → RH + A•
(eq. 1.4)
R• + A• → RA
(eq. 1.5)
During the oxidation processes, hydrogen atoms are transferred from a substrate to an oxidizing agent with the production of free radicals (R•), which are generally very
1
unstable and can rapidly react with oxygen to form peroxyl radicals (ROO•). The resulting peroxyl radicals then react with other compounds to obtain the necessary hydrogen atoms. When attacked by free radicals, the new molecule loses a hydrogen atom and itself becomes a free radical, in a chain reaction (eq. 1.3). Antioxidants can remove free radical intermediates and terminate these chain reactions by donating electrons or hydrogen atoms to the free radicals (eq. 1.4), and/or by combining with the free radical (eq. 1.5). The antioxidant radical (A•) generated as a result of the loss of hydrogen is generally much more stable and will not continue the chain reaction. Being electron or hydrogen donors, antioxidants are often reducing agents. As a “sacrificial” reactant, antioxidants protect other components from damage by free radicals.
In a biological context, free radicals are atoms or groups of atoms with an unpaired electrons that react easily with vital macromolecules, such as proteins, lipids and DNA5, and can cause cellular injury and even diseases leading to death6, 7. Dietary antioxidants, such as polyphenols, carotenoids, vitamin E and vitamin C, are effective free radical scavengers, and have been implicated in the prevention of many human diseases, including arthritis, atherosclerosis, cancer, aging, etc.8-10
Various natural and commercial antioxidants have been reported to exhibit free radical scavenging activities11 and these have been used as preservatives in various food products for retarding undesirable oxidative changes12,
13
. In this context, free radicals are
considered to be a major cause of the deterioration and rancidity of foodstuffs, particularly those rich in polyunsaturated fats. It has been reported that containers treated
2
with antioxidants, such as vitamin E and butylated hydroxytoluene (BHT), can extend the shelf life of the food products they contain14-16. Antioxidant effects are therefore of great interest in the food and beverage industries.
1.2 Determination of antioxidant capacity There are a number of methods available for determining the antioxidant capacity of compounds. These can be based on metal reducing power17-19, quantification of products formed during lipid peroxidation13 and various free radical scavenging methodologies2022
. Free radical scavenging methods have been commonly used to evaluate the
antioxidant activity of compounds due to their simple, rapid, sensitive and reproducible nature. The major antioxidant capacity assays can be classified into two categories: single electron transfer (ET) reaction assays and hydrogen atom transfer (HAT) reaction assays. In fact, both HAT and ET reactions can occur together in a given system2, and the dominating mechanism is determined by the properties of the antioxidant in question. Factors such as bond dissociation energy are important in determining the mechanism and efficacy of each antioxidant. Normally, ET–based methods detect the ability of an antioxidant to transfer electrons to a test compound, while HAT–based methods measure the capacity of an antioxidant to donate hydrogen to free radicals. Compared to HAT methods, ET reactions are usually slow and require a longer time to complete2, 3.
The DPPH assay is a method based on electron transfer (ET) between two constituents, the antioxidant and the blue coloured DPPH• radical (a weak oxidant). This method is used to evaluate the reducing ability of antioxidants toward DPPH•. When the ET
3
reaction takes place, DPPH• accepts an electron from the antioxidant, causing a color change in the system, and the rate and extent of colour loss provides a measure of the efficiency of the antioxidant. The absorbance of the remaining DPPH• is examined at 516 nm and the percentage of the DPPH• remaining can be calculated as follows: %DPPH rem = 100 × [DPPH]rem/ [DPPH]i
(eq. 1.6)
where [DPPH]rem is the concentration of DPPH• remaining at a certain time, and [DPPH]i is the initial DPPH• concentration. The DPPH assay also involves a hydrogen atom transfer reaction2, 3, 17, 23. It was found that the reaction consists of a fast electron transfer process and a slow hydrogen atom transfer reaction from antioxidants to DPPH radicals, especially when it occurs in strong hydrogen bond-accepting solvents, such as methanol and ethanol. The half-reaction for the DPPH radical is as follows: DPPH• + H+ + e- → DPPHH
(eq. 1.7)
One of the advantages of this method is that DPPH• is a quite stable organic nitrogen radical and does not have to be generated before use, which is why this method is widely used17. However, many antioxidants that react fast with other free radicals may only react slowly with DPPH• because of the radical’s high stability. Moreover, steric inaccessibility might make some antioxidants effectively inert to the radical2.
The ABTS assay is also an ET-based method. The assay was originally reported by Miller and Rice-Evans24. Due to the simplicity of its operation, this method has been used by many researches for studying the antioxidant capacity of compounds and foodstuffs. In this method, the blue/green ABTS radical is formed through a reaction between ABTS 4
and ferrylmyoglobin radical species, itself produced through reaction of metmyoglobin and H2O2. During the reaction, ABTS donates an electron to the ferrylmyoglobin radical, +
forming the ABTS• radical and regenerating metmyoglobin (Scheme 1.1). The tested antioxidant is added to the reaction medium before the radical is generated. In the +
presence of an antioxidant, the ABTS• radical cations can be quenched, causing inhibition of the colour production and a lower absorbance at 753 nm for the test solution25, 26. The percent loss in absorbance of the reaction mixture can be used to evaluate the antioxidant capacity of the tested samples.
+
H2O2
HX - Fe 3+
Metmyoglobin
.X-[Fe 4 + =O] Ferrylmyoglobin Antioxidant
ABTS
No antioxidant
.+
More ABTS
+
.+
Less ABTS
HX - Fe 3+
+
Scheme 1.1 Formation of the ABTS• radical cation in the presence and absence of an antioxidant. Ferrylmyoglobin is produced by oxidation of metmyoglobin.
5
However, because the antioxidant can also react with the ferrylmyoglobin radical, and lead to an overestimation of the antioxidant capacity, the method has been modified to allow formation of the ABTS radical via a reaction with potassium persulfate (K2S2O8) +
prior to the addition of the antioxidant27. The ABTS• generated in the ABTS / K2S2O8 system is present as follows28:
S2O82- + ABTS → SO42- + SO4-• + ABTS+•
(eq. 1.8)
SO4-• + ABTS → SO42- + ABTS+•
(eq. 1.9)
Overall reaction: S2O82- + 2ABTS → 2SO42- + 2ABTS+•
(eq. 1.10)
After the addition of the test sample (antioxidant) to the free radical solution, the absorbance can be measured at various times. The antioxidant capacity can then be evaluated as the ability of the tested compound to decrease the color of the solution by reacting directly with the ABTS radical (Scheme 1.2)29. The method is a decolorization +
assay applicable to both lipophilic and hydrophilic systems, since ABTS• is soluble in +
both aqueous and organic solvents30-32. Also, ABTS• normally reacts rapidly with antioxidants and can react with many antioxidants with low redox potentials. In addition, +
the ABTS• radical cation can be rapidly generated by an enzyme reaction or using an electrochemical method33, 34. One disadvantage of the method is that the ABTS radical is not found in biological systems and thus only represents a “nonphysiological” radical source.
6
+ 4 H N 3 O S
N
N
S
S
S 3 O 4 H N +
N
N S T B A
C2H5
C2H5
-
-e-
+e
+ 4 H N 3 O S
S
S
S 3 O 4 H N +
N
N
+ N
N
.
C2H5
S T B A
.+
C2H5
) H A ( t n a d i x o i t n A + 4 H N 3 O S
S N
N
+ S HN
S 3 O 4 H N +
N C2H5 H S T B A
C2H5
+
+
Scheme 1.2 The production of ABTS• from the ABTS diammonium salt and reaction of +
ABTS• with an antioxidant.
7
Although the DPPH and ABTS assays are normally considered as ET reactions, these two radicals can also receive a proton from the antioxidant, after direct reduction via electron transfer, which is similar to a HAT reaction, with H atom transfer from the antioxidant to the quenched radical35. Therefore, it is difficult to completely distinguish ET and HAT reactions, since hydrogen atom transfer reactions can be the result of proton-coupled electron transfer, an aspect also stressed by other researchers2, 3.
The oxygen radical absorbance capacity (ORAC) assay, which measures antioxidant scavenging activity against peroxyl radicals generated by the decomposition of 2-2’azobis(2-amidinopropane) dihydrochloride (AAPH) at 37oC, was initially developed by Cao et al36,
37
. In this assay, β-phycoerythrin (β-PE), a protein isolated from
Porphyridium cruentum, was chosen as the fluorescent probe. The loss of fluorescence of β-PE can indicate the extent of damage from its reaction with the peroxyl radical. The protective effect of an antioxidant is measured by assessing the area under the fluorescence decay curve (AUC) of the tested sample as compared to that of the blank in which no antioxidant is present. However, β-PE was found to produce variable reactivity to peroxyl radicals38 and not to be photostable when exposed to light excitation for a certain time36. Because of the disadvantages of β-PE, fluorescein, a more stable fluorescence source, was therefore used to replace β-PE as the probe39, 40. The ORACfluorescein (ORAC- FH) assay was first proposed by Ou et al.41. Among the methods for measuring the antioxidant capacity of food, beverages and biological samples, the ORAC assay is of great interest because it deals with peroxyl free radicals, which are the predominant radicals found in the oxidation of lipids in foodstuffs and in biological
8
systems2. This method is based on a hydrogen atom transfer reaction (HAT) and competitive reaction kinetics. Generally, the azo radical initiator offers a steady flow of peroxyl radicals in the fluorescein-containing solution, which is used as a molecular probe for monitoring reaction progress by the change in fluorescence intensity. The added antioxidant competes with fluorescein for the radicals and retards the decay in fluorescence (Scheme 1.3). The competitive reaction scheme for this assay is as follows (R2N2 = AAPH; FH = fluorescein; AH = antioxidant): R2N2 + 2 O 2 → 2 ROO• + N2
(eq. 1.11)
ROO• + FH → ROOH + F•
(eq. 1.12)
ROO• + F• → ROOF
(eq. 1.13)
The antioxidant acts as a chain-breaking inhibitor, which traps peroxyl free radicals by transferring hydrogen atoms to the radical. ROO• + AH → ROOH + A•
(eq. 1.14)
To be an effective chain breaking antioxidant, the rate constant of reaction (eq. 1.14) must be much greater than that of reaction (eq. 1.12)2.
Quantification is achieved by
calculating the area under the kinetic curve (AUC) compared to the net AUC, and is obtained by AUCsample – AUCblank (Figure 1.1). The area under curve (AUC) is calculated as follows for fluorescence readings (f) at different times (taking 99 readings across the measurement period; about once every 2 minutes for most CP samples)41: AUC = (1 + f1/f0 + f2/f0 + f3/f0 + f4/f0 + …. + fn/ f0) × Δt
(eq. 1.15)
Where f0 is the fluorescence reading at the beginning of the reaction, fn is the final measurement and Δt is time interval (2 mins).
9
Relative Fluorescence Intensity
1.2 1.0 0.8 0.6
(a) (b)
0.4 0.2 0.0 0
10
20
30
40
50
Time / min
Figure 1.1 Fluorescence decay curve of (a) blank (b) sample. The area between curve a and curve b provides the net AUC of the tested sample.
The ORAC assay can estimate both the inhibition time and the inhibition degree, which together reflect the radical chain breaking antioxidant activity 19, 38. Another advantage of this method is that the ORAC-FH measurement has been successfully automated using a multichannel liquid handling system combined with a microplate fluorescence reader in a 96-well format, which improves efficiency for large scale analyses.
10
AAPH Decompose at 37oC
O2
ROO
Fluorescent probe
Fluorescent probe
(In absence of antioxidant)
(In presence of antioxidant)
Slower decay of f luorescence
Faster decay of f luorescence
AUCantioxidant
AUCblank
Scheme 1.3 Illustration of the principle of the ORAC assay.
1.3 Conducting polymers Conducting polymers (CPs) were discovered in 1977, when a “doped” form of polyacetylene was found to show high conductivity42, and for which three scientists, Alan J. Heeger, Alan G. MacDiarmid and Hideki Shirakawa, were awarded the 2000 Nobel Prize in Chemistry. Unlike regular plastics that are typically insulators, conducting
11
polymers exhibit electrical and optical properties of metals and semiconductors, while retaining the mechanical properties of conventional polymers42, 43. These polymers are therefore called “synthetic metals” to signify their organic characteristic and metal-like properties.
The polymers typically contain a π-conjugated system, which has single and double bonds alternating along the polymer chain. However, it has been found that conjugation is not enough to make the polymer conductive. Added dopants and the formation of charge carriers in the polymer chains are also essential features to enable the polymers to exhibit conductive properties. In general, conducting polymers can be p-doped or n-doped through reaction with either an oxidant or a reductant. p-doping is partial oxidation, while n-doping is partial reduction, of the π-backbone of the polymer. The dopant either removes (p-doping) or adds (n-doping) electrons to the polymer, which adds extra holes or electrons to the polymer chain, which can move freely along the polymer and thus cause the polymer to become conductive. If an electron is removed locally from one carbon atom, a radical cation, called a polaron, is obtained and a counter “dopant” ion is introduced to stabilize the charge on the polymer backbone. When a second electron is removed from an oxidized section, a second polaron or a bipolaron is formed (Figure 1.2A). In p-doped conducting polymers, polarons and bipolarons are the main charge-carriers. Generally, a high density of charge-carriers and mobility of chargecarriers along the polymer chains, are the reasons for doped conjugated polymers being good conductors43. The polaron migration of all-trans-polyacetylene is shown in Figure 1.2B, as an example.
12
A -
Neutral
-e +
-
.
Polaron
-e +
Bipolaron
+
B
+
.
+
. +
.
Figure 1.2 (A) Formation of a polaron and a bipolaron, and (B) charge-carrier delocalization, for all-trans polyacetylene.
Conducting polymers can typically be synthesized electrochemically or chemically. In electrochemical polymerization, the polymerization process is initiated by placing working, counter and reference electrodes into a solution containing the relevant monomer and electrolyte and applying a suitable voltage. The polymer film then starts to form on the working electrode, which is normally a metal plate such as platinum or stainless steel. The advantage of an electrochemical synthesis is that the thickness of the 13
film can be controlled by varying either the potential or the current with time, while the time required for polymer generation is typically less than that for chemical polymerization.
However,
low
yields
restrict
the
large-scale
application
of
electrochemical syntheses. With chemical polymerization, monomers react with an excess of oxidant, which is dissolved in a suitable solvent, for a period time with constant stirring. Chemical polymerization is normally applied when attempting to generate bulk polymer samples, from which the products are normally powders.
Although polyacetylene was the first conducting polymer (CP) to be investigated, the application of this prototype conducting polymer was limited by its high environmental instability, as it degrades readily in air. Among intrinsically conducting polymers, polypyrrole, polyaniline and poly (3,4-ethylenedioxythiophene) (PEDOT) (Scheme 1.4) have been intensively investigated because of their excellent stabilities, specific properties and wide applications in technology, which are attracting increasing interest from researchers.
Polypyrrole (PPy) was first prepared electrochemically by Dall’Olio et al. in 196844. The polymer is now attractive due to its ease of preparation, high electrical conductivity (from 10-5 to 101 S cm-1)45, environmental stability and electrochemical properties. PPy is therefore being considered for a range of applications, such as an electrode for rechargable batteries46, sensors47, 48, corrosion-protecting materials49, 50 and actuators51. Great interest has also been generated in bioapplications of this conducting polymer owing to its good biocompatibility. PPy has been examined for use in the controlled
14
release of drugs by incorporating target species within the conducing polymer52, 53. PPy has also proven to be a suitable material for in vitro nerve cell culture and for in vivo implantation52, 54. Researchers have demonstrated a low toxicity for PPy when in contact with biological tissues. In addition, PPy particles did not induce a cytotoxic effect, including an allergic response, in experiments on mouse cells55, while a PPy extract solution showed no evidence of systemic toxicity when used to bridge the gap of a rat sciatic nerve 56.
Polyaniline (PANI) was first synthesized in the second half of 19th century57 and was studied at the beginning of the 20th century58-61. This conducting polymer has attracted interest as a conducting material (with the conductivity from 10-8 to102 S cm-1)62 owing to its inexpensive monomer, high yield, unique chemical and physical properties and remarkable stability63,
64
. It is stable to air, to moisture and to relatively high
temperatures; under these conditions most conducting polymers loose their interesting properties. Polyaniline (PANI) has also been widely investigated as a material for various applications, such as in batteries65, 66, electronic display devices67, corrosion coatings68, 69, chemical sensors70,
71
and for other uses72-75. It has been reported that polyaniline is
biocompatible and is suitable for tissue engineering applications76. PANI-based biosensors have been developed for DNA hybridization detection and for the examination of saccharides, such as glucose77, 78.
Polythiophene derivatives have received increasing attention because of their useful properties, including high stability. PEDOT, one of the most investigated polythiophene
15
derivatives, was developed in the late 1980s in Germany79-81. Various polythiophene derivatives show very high stability and conductivity in the oxidized state80-82 because of an alkylenedioxy substituent, which leads to a lowering of the electronic π → π* band gap and thus to an increase in conductivity (ca. 300 S cm-1)83 and stability82, 84, 85. Due to their unique properties, PEDOT has been reported to be suitable for many applications in materials science, such as an anticorrosive additive86, antistatic and conductive coating87, 88
. In bioapplications, PEDOT-based biosensors have been proven to be excellent for
long-term glucose measurements, because of their high electrochemical stability89, 90. It has also been demonstrated that PEDOT can be applied for controlled drug release by external electrical stimulation91.
16
H
H N
N H
H
H
N
N
N
Polypyrrole (
H N
H N︶
(
N︶
N
quinoid ring
Benzenoid ring
Polyaniline O
O
O
O
S
S
S
S
S
O
O
O
O
O
O
PEDOT Scheme 1.4 Structures of polypyrrole, polyaniline and PEDOT.
When they react with an oxidizing agent, neutral CPs lose electrons to form a polaron or a bipolaron. The oxidation of PEDOT is shown in Scheme 1.5, while the redox chemistry of PPy and PANI is discussed further in Chapter 3, p.63 and p.73.
17
O
O
O
O
-
O
-e
S S
O
O
O
O
+
+
S
O
S S
O
S O
O
-
O
O
O
S S
O
O
O
.
S
S
O
O
+
S
O
-e
S O
O
Scheme 1.5 Oxidation of PEDOT.
The morphology of conducting polymers has attracted a great deal of interest in recent years, because nanostructures of conducting polymers, such as nanowires, nanofibers and nanotubes have been reported to exhibit new chemical and physical properties92, 93, which can be useful in the materials sciences and for micro/nanodevices. Nano-structured CPs have also been reported to show superior chemical and physical properties to their conventional forms94-96. The large surface area of these nanostructures seems beneficial to their response and in prospective applications. 1.4 Conducting polymer composites Polymer composites are of great interest because they can combine the properties of various compounds97-99. Various combinations of different components added to polymers have been investigated to obtain materials with desirable new properties100. The methods for producing conducting polymer containing composites can be mainly 18
classified into two types: synthetic methods and blending methods. The former approach is based upon synthesis of CPs in the presence or inside a matrix of another polymer, which can be undertaken either by electrochemical or chemical approaches. However, similar to the preparation of CPs, electrochemical polymerization is only suitable for the preparation of thin layers of materials71,
101
. The blending method involves mixing a
previously prepared CP, usually as a powder, with a matrix polymer (eg. ethyl cellulose) 71, 100
. This can take the form of solution blending of the CP with a soluble matrix
polymer, or dry blending by melt processing. For large-scale production, the blending method is more important than the synthetic method. Of the blending techniques available, solution processing is generally simpler than melt blending.
The antioxidant activity of CPs has been studied and reported previously. PANI and its derivatives have been found to show antioxidant ability in the protection of rubber against oxidation, where the polymers were incorporated into both natural and synthetic rubbers102, 103. Conducting polymers, such as soluble forms of PPy and polyaniline that are commercially available, have been shown to exhibit reducing ability and DPPH free radical scavenging activity104, 105. The results indicated that the CPs act as free radical scavengers and are suitable for termination of free radical chain reactions that are the main causes of oxidations in biological system. However, for broadening the application of CPs, such as the development of packaging materials, it is important to investigate antioxidant activity of solid CPs and their composites. The antioxidant activity of polyaniline nanofibers has been investigated and it has been demonstrated that the CP nanofibers are efficient DPPH free radical scavengers, and more so with smaller diameter
19
nanofibers of a higher surface area95. Composite materials containing CP nanostructures possess hybrid properties that reflect both the incorporated CP nanostructures and the host matrix, which might provide synergistic effects and bring novel properties to the materials. This is the reason why the preparation and investigation of CP structures and their composites are a crucial part of this thesis.
Although the DPPH radical scavenging activity of CPs has been demonstrated, the reaction of CPs with other free radicals, including ABTS and peroxyl free radicals, has not been previously investigated, and is important because different antioxidants respond in various ways to different test free radicals. Therefore, a range of methods, based on different test mechanisms, have to be used to evaluate antioxidant capacity, a point stressed by other researchers2,
3, 22
. CPs and their composites have attracted much
attention in a range of proposed applications. In the food industry, the application of CPbased biosensors may be an alternative to conventional testing methods106, 107. Other uses of CPs and their composites in food science can also be considered, and the focus of the present thesis is the development of CP-based packaging materials with antioxidant properties.
20
1.5 Packaging Packaging is expected to not only to increase the economic value of products but also to preserve their qualities. Food packaging is traditionally used for protection, preservation and storage of sensitive food items. Intelligent packaging and active packaging are two modern concepts that are making exciting advances compared to conventional packaging. The first technology (intelligent packaging) applies to materials able to respond to changing environmental condition and to signal changes, such as temperature indicators and leakage indicators. The second (active packaging) relates to changes that can be brought about in the condition of packaged food by altering the package properties, such as antimicrobial, odor absorbing and oxygen scavenging108-110.
Active packaging is of great interest because of the increasing demand for better quality and longer shelf life with food products. The most widely used active packaging technologies for foods are in the removal of undesirable substances through absorption or/and scavenging, as with the widely used oxygen-scavengers (eg. iron-based oxygen scavenging sachet)109. Small molecule antioxidants have been commonly used as food additives to help protect food against oxidation because of their ability to scavenge free radicals. A few reports indicate that addition of an antioxidant, such as BHT, an artificial antioxidant, delays the oxidation of oil products111,
112
. However, the health risk of
synthetic antioxidants and a low thermal stability of natural antioxidants, have led to a consideration of alternative antioxidants113,
114
. Moreover, addition of low molecular
weight antioxidants may affect the taste and smell of a food.
21
Antioxidant packaging materials can be considered for use as active packaging to prevent the oxidation of foodstuffs.
The oxidation process normally occurs at the surface
between air and the foodstuff. If the packaging surface can include an antioxidant material, oxidation might be retarded. If a packaging material (of a high molecular weight) can itself exhibit antioxidant activity, it may be excellent for food packaging applications. Conducting polymers with high molecular weight (and thus not liable to leach into the food product) and tailored antioxidant properties, can be considered as antioxidant packaging materials.
1.6 Objectives of this thesis The focus of this PhD thesis is to develop conducting polymer antioxidant packaging materials, which are expected to act as free radical scavengers. The main aims of this project are: 1. Preparation of conducting polymer powders 2. Structural determination of CPs 3. Evaluation of antioxidant properties of conducting polymer powders 4. Preparation of conducting polymer films 5. Evaluation of antioxidant effects of conducting polymer films
PPy, PANI and PEDOT have been intensively investigated as conducting polymers with interesting properties and have proven suitable for bioapplications, and were therefore chosen for this project. Given the need to consider large scale production for packaging applications in the future, the CPs were prepared by chemical polymerizations and the
22
CPs were initially obtained as powders. To understand the influence of oxidants on the synthesis of CPs, different concentrations of ammonium persulfate were used in the polymerizations and the ratio required to prepare a more efficient antioxidant CP has been determined.
Scanning electron microscopy (SEM) has been used to study the surface morphology of the conducting polymers, since CP morphology plays an important role in the properties of both pure conducting polymers and their composites115-117. The measurement also provides information on particle size, which is related to the surface area of the conducting polymers and potential efficiency of the materials.
FTIR and Raman spectroscopic analyses has been applied as a diagnostic technique to identify the structures of the various CPs and to track changes in specific chemical groups118. The techniques also give special information that characterizes the state of oxidation or reduction of the CPs. In this project, FTIR and Raman spectra have been used to identify the chemical structures within the conducting polymers, particularly after various concentrations of oxidant have been used in their preparation, and to track changes in particular groups after reaction with the free radicals. For instance, when polymerizations are performed at high concentrations of the ammonium persulfate oxidant, the presence of C=O groups can be detected by IR (Figure 1.3A) and a shift in Raman peaks can also be observed, due to overoxidation (Figure 1.3B).
23
B Signal Intensity
Absorbance
A
C=O
(a)
(b)
600
750
900
1050
1200
1350
Wavenumber / cm
1500
1650
1800
1500
1550
-1
1600
1650
Wavenumber / cm
1700
-1
1600
C
D
Signal Intensity
1400
CPS
1200 C-N 1000 +
C-N C=N
800
600 332.5
335.0
337.5
340.0
394
342.5
396
398
400
402
404
406
Binding Energy / eV
Magnetic Field / mT
Figure 1.3 Representative conducing polymer spectra: (A) FTIR spectrum of overoxidised PPy; (B) Raman spectra of (a) regular and (b) overoxidised PPy; (C) ESR spectra of PPy; (D) XPS (N1s) spectra of PANI.
Electron Spin Resonance (ESR) spectroscopy is commonly used to detect electrontransfer processes in specific systems. The ESR signals of the samples indicate the presence of free radicals and polarons. ESR has been applied for evaluating the level of radicals in CPs prepared using various ratios of oxidants to monomers (Figure 1.3C).
24
Moreover, the technique has also been used to measure the percentage of remaining free radicals after reaction with antioxidants. In this project, the technique has also been used to predict the main charge carrier within the CPs, namely polarons or bipolarons, alongside a consideration of the doping level of the CPs, as discussed further in chapter 3, p. 54 and p. 82.
X-Ray Photoelectron Spectroscopy (XPS) is a very common analytical technique applied to CPs, which is used to analyse the surface (a few nanometers) chemistry of the tested materials. The wide-spectrum scan is utilized for elemental analysis to determine the percentage of each atom in the tested sample, while high resolution (core-level) XPS is very useful for determining the oxidation states of polymer segments, hence elucidating the redox state of the polymer. It is therefore a useful technique to study the structural changes of PANI powders during reactions with free radicals. In the case of PANI, using core-level XPS, the N1s spectra can be deconvoluted into three main environments: C-N, C=N and C-N+ (Figure 1.3D), and the areas under each curve can provide a quantification of the three components. The [C=N] / [C-N] ratio can also be used to evaluate the ratio of quinonoid to benzenoid rings and therefore provide information on structural changes occurring with PANI under various circumstances.
The antioxidant activity of PPy, PANI and PEDOT powders has been determined by considering the free radical scavenging property of these CPs. The DPPH and ABTS assays have been applied to evaluate the free radical scavenging effects of CPs. The
25
results are compared and the most efficient conducting polymer samples have then been used for the preparation of antioxidant packaging materials. Considering a simple process for the preparation of conducting polymer packaging materials, blends that combine a CP and a processable plastic have been prepared mainly using a solution blending method. In some cases melt processing and a synthetic method have also been employed to produce conducting polymer composites. When using a blending method, the previously prepared conducting polymer powder has been mixed with the conventional polymer. For the synthetic method, a conducting polymer has been synthesized in a matrix of a conventional polymer containing an oxidant. The free radical scavenging activity of the conducting polymer packaging materials has then been evaluated using the ORAC assay.
The main reason for using various methods, including ET based methods, such as the DPPH and ABTS assays, and an HAT based method, such as the ORAC assay, to determine the antioxidant capacity of CPs is to understand the various reaction possible mechanisms between CPs and the scavenged free radicals. In addition, investigations have been performed using different systems, including hydrophilic and hydrophobic phases, to allow an understanding to develop of the most suitable circumstances for applying CPs as antioxidants.
Foodstuffs that are easily attacked by oxygen free radicals have also been examined. Fish oils, such as Ropufa oil, are rich sources of polyunsaturated fatty acids, especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), which are highly
26
unsaturated fatty acids and are easily attacked by oxygen free radicals, and these have been tested with CP antioxidant samples. In order to investigate the influence of conducting polymer materials on the retardation of the oxidation of foodstuffs in contact with the packaging material, the level of peroxides in the fish oil has been monitored by measuring the peroxide value (PV).
Refereed publications from the research to date: 1. C. F. Hsu, L. Zhang, H. Peng, J. Travas-Sejdic, and P. A. Kilmartin, Scavenging of DPPH free radicals by polypyrrole powders of varying levels of overoxidation and/or reduction, Synthetic Metals 158 (2008) 946 2. C. F. Hsu, L. Zhang, H. Peng, J. Travas-Sejdic, and P. A. Kilmartin, Free radical scavenging properties of polypyrrole and poly (3,4-ethylenedioxythiophene), Current Applied Physics 8 (2008) 316 3. C. F. Hsu, H. Peng, L. Zhang, J. Travas-Sejdic, and P. A. Kilmartin, Structural changes in polyaniline upon reaction with DPPH•, e-Journal of Surface and Nanotechnology 7 (2009) 269
27
Chapter 2 Experimental Methods
2.1 Introduction In this research, conducting polymers (CPs) were prepared using both chemical and electrochemical methods.
To make films of conducting polymers blended with
conventional polymers, both solution-based and melt-processing methods were tried. The antioxidant activities of the resulting CPs, including powders and films, were evaluated using the DPPH, ABTS and ORAC assays, which have been commonly used for the evaluation of the antioxidant capacity of beverages and food stuffs. In these assays, UVvisible spectroscopy was typically used to monitor changes in concentrations of free radicals as they reacted with the conducting polymer antioxidants. The influence of CPs on the oxidation of oils was investigated using gas chromatography (GC) analysis of fatty acid profiles, and by measuring the peroxide value (PV) of the tested oil. In order to examine the structures of the CPs before and after reaction with free radicals, various spectroscopic methods, including FTIR, Raman, X-ray Photoelectron Spectroscopy (XPS) and Electron Spin Resonance (ESR), were applied. Cyclic voltammetry (CV) was also used to evaluate the internal redox properties of the CPs. The conductivity of the CPs and the mechanical properties of the conducting polymer films were also determined.
28
2.2 Materials Pyrrole, aniline, 3,4-ethylenedioxythiophene (EDOT), hydrazine, ammonium persulfate (APS), potassium persulfate, 2,2’-diphenyl-1-picrylhydrazyl(DPPH), dimethyl sulfoxide (DMSO), dodecylbenzenesulfonic acid (DBSA), 2,6-Di-tert-butyl-4-methyl-phenol (BHT), α-tocopherol and tridecanoic acid were obtained from Sigma-Aldrich. Fluorescein
(3’,6’-dihydroxyspiro[isobenzofuran-1[3H],9’[9H]-xanthen]-3-one)
was
purchased from BDH Chemicals Ltd, Poole, England. Ethyl cellulose (100 cps) and 2-2’Azobis(2-amidinopropane) dihydrochloride (AAPH) was purchased from Acros, USA. 3,4-dihdro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-carboxylic acid (Trolox) was purchased from Fluka, Dänemark. 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was obtained from Fluka, Germany. Ropufa oil and avocado oil were obtained from DSM Nutritional Ltd. FAME fatty acid standards were obtained from Supelco, USA.
Table 2.1 Formula and molecular weight of the chemicals. Chemical Pyrrole Aniline EDOT Ammonium persulfate Potassium persulfate Hydrazine DPPH ABTS AAPH Fluorescein Trolox BHT α-Tocopherol
Formula C4H5N C6H7N C6H4O2S (NH4)2S2O8 K2S2O8 N2H4 C18H12N5O6 C18H18N4O6S4 C8H18N6 · 2HCl C20H12O5 C14H18O4 C15H24O C29H50O2
29
Molecular Weight (g mole-1) 67.09 93.13 142.18 228.18 270.32 32.05 394.32 514.62 271.19 332.31 250.29 220.36 430.69
HN
NO 2
.
N
O 2N
H2N
N
N=N
NO2
NH2 NH
AAPH
DPPH H3C N O 3S
OH
N
S
O
SO 3
S
N N CH3
HO
O
ABTS
Fluorescein
CH3
OH
HO
O
H3C
O CH3
OH CH3
BHT
Trolox HO O
α-Tocopherol
Scheme 2.1 Chemical structures of some of the key chemicals used in this research.
30
O
2.3. Polymerization of polypyrrole, polyaniline and poly(3,4-ethylenedioxythiophene) powders Pyrrole (Py) was distilled under reduced pressure and stored in the dark under nitrogen. PPy powders were synthesized chemically at room temperature by adding 25 mL of different concentrations (0.2M, 0.3M, 0.4M and 0.5M) of an APS solution to Milli-Q water (25 mL) containing pyrrole (5 mmol), with vigorous stirring. After reacting for 2 hr, the PPy powders were filtered, washed several times with distilled water and methanol, and dried in a vacuum oven at 35 oC. PANI and PEDOT powders were polymerized using the same method as for PPy but using a 24 hour reaction time for PANI and 48 hours for PEDOT. Using PANI as an example, the procedure is illustrated in Figure 2.1.
Figure 2.1 Preparation of PANI powder.
31
2.4 Reduction of PPy, PANI and PEDOT powders and preparation of dedoped PANI powder To gradually reduce PPy, PANI and PEDOT powders (30 mg), these were reacted with hydrazine which was added dropwise into Milli-Q water (10 mL) for 30 min. For more complete reduction, a larger addition of 2 mL of hydrazine was added and for a 48 hour reaction period. PANI powder (30 mg) was dedoped (deprotonated) with 10 mL of 15 % ammonium water for 48 hrs.
2.5 Elemental analysis Elemental analyses of the PPy samples were carried out at the University of Otago, Dunedin, New Zealand. The results were used to calculate the doping levels of the samples, given by the level of bisulfate (HSO4-) and sulfate (SO42-) dopant, the ratio of which was estimated using the pH of the solution containing the prepared powders. The doping level is defined as the average number of positive charges per monomer unit in the polymer, so that the doping level with a doubly charged anionic dopant, such as SO42-, is thus twice the number of anions per monomer unit.
2.6 Preparation of PANI EC film 2.6.1 Solution blending of EC and previously prepared PANI powders Ethyl cellulose (EC) powder (5 g) was dissolved in 60 mL of ethanol by stirring for 1 hr. As-prepared polyaniline or reduced PANI powder (500 mg) dispersed in 40 mL ethanol was sonicated for 5 min and mixed with the ethyl cellulose solution with stirring for 3 hrs. The mixture was then concentrated to around 60 mL using a vacuum rotator (Büchi
32
Rotavapor R-114) at 30 oC for 30 min. The blended film was made by casting the mixture on a Teflon sheet and evaporating the casting solvent at room temperature for ca. 3 hrs. 2.6.2 Reduction of PANI EC film PANI EC film was prepared as described in section 2.6.1. The film was reduced by reacting with hydrazine of the same concentration for CPs powders, as given in section 2.4 above, for 48 hrs.
2.6.3 Preparation of an in situ oxidatively polymerized PANI(OX)EC film One gram of ethyl cellulose (EC) powder was dissolved in ethanol by stirring for 1 hr. Aniline (0.2 g) was then added into the EC ethanolic solution with stirring for 10 min. APS (0.74 g) was suspended in ethanol and then added into the mixture of EC and aniline. The molar ratio of APS to PANI was 1.5:1. After reaction for 24 hrs, the mixture was concentrated and degassed using a vacuum rotator and cast onto a Teflon sheet. The resulting film was then rinsed with plenty of milli-Q water to remove the residual APS as illustrated in Figure 2.2.
APS
aniline +EC
24 hr
Air dry
Stirring
Figure 2.2 Preparation of PANI(OX)EC film.
33
Rinse with water
Air dry
Using this method, and assuming that the percentage of ANI applied for synthesizing PANI is the same as in the polymerization described in section 2.3, about 76 % of the ANI should be converted to PANI, i.e. about 0.15 g (Table 3.4 and 3.5). Associated with this will be (bi)sulfate dopants, typically a third of the weight of PANI itself, i.e. a further 0.05 g. Upon combination with 1.0 g of ethyl cellulose, the 0.2 g of doped PANI represents about 17 % of the blend, with 83 % ethyl cellulose.
2.7 Preparation of PANI PE film PANI powder prepared with an APS/PANI ratio of 1.5 was dedoped with ammonium water (15 %) for 24 hrs and redoped with DBSA (90 %). The ratio of DBSA to dedoped PANI was 3:1 (by weight). 50 g of redoped PANI was then blended with 450 g of polyethylene (PE) using a blender (Brabender, Model: FDO 234S, Germany) and pressed using compression molding (Carver, Model: 4332, IN, USA) to form a film of 120 ± 10 μm in thickness. The temperature used for the blending was 180 oC.
2.8 Preparation of PANI and PPy films electrochemically A PANI film was prepared in a 0.1 M H2SO4 solution with 0.1 M ANI using a stainless steel as working electrode, Ag/AgCl as reference electrode and a platinum counter electrode, and operated at 1.1 V for 4 hours. A PPy film was prepared in a 0.1 M sodium bisulfate solution with 0.1 M of Py using the same procedure but conducted at 0.8 V.
34
2.9 Evaluation of antioxidant capacity of CPs powders and films 2.9.1 DPPH assay The DPPH free radical scavenging capacity of each sample was carried out by adapting methods used previously for soluble forms of conducting polymers104, 105. Details of the method development are provided in section 3.3. The final testing procedure involved adding 1.0 mg of each test powder, measured using a 5 digit balance to ensure a weight in the range between 1.00 and 1.05 mg (weighing error probe spacing) for conductivity measurements at a Jandel Multi Height four-point probe with a DC current
38
source at ambient temperature. The probe spacing of this device is (6.35 ± 0.02) ×10-2 cm. The conductivity can be calculated by the equation below:
σ ( S cm −1 ) =
1 2π s
×
I V
(eq. 2.2)
where s is the spacing of the probe, I is the test current and V is the measured voltage.
2.10.4 Cyclic Voltammograms of PANI powders The electrochemical properties of the PANI powders were examined using a BAS100B electrochemical analyzer. The sample powders were dispersed in CHCl3 (2 mg in 5 mL) and the dispersion was then dropped onto a 3 mm diameter glassy carbon plate working electrode to form a film. The cell was filled with 0.1 M HCl was purged with N2 for approximately 10 min. Cyclic voltammograms were recorded in the potential range from -300 to +850 mV at a scan rate of 20 mV s-1, using an Ag/AgCl (+207 mV vs. SHE) reference electrode and a Pt counter electrode.
2.11 Mechanical properties of PANI films
Tensile tests (ASTM D638) of the PANI EC films were performed on film strips of 8.0 × 1.5 cm in dimension at room temperature using an Instron 1185 Universal Tensile Machine. The tensile modulus, between 0.05% and 0.25% strains, and ultimate tensile strength were conducted at a crosshead speed of 5 mm/min. For each test, at least three sample measurements were averaged.
39
2.12 Measurement of the peroxide value (PV) of oil samples
3.0 g of fish oil was dissolved in 30 mL of mixed solution of chloroform and glacial acetic acid (2:3). 0.5 mL of freshly prepared saturated potassium iodide (KI) solution, which was prepared by dissolving excess KI (about 10 g) in 6 mL of deionised water, was added to the fish oil solution and occasionally shaken for exactly 1 minute. After the immediate addition of 30 mL of deionised water, the liberated iodine was then titrated with 0.05 N of sodium thiosulfate solution with constant agitation using soluble starch solution as an indicator. Each experiment was conducted in triplicate. The PV was calculated as following:
PV (mEq peroxides / Kg oil ) =
( S − B) × N × 1000 mass of sample ( g )
(eq. 2.3)
where B is the volume of titrant for the blank run (mL), S is the volume of titrant for the sample (mL), and N is the normality of the Na2S2O3 solution.
2.13 Determination of fatty acids in fish oil
30 mg of Ropufa oil with 300 μL of internal standard (tridecanoic acid) were saponified by reaction with 1 mL of saponifying agent at 65 oC in a water bath for 20 min and then methylated by reaction with 3 mL of methylating agent at the same temperature for 10 min. After cooling down to room temperature, the fatty acid methyl esters were extracted with 10 mL of hexane with shaken well for 2 min. Using anhydrous sodium sulfate to remove moisture, the hexane layer was analyzed by gas chromatography (GC) (Hewlett Packard GC series (11) Paolo Alto, CA), which is equipped with FID detector and a DB 40
225 column (J & N Scientific, USA) with 0.25 mm internal diameter, 0.25 μm film thickness and 15 m length. The injection volume was 1 μL. The temperature of the injector and detector was maintained at 230 oC and 250 oC, respectively. The oven was heated from 50 oC to 175 oC at a rate of 25 oC / min and further heated to 225 oC at a rate of 4 oC / min and held at 225 oC for 5 minutes. The inlet pressure of the carrier gas (helium) was held at 10 psi. Internal standard, saponifying agent and methylating agent were prepared as described below.
2.13.1 Preparation of the GC internal standard Tridecanoic acid was dissolved in hexane to a concentration of 4 mg/mL.
2.13.2 Preparation of saponifying agent 1 g of sodium hydroxide was added into 50 mL of methanol and sonicated to dissolve completely.
2.13.3 Preparation of methylating agent 60 mL of methanol and 3 mL of conc. sulfuric acid were added to 2 g of ammonium chloride in a conical flask. The mixture was then heated at 50 oC in a water bath for 20 minutes with a cold finger on the top.
41
Chapter 3 DPPH scavenging activity of conducting polymers
3.1 Introduction
Polypyrrole (PPy), polyaniline (PANI) and poly (3,4-ethylenedioxythiophene) (PEDOT) have received much attention due to their interesting properties. Soluble PPy and PANI forms available commercially from Sigma-Aldrich have been reported to show free radical scavenging activity104. Investigation of the antioxidant capacity of these conducting polymers (CPs) in solid powder forms is then of great interest, owing to the possibility of using the CP powders as an antioxidant material in biomedical or food packaging applications, and is the focus of the present study.
In this chapter, the ratio of ammonium persulfate (APS) oxidant to pyrrole (Py), aniline (ANI) and 3,4-ethylenedioxythiophene (EDOT) monomer in the preparation of PPy, PANI and PEDOT powders, to provide optimum free radical scavenging, has been examined. The radical scavenging capacity has been investigated using the α,α-diphenylβ-picrylhydrazyl (DPPH) assay, adapted for use with conducting polymer powders, an assay which is commonly used to test the antioxidant capacity of molecules 3, 123-125.
3.2 Experimental
PPy, PANI and PEDOT powders were prepared as described in section 2.3. Reduced PPy and PANI, and dedoped PANI were prepared as described in section 2.4. The DPPH free radical scavenging capacity of each sample was carried out by modifying methods used
42
previously for soluble forms of conducting polymers 104, 105, as described in section 2.9.1. SEM images of these sample powders were determined, as described in section 2.10.1. Characterization of polypyrrole, polyaniline and PEDOT powders were carried out by using FTIR, Raman, Electron Spin Resonance (ESR) and X-ray photoelectron spectroscopy (XPS), as described in section 2.10.2. The conductivity of the powders was measured as described in section 2.10.3, and cyclic voltammograms of the PANI samples were obtained as described in section 2.10.4.
3.3 Development of a DPPH assay for conducting polymer powders
In our previous reports on the use of the DPPH assay to examine the radical scavenging ability of conducting polymers, mainly in soluble forms, 1.5 mL of a 72 μM solution of DPPH radicals in methanol was employed, which showed an initial absorbance reading of about 0.65 units at 516 nm 104. During the reaction between a conducting polymer and DPPH•, proton and electron transfer are expected to occur to the DPPH radical leading to decolorisation 104, 126:
DPPH• + H+ + e- → DPPHH
(eq. 3.1)
However, it was found that 1.0 mg of a PPy powder (APS/pyrrole ratio of 1.5) removed all of the DPPH radicals in a very short time, which did not allow comparisons of the radical scavenging properties between different PPy preparations to be made. Instead 20.0 mL of a 255 µM methanolic DPPH• solution was found to produce final absorbance
43
values in the 0.5 to 1 range for 1.0 mg samples, while the high initial absorbance reading of around 2.6 units was still within the range of the spectrophotometer.
In Figure 3.1 the decline of the absorbance at 516 nm (and thus in the level of DPPH free radicals) in the presence of 1.0, 2.0 and 5.0 mg of PPy powder over a 24 hour period is presented. In the absence of any conducting polymer powder, the level of DPPH radicals was found to decline by only a small amount during the first two hours, but after 24 hours the absorbance reading had declined from 2.6 to around 2.1 units, representing a 20% loss during the 24 hour period. The decrease of the absorbance might be the result of a termination reaction (DPPH• + DPPH• → nonradical products) involving the DPPH radicals3. The scavenging of DPPH radicals due to the conducting polymer was therefore determined using the difference between the control run with no added conducting polymer and the absorbance reading with the polymer present. The addition of 1.0 mg of PPy lowered the absorbance reading to below 2 units within an hour (a loss of 0.64 units), and to a reading of 0.93 after 24 hours (1.2 units different from the control run). This result indicates that part of the PPy polymers exhibit a high reducing strength and can react rapidly with the DPPH radicals, while other parts are either less accessible (in the interior of the powder particles) or require a higher potential to be oxidised and thus react more slowly with DPPH•, previously shown to be a weak oxidising agent
126
. The
addition of 2.0 mg of the PPy powder led to nearly twice the loss of DPPH• in the first hour (absorbance drop by 1.20 units) and a final decline of 1.8 units after 24 hours. By contrast, when 5.0 mg of PPy was added, the absorbance reading fell to a value of less
44
than 0.2 units within 2 hours, showing near complete removal of DPPH• in a short period of time, with a final reading of 0.094 units after 24 hours.
It was thus concluded that with 20.0 mL of the 255 µM methanolic DPPH• solution, 1.0 mg PPy samples could be reliably assessed for their radical scavenging abilities. When the reaction was continued for longer periods, the absorbance values were found to decrease by no more than 0.1 units after a further 5 days, with a similar decrease seen in the DPPH• solution in the absence of added conducting polymer, hence a 24 hour test period was used to evaluate the radical scavenging properties.
Absorbance at 516nm
2.5
(a)
2.0
1.5
1.0
(b)
0.5
(c) (d)
0.0 0
5
10
15
20
25
30
Tim e / hr Figure 3.1 Decline in the absorbance at 516 nm of a 255 µM methanolic DPPH• solution with (a) nil, (b) 1.0, (c) 2.0 and (d) 5.0 mg of PPy powder added (formed using an APS/pyrrole ratio of 1.5).
45
Two further control experiments were undertaken. In the first procedure, an excess of ascorbic acid (2.2 mg) was added, as required to react fully with the 255 µM of DPPH•. Within a few minutes the absorbance reading declined to a value of 0.07 units, where it remained steady even after 24 hours. This absorbance reading was the most that would be contributed by the fully reduced DPPHH produced in the reaction, and no attempt was made to correct for the small absorbance contribution of DPPHH in later experiments. In a further experiment, 1.0 mg of a PPy powder was left in methanol for 24 hours to see if any soluble PPy forms would develop and contribute to the absorbance reading, but this value only reached 0.006 units after 24 hours. Therefore any dissolution of PPy into the methanolic solution is not expected to interfere with the radical scavenging result.
3.4 Antioxidant activity of PPy
3.4.1 Polymerization of PPy PPy powders were synthesized chemically by oxidative polymerization of pyrrole in an aqueous solution containing various levels of the APS oxidant. Similar to the polymerization mechanism of most conducting polymers, pyrrole is oxidized to a polymer and further oxidized to a cationic form of PPy doped with anions, with a positive charge on at most every 3-4 pyrrole units
127
. The equations given in scheme 3.1
summarise the polymerization processes for PPy; in theory an APS to pyrrole ratio of 1.15 is required for complete oxidation of pyrrole to a fully doped polymeric form.
46
+ 2H+ + 2e-
1/n N H
N H S2O82- + 2e-
n
2SO42+
N H
N H 1/2S2O82- + eSO42- + H+
+ e-
SO42HSO4-
Scheme 3.1 Chemical polymerisation of pyrrole.
With APS as the oxidant, bisulfate, HSO4-, and sulfate, SO42-, are expected to be the main dopant anions, formed from the reduction of APS during the chemical polymerisation, with sulfate able to act as a dopant for two positive PPy centres, but bisulfate only one. A mixture of bisulfate and sulfate is expected given that pKa2 for sulfuric acid is 1.99128, and a final solution pH in the range of 1-2 units was obtained (Table 3.1). A lower pH was obtained for a higher level of APS as more protons were released when more pyrrole groups were oxidised. The predicted ratio of [HSO4-]/[SO42-] was determined using the following equation: pKa – pH = log [HSO4-]/[SO42-]
(eq. 3.2)
47
Table 3.1 pH value of the PPy reaction mixture after 2 hours. APS/pyrrole for preparing PPy
0.25
0.5
1.0
1.5
2.0
Final pH value
1.61
1.44
1.26
1.23
0.99
Predicted [HSO4-]/[SO42-]
2.40
3.54
5.37
5.75
10
Table 3.2 shows the results of an elemental analysis of the PPy powders, including the molar percentage of sulfur to pyrrole units.
In each case the S/pyrrole ratio was
calculated based on the molar ratios of sulfur to nitrogen.
Table 3.2 Elemental analysis results for PPy samples prepared with different APS/pyrrole ratios. APS/pyrrole
0.25
0.5
1.0
1.5
2.0
C (mass %)
52.65
52.75
51.95
50.92
48.75
H (mass %)
3.78
3.89
3.97
3.77
3.61
N (mass %)
14.90
15.14
15.02
14.66
14.03
S (mass %)
3.28
3.3
3.14
2.07
1.03
O (mass %)
25.39
24.92
25.92
28.58
32.58
S/pyrrole (mol %)
9.7
9.6
9.2
6.2
3.2
48
The doping level of PPy samples was then calculated making use of the [HSO4-]/[SO42-] ratio at the final pH of the preparative solution; the doping level of PPy is equated to the sum of the number of bisulfate and twice the number of sulfates present per monomer unit.
The values are plotted in Figure 3.2 versus the APS/pyrrole ratio employed to form the polymers. Despite evidence for more pyrrole oxidation with a higher APS/pyrrole reaction mixture, a higher doping level was obtained when less APS was employed. Likewise the conductivities of the PPy powders were found to decrease markedly for the higher APS/pyrrole preparations (also given in Figure 3.2); the conductivity was less than 10-3 S/cm for the APS/pyrrole ratio of 2.0. The findings are consistent with earlier reports that a high oxidant concentration leads to low conductivity in PPy
129
, that a higher
conductivity in PPy is associated with a higher doping level 118, and that properties of the oxidant solution influences the conductivity of PPy 130.
49
8
-2
4
-4
-1
0
log Conductivity / S cm
Doping Level / % charged pyrrole units
12
0 0.0
0.5
1.0
1.5
2.0
APS/Pyrrole Ratio
Figure 3.2 Doping level (■) and conductivity (▲) of PPy powders prepared with various ratios of APS/pyrrole.
SEM images of the PPy powders obtained from different APS/pyrrole ratios are shown in Figure 3.3. The polymer particles cluster together with diameters of 70 to 250 nm and are spherical in shape, similar to previous SEM images prepared for PPy 129, 131, 132. However, no clear difference in PPy particle size was evident as the APS/pyrrole ratios used to prepare the PPy powders was varied in these experiments.
50
A
C
B
D
E
Figure 3.3 SEM images of PPy prepared with APS/pyrrole ratio of (A) 0.25 (B) 0.5 (C) 1.0 (D) 1.5 (E) 2.0.
3.4.2 Spectroscopic characterisation The FTIR spectra of the five PPy powders are presented in Figure 3.4. Typical PPy bands are seen at 1550 cm-1 (C=C stretching), 1184 cm-1 (C-N stretching), 1298 cm-1 (CC stretching), and at 1042 cm-1 and 1090 cm-1 due to C-H in-plane vibrations. On the other hand, the peak at 1690 cm-1 is induced by a C=O absorption, ascribed to overoxidation of PPy 133. This band was strongest for the PPy powders prepared with an APS/pyrrole ratio of 1.5 or 2, indicating that PPy was susceptible to overoxidation when the higher levels of APS were used.
51
Absorbance
ν C-N
ν C=C ν C-C
(a) (b)
δ C-H
(c) ν C=O
(d) ν C=N
(e)
(f) 600
800
1000
1200
1400
W avenum ber / cm
1600
1800
2000
-1
Figure 3.4 Comparison of the FTIR spectra of PPy powders prepared with different APS/pyrrole ratios, from top to bottom: (a) 0.25, (b) 0.5, (c) 1.0, (d) 1.5, (e) 2.0 and (f) reduced PPy 2.0.
The overoxidation of PPy is shown in Scheme 3.2A, which has been reported to occur in two stages: a ß-C group is initially oxidized to C-OH, and then further oxidised into C=O, accompanied by a loss of positively charged -NH+- groups and the removal of counter anions
134-137
. This also provides an explanation for the very low doping level exhibited
by the PPy powders prepared with a high APS/pyrrole ratio (Figure 3.2).
52
O
OH
A
N XH
B
N H
N XH O
NNH2 H2NNH2
N H
-H2O
N H
Scheme 3.2 A: Overoxidation processes for PPy. B: Initial reaction phase for a WolffKishner reduction of the carbonyl group of overoxidised polypyrrole to a hydrazone.
As seen in Figure 3.5A, there was little immediate difference observed in the Raman spectra of the five PPy samples.
However, on closer examination the main C=C
stretching vibration was seen to move from 1597 cm-1 for PPy0.25 through to 1608 cm-1 for PPy2 (Figure 3.5B). According to previous research 138, the upwards shift of the 1600 cm-1 peak can be due to overoxidation, which leads to instability of the pyrrole rings owing to the presence of carbonyl groups 139.
53
Signal Intensity
A
200
400
600
800 1000 1200 1400 1600 1800 2000
Wavenumber / cm
-1
Signal Intensity
B (a) (b) (c) (d) (e) 1400
1500
1600
1700
Wavenumber / cm
1800
-1
Figure 3.5 Raman spectra of PPy powders formed with different APS/pyrrole ratios, from top to bottom: (a) 0.25, (b) 0.5, (c) 1.0, (d) 1.5 and (e) 2.0; (A) in the range of 200-2000 cm-1, and (B) highlighting the peak at around 1600 cm-1.
54
ESR spectrometry has been commonly used to monitor electron-transfer processes
140
,
and the ESR signal indicates the level of free radicals/ polarons in conducting polymers. Scheme 3.3 shows the polaron and bipolaron structures which are believed to be the main charge carriers within polypyrrole. ESR measurements were used to examine the level of free radicals in the PPy powders.
HN
.
HN NH
NH
+
NH
NH
HN
HN
NH
NH
+
NH
NH
+
HN
HN
HN
HN
polaron
bipolaron
Scheme 3.3 Polaron and bipolaron forms of oxidised PPy.
As shown in Figure 3.6, the PPy powders prepared using a lower level of the APS oxidant, but which were shown above to have a higher doping level (Figure 3.2), displayed a small ESR signal and thus a lower level of free radicals.
The likely
explanation is that spinless bipolarons were the main charge carriers in this case. The presence of bipolarons was confirmed by changing them into polarons using hydrazine, a strong reducing agent. As observed in Figure 3.7, the ESR signal became stronger upon the dropwise addition of hydrazine, indicating that polarons were being formed at the
55
expense of some of the bipolarons. As the addition of hydrazine continued, a lowering of the signal resulted due to the reduction of some of the polarons. The results demonstrate that bipolarons were the main charge carriers in PPy prepared with a lower APS/pyrrole ratio.
Signal Intensity
(a)
332.5
(b) (c) (d)
(e)
335.0
337.5
340.0
342.5
Magnetic Field / mT
Figure 3.6 ESR spectra of the PPy powders formed using different APS/pyrrole ratios, from top to bottom: (a) 1.5, (b) 2.0, (c) 1.0, (d) 0.5 and (e) 0.25.
56
Integral of Signal Intensity
4000
3000
2000
1000
0 0
100
200
300
400
500
600
700
Concentration of NH2NH2 (mM)
Figure 3.7 The change in the EPR signal for 30 mg of a PPy powder in 10 mL of Milli-Q water upon reduction with various amounts of hydrazine.
3.4.3 DPPH free radical scavenging activity of PPy powders The DPPH free radical scavenging ability of the PPy powders prepared using different APS/pyrrole ratios are shown in Figure 3.8. The PPy powders prepared with an APS/pyrrole ratio of 0.25 or 0.5 (PPy0.25 and PPy0.5) showed the highest radical scavenging capacity. PPy powders from an APS/pyrrole ratio of 1 or 1.5 showed a less intense response (more apparent after 24 hours than after the first hour of the reaction),
57
while the PPy prepared with a ratio of 2 (PPy2) had the lowest response. Very good repeatability was obtained with the duplicate analyses, with an average relative difference of 5 %, and no more than 9 %. A lower level of oxidant in the polymerisation thus led to
Absorbance at 516nm
powders which showed more effective free radical scavenging.
2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
(a) *
(b) (c) (d) (e) (f) 0
5
10
15
20
25
Time / hr
Figure 3.8 DPPH• scavenging capacity of PPy synthesized with different APS/pyrrole ratios: (a) DPPH• only, (b) 2.0, (c) 1.5, (d) 1.0, (e) 0.25, (f) 0.5. uncertainty.
58
*: experimental
The 1.0 mg of PPy sample represents 1.08 × 10-5 moles and 1.00 × 10-5 moles of pyrrole units for PPy0.5 and PPy2 respectively (using the N level in Table 3.2 to exclude the mass contribution of the dopant), while 20.0 mL of 255 µM DPPH• solution represents 0.51 × 10-5 moles of DPPH•. As seen in Figure 3.8, the PPy0.5 powder reduced 70 % of the DPPH• in 24 hours (or a 3 to 1 ratio of pyrrole units to DPPH•). By contrast the PPy2 powder reduced 50 % of the DPPH• in 24 hours (a 4 to 1 ratio of pyrrole units to DPPH•).
3.4.4 Properties of fully reduced PPy powders Additional PPy samples, prepared using APS/pyrrole ratios of 0.5 and 2.0, were subject to a fuller reduction process by treatment of 30 mg samples with 2 mL of hydrazine for 48 hours. As a result of the reduction process, the ESR signal was found to decline in both cases, the RedPPy0.5 sample falling to an integrated signal intensity value of 480 (34 % of the initial value), showing that the increased signal seen in Figure 3.7 for small initial additions of hydrazine, and conversion of bipolarons to polarons, was followed by a large drop in the concentration of polarons during the fuller reduction process. With PPy2, the high initial ESR signal fell to 30 % of its starting value after reduction with hydrazine.
The dedoping achieved through the hydrazine reduction was also seen in the results of elemental analyses (Table 3.3). There was a complete loss of the (bi)sulfate dopant in each case consistent with the reduction of positively charged pyrrole units and the release of counter anions from the powders. On the other hand, an increase in the nitrogen content (relative to carbon) was observed, by 15 % in the case of PPy0.5, and by 55 % for
59
the PPy2 sample.
While some sample degradation cannot be excluded, the likely
explanation is a conversion of carbonyl groups, produced due to overoxidation and present in larger amounts in the PPy2 sample, to a hydrazone as indicated in Scheme 3.2B, the initial step in the well-known Wolff-Kishner reduction involving hydrazine 142
141,
. This result would indicate that a carbonyl group would have been present on 27 % of
the pyrrole groups in the PPy2 sample, and 8 % of the pyrrole groups for PPy0.5 At the same time, FTIR scans of the reduced powders revealed an absence of the 1690 cm-1 band seen weakly for PPy0.5 and prominently for PPy2 (Figure 3.4), while a new band at 1640 cm-1 was seen in the RedPPy2 sample (Figure 3.4(f)), which can be ascribed to the hydrazone structure
143, 144
. Further, the loss of oxygen expected from the removal of
(bi)sulfate dopant and the conversion of carbonyl groups to hydrazones accounted for the majority of the actual decline in oxygen content (relative to carbon) in the samples (Table 3.3), with further oxygen loss expected from loss of water which can accompany the counterion dopants. The small decrease in hydrogen seen in PPy0.5 can be associated with losses of both water and bisulfate, while the increase in hydrogen seen in PPy2 can be related to the larger conversion of carbonyl to hydrazone groups.
60
Table 3.3 Elemental analysis results (mass %) for PPy samples prepared with an APS/pyrrole ratio of 0.5 or 2.0, before (PPy0.5 and PPy2) and after reduction with hydrazine (RedPPy0.5 and RedPPy2), with molar ratios given in brackets, normalised to a value of 4 for carbon in each case. APS/pyrrole
PPy0.5
RedPPy0.5
PPy2
RedPPy2
C (mass %)
52.75 (4)
58.53 (4)
48.75 (4)
50.42 (4)
H (mass %)
3.89 (3.52)
4.06 (3.31)
3.61 (3.53)
4.07 (3.85)
N (mass %)
15.14 (0.98)
19.34 (1.13)
14.03 (0.99)
22.54 (1.53)
S (mass %)
3.3
0
1.03 (0.03)
0
O (mass %)
24.92 (1.42)
18.07 (0.93)
32.58 (2.01)
22.97 (1.37)
(0.094)
Losses in O content upon reduction Actual O loss
(0.49)
(0.64)
(bi)sulfate O loss
(0.37)
(0.13)
C=O to C=NNH2
(0.075)
(0.27)
The DPPH• scavenging capacity of the reduced powders was also determined and the results presented in Figure 3.9. In the case of RedPPy2, the reaction with DPPH• was slower over the first two hours compared to the original PPy2 powders (a consistent result for several replicates), showing some inhibition of the faster acting PPy segments as a result of the reduction process. However, after 5 hours of reaction time, and through to 24 hours, the RedPPy2 showed a greater DPPH• scavenging capacity. In the case of the PPy0.5, the reduced form showed both a faster initial reaction and a greater overall
61
scavenging capacity. This result served to confirm the hypothesis that the conducting polymer is acting as a reducing agent in scavenging DPPH•, itself a weak oxidant, with
Absorbance at 516nm
reduced polypyrrole able to react with a greater number of DPPH radicals.
2.5
(a)
2.0 1.5 1.0
(b) (c) (d) (e)
0.5 0.0 0
5
10
15
20
25
Time / hr Figure 3.9 DPPH• scavenging capacity of PPy synthesized with two APS/pyrrole ratios, before and after reduction of 30 mg samples in 10 mL Milli-Q water with 2 mL of hydrazine for 48 hours: (a) DPPH• only, (b) PPy2, (c) RedPPy2, (d) PPy0.5, (e) RedPPy0.5.
62
3.4.5 Comparison of DPPH• scavenging activity of PPy and PNMPy A possible reaction sequence for PPy with DPPH free radicals is showed in Scheme 3.4. An electron and proton from the polypyrrole N-H group were removed when the polymer reacts with DPPH free radicals; electrons are transfered from the conducting polymer to the DPPH free radical initially, followed by proton transfer145, 146. A study by Chepelev et al. indicated that pyrrole oligomers containing a free N-H group were stronger free radical scavengers than those that lacked the group145. It seems that the N-H group is important for the conducting polymer to be able to act as an antioxidant. In order to confirm this, PolyN-methylpyrrole (PNMPy) was prepared, using APS as an oxidant with the addition of sulfuric acid (0.1 M), and the free radical scavenging activity of PNMPy was compared with that of as-prepared PPy and PPy made with addition of acid (PPya), which followed the same procedure as for the preparation of PNMPy.
1 mg of each sample powder reacted with DPPH• for various times, and the decline of the level of DPPH free radicals is shown in Figure 3.10. It can be seen that both as-prepared PPy and PPya show great DPPH free radical scavenging activity, while PNMPy showed only a very weak effect upon DPPH radicals. This result supports the idea that the N-H group is important for such polymers to act as free radical scavengers, and supports the findings of Chepelev et al.
63
H N N H
N H -H+ -e-
DPPH
N N H
N H
N N H
N H
Scheme 3.4 Reaction scheme for polypyrrole with DPPH•.
64
+ DPPHH
3.0
Absorbance at 516nm
2.5 2.0
(a)
1.5
(b)
1.0 0.5
(c) (d)
0.0 0
4
8
12
16
20
24
28
Time / hr
Figure 3.10 Decline in the absorbance at 516 nm of a 255 µM methanolic DPPH• solution with (a) nil, and 1 mg of (b) PNMPy, (c) PPya and (d) PPy powders.
3.5 DPPH scavenging activity of PANI
3.5.1 Polymerization of PANI PANI powders were synthesized using the same method as used for the PPy powders, and without addition of a strong acid at the beginning of the chemical polymerisation. The preparation mechanism is shown in scheme 3.5. Similar to PPy, aniline is initially oxidized to a polymer and then further oxidized to an oxidized form. Elemental analysis results, and the molar percentage of sulfur to aniline units calculated based on the molar ratios of sulfur to nitrogen, are presented in Table3.4. Bisulfate, HSO4-, and sulfate, SO42-, are also expected to be the main dopant anions, as APS was used as the oxidant. The
65
doping level of PANI samples was calculated, using the method for PPy described in section 3.4.1, and values are presented in Table 3.5.
Scheme 3.5 Chemical polymerisation of aniline.
Table 3.4 Elemental analysis results for PANI samples prepared with different APS/aniline ratios. APS/aniline
1.0
1.5
2.0
2.5
C (mass %)
61.98
60.93
61.71
59.95
H (mass %)
4.62
4.72
4.13
3.89
N (mass %)
11.61
11.24
10.62
10.05
S (mass %)
3.95
3.91
2.47
2.18
O (mass %)
17.84
19.20
21.07
23.93
S/aniline (mol %)
14.8
15.2
10.1
9.5
66
Table 3.5 shows pH value of the PANI reaction mixture after 24 hours. The pH value decreased as the level of APS increased, indicating that more protons were released when more aniline groups were oxidised.
Table 3.5 Conductivity and pH values of the PANI reaction mixture after 24 hours. APS/aniline for preparing PANI
1
1.5
2.0
2.5
pH value
1.55
1.53
1.38
1.30
Predicted [HSO4-]/[SO42-]
2.75
2.88
4.07
4.9
Doping level
18.8
19.1
12.1
11.1
Conductivity (mS cm-1)
0.106
0.184
b
b
Yields (g) (experimental)
0.31
0.49
0.42
0.38
Yields (g) (theoretical)
0.54
0.54
0.52
0.51
% yielda
58
91
81
74
a: including HSO4- / SO42- dopants b: too low to measure (i.e. less than 10-3 mS cm-1)
As seen in table 3.5, polymerisation with the two lower APS concentrations led to a higher PANI doping level. The conductivities of the PANI powders also decreased markedly when a higher APS/aniline ratio was used for preparations (Table 3.5), and was less than 10-3 mS/cm for the APS/aniline ratios of 2.0 and 2.5. The findings also indicate that a higher conductivity in CPs is associated with a higher doping level.
67
3.5.2 SEM morphology of PANI SEM images of the as-prepared PANI powders are shown in Figure 3.11. As seen in the impages, typical PANI granular units 45 to 70 nm in diameter, can be seen, particularly in Figures 3.11A and 3.11D. On the other hand, the powder prepared with an APS/aniline ratio of 1.5 (PANI1.5) contained a considerable amount of nanotubes, which from Transmission Electron Microscopy were shown to be hollow in nature (Figure 3.12). The presence of PANI nanotubes is a common result when a high initial pH chemical polymerization of aniline is undertaken147-152, and may contain chemical structures such as phenazine units in addition to regular PANI153. These nanotube structures potentially provide more surface area for subsequent chemical reactions than the other PANI powder forms, including the scavenging of DPPH radicals.
68
A
B
C
D
Figure 3.11 SEM images of PANI prepared with various ratios of APS/aniline ratio (A) 1.0 (B) 1.5 (C) 2.0 (D) 2.5.
69
Figure 3.12 TEM image of PANI 1.5.
Figure 3.13 shows the SEM morphology of PANI powders (of a APS/aniline ratio of 1.0) prepared with addition of various concentration of sulfuric acid. When the oxidation of aniline starts in a strong acidic condition, granular particles were obtained without nanotubes, as was also observed by Stejskal et al. However, the particles (with diameter of 70 to 95 nm) tended to be larger than those prepared without the addition of the strong acid (Figure 3.11A).
70
A
B
C
D
Figure 3.13 SEM image of PANI prepared with the APS/aniline/H2SO4 of (A) 1:1:0.5 (B) 1:1:1 (C) 1:1:1.5 (D) 1:1:2.
3.5.3 Cyclic Voltammetric investigation The electrochemical properties of the PANI powders were evaluated by cyclic voltammetry of the powders cast onto a glassy carbon electrode, and results are presented in Figure 3.14. The voltammograms of the powders prepared using the ratio of APS/ANI of 1 and 1.5 showed oxidation peaks at +121, +247 and +446 mV and reduction peaks at
71
+62 and +420 mV at a scan rate of 20mVs-1. The first anodic peak at 121 mV is assigned to the oxidation of the leucoemeradine to the emeraldine form of PANI (Scheme 3.6(a)), and the peak at 446 mV to the oxidation from emeraldine to pernigraniline (Scheme 3.6(b)). The presence of an intermediate oxidation peak at 247 mV points to the presence of branched chains and/or shorter oligomeric species, which are oxidized in the 100-300 mV range. This peak is very small, or largely absent, in PANI formed electrochemically in the presence of a strong acid and not subject to a high electrode potential (E < 0.8 V) (Ag/AgCl)154, but appears more prominently in cyclic voltammograms taken of the highpH synthesis nanotube forms, pointing to a larger presence of branched structures formed during the synthesis155.
It was found that polyaniline powder prepared with a low concentration of APS showed distinct PANI redox peaks. However, as APS level increased, the cyclic voltammograms of the conducting polymers began to loose their shape and electroactivity (which can be related to their low conductivity values). The anodic peaks became featureless and showed little sign of internal redox processes. The change of the peaks can be due to the change of PANI structure because of occurrence of side reactions, such as overoxidation. The variation in the APS concentrations used to prepare polyaniline powders thus clearly affects their redox activity, indicating that high level of APS might lead to structural changes in the polymer powders.
72
40 30 (a)
Current / μA
20 (b) 10
(c)
(d) 0 -10 -20 -30 -40 -0.4
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
Potential / V
Figure 3.14 Cyclic voltammograms of PANI powders, prepared with various APS/aniline ratios: (a) 1.5 (b) 1.0 (c) 2.0 (d) 2.5, cast on a glassy carbon working electrode and cycled in 0.1 M HCl at a scan rate of 20 mV s-1.
73
NH )n
-
e +
NH
e
(
-
-
NH
(
Reduced Form (leucoemeraldine)
+
-
-
e +
.
Polaron (protonated emeraldine) e
NH
(
NH )n
.
NH )n (a) +
-
(
NH +
)n (b) NH +
Bipolaron (protonated pernigraniline)
+
NH )n +
-2H
+
NH
(
+
(
2H +
N
N )n
(c)
Oxidized Form (pernigraniline base)
Scheme 3.6 Oxidation of polyaniline.
3.5.4 FTIR spectroscopy FTIR spectra of the PANI powders were recorded using KBr pellets (Fig 3.15). The peaks at 1576 and 1498 cm-1 are assigned to C=C stretching vibration of quinoid (C=C (Q)) and benzenoid ring (C=C (B))
149, 155, 156
. The absorbance at 1140 cm-1 is due to the
stretching vibration of N=Q=N (Q here represents the quinoid ring)156. The band at 1294 cm-1 corresponds to the stretching vibration of the imine group C=N157 and the band at 822 cm-1 to the bending vibration of C-H. The peak at 1670 cm-1 at a higher APS level indicates the occurrence of overoxidation because of the formation of carbonyl groups (C=O). The peaks at 1294 and 1140 cm-1 declined with the ratio of APS to aniline was increased, pointing to destruction of the quinoid rings, which might be caused by overoxidation of polyaniline during polymerization. Moreover, the diminution of peak for the C=C
74
stretching vibration of the quinoid and benzenoid rings at 1576 and 1498 cm-1 can also be observed at a high APS level. Summarily, overoxidation might cause the destruction of both benzenoid and quinoid rings in PANI.
C =C (B ) C =C (Q )
N =Q = N
Absorbance
C -N C=N
(a ) (b)
(c) C =O (d)
600
800
1000
1200
1400
W avenum ber / cm
1600
1800
-1
Figure 3.15 FT-IR spectra of polyaniline prepared with various ratios of APS/aniline: (a) 1.0 (b) 1.5 (c) 2.0 and (d) 2.5.
3.5.5 Raman spectroscopy Figure 3.16 presents Raman spectra of the polyaniline samples. The bands at 1604 and 1395 cm-1 correspond to the C-C stretching vibration of benzenoid and quinoid rings; the peaks at 1514 and 1341 cm-1 to N-H and C-N stretching vibrations, and at 1178 cm-1 to C-H in-plane vibrations 158. It can be observed that the band at 1395 cm-1 declined when a
75
higher concentration of APS was used for polymerization of polyaniline powders, meaning that a high level of APS leads to the destruction of the quinoid ring. At the APS/aniline ratio of 2.5, a weakness in of all peaks can be observed, indicating destruction of the structure of PANI.
C -C (B )
Signal Intensity
C -C (Q )
(a ) (b )
(c ) (d )
400
800
1200
W avenum ber / cm
1600
2000
-1
Figure 3.16 Raman spectra of polyaniline prepared with various ratios of APS to aniline : (a) 1.0 (b) 1.5 (c) 2.0 and (d) 2.5.
3.5.6 ESR spectroscopy ESR spectra of the polyaniline samples are shown in Figure 3.17. PANI prepared with an APS/aniline ratio of 1.5 showed the strongest signal, pointing to a high level of free 76
radicals. In contrast, using a higher concentration of APS, PANI showed a very low level of free radicals. The strength of ESR signal was correlated to the doping level of PANI sample, which are based on molar ratio of sulfur and nitrogen (S/N) calculating according to elemental analysis results, where polyaniline prepared using a APS/aniline ratio of 1.5 shows highest doping level (see Table 3.5 above). It is possible that polarons are the main charge carriers in the PANI powders. According to previous reports, when overoxidation takes place, ß-C was initially oxidized to C-OH, and then further oxidised into C=O accompanied by the loss of positive charges, as counter anions were therefore removed 134-137
. This gives a possible explanation for PANI prepared with a high APS/aniline
presenting only a low doping level.
Signal Intensity
a
3 3 0 .0
b c
3 3 2 .5
3 3 5 .0
d
3 3 7 .5
3 4 0 .0
3 4 2 .5
3 4 5 .0
M a g n e tic F ie ld / m T
Figure 3.17 ESR spectra of polyaniline with various ratios of APS to aniline, from a to d, the ratios are: 1.5:1, 1:1, 2:1and 2.5:1.
77
3.5.7 DPPH• scavenging activity of PANI powders The DPPH free radical scavenging activity of the polyaniline powders prepared with various concentrations of APS is shown in Figure 3.18A. It was observed that PANI1.5 was the greatest DPPH• scavenger among the PANI powders, followed by the powder prepared with an APS/aniline ratio of 1.0. PANI prepared with a higher ratio of APS to aniline (PANI2.5) showed only weak DPPH free radical scavenging activity. After reaction with DPPH radicals for 24 hrs, PANI2.5 scavenged only 37% of DPPH radicals, while PANI1.5 scavenged 71% of DPPH radicals, with a ratio of 2.2 aniline units to each DPPH radical scavenged.
From the SEM image of PANI1.5, it was found that there were a considerable number of nanotubes present in the powder (Figure 3.11B above). The increase of surface area due to the presence of the nanotubes might be one of the reasons for PANI1.5 being such an efficient free radical scavenger. The results agree with the finding by Wang et al.95, which showed that the antioxidant activity of PANI nanofibers is higher than that of conventional PANI and increased with an increase in the nanofiber surface area95. Polyaniline prepared with an APS/aniline ratio of 1.0 (PANI1.0), which was not dominated by nanotubes, scavenged a similar amount of DPPH radical as PANI1.5 after reaction for 24 hr. The high DPPH• scavenging activity of this powder might be due to the type of nanoparticles present (Figure 3.11A), which also provides large surface area for the scavenging reaction.
78
A
Absorbance at 516nm
2.5
2.0
(a) *
1.5
1.0
(b)
0.5
(c) (d) (e)
0.0 0
4
8
12
16
20
24
28
Time (hr)
B
Absorbance at 516nm
2.5 2.0
(a) *
1.5 1.0 0.5 0.0
(b) (c) (d) (e)
0
4
8
12
16
20
24
28
Time (hr)
Figure 3.18 (A) DPPH• scavenging activity of PANI powders prepared with various APS/aniline ratios: (a) DPPH• only, (b) 2.5, (c) 2.0, (d) 1.0 and (e) 1.5. (B) Decline in the absorbance at 516 nm of a 255 µM methanolic DPPH• solution with (a) nil and PANI prepared with molar ratio of APS: aniline: H2SO4 of (b) 1:1:1, (c) 1:1:1.5, (d) 1:1:0.5 and (e) 1:1:0. *: experimental uncertainty.
79
Figure 3.18B shows DPPH• scavenging activity of PANI1.0 prepared with addition of various concentrations of sulfuric acid. All of the samples exhibited excellent DPPH• scavenging ability, but the rate of scavenging was slower than the PANI1.0 prepared in the absence of added sulfuric acid over the first few hours. After 24 hours of exposure to the DPPH• solution all of the PANI1.0 samples had scavenged a similar total number of DPPH radicals. As seen in Figure 3.13, the larger particles with a smaller surface area for the PANI1.0 prepared in the presence of sulfuric acid, might explain the slower initial rate of radical scavenging.
3.5.8 Structural changes in PANI upon reaction with DPPH• The DPPH free radical scavenging activities of reduced and dedoped PANI1.5 powders were also determined by reaction of 1.0 mg of each sample powder with 20 mL of 255 μm of DPPH• solution, which contained 5.1 μmole of DPPH radicals. As seen in Figure 3.19, the reduced PANI and as-prepared PANI powders removed 76 % and 72 % of the DPPH radicals, respectively, over 24 hours, while the dedoped (low conductivity) PANI removed only 54 % of DPPH•. The results indicate that C–N and C-N+ groups are important units for PANI when it acts as a DPPH free radical scavenger. A possible mechanism for this reaction is that the C–N and C-N+ groups are oxidised through to C=N species 159, 160. That the change in the structure of PANI is an oxidation process has been shown in previous studies126, 161.
80
DPPH Scavenged / μmole
4.0
(a) (b)
3.5
3.0
(c) 2.5
2.0
1.5 0
4
8
12
16
20
24
28
Time / hr
Figure 3.19 DPPH• scavenging capacity of PANI powders synthesized with an APS/ANI ratio of 1.5: (a) reduced PANI, (b) as-prepared PANI, (c) dedoped PANI.
The FTIR spectra of polyaniline powders before and after reaction with DPPH radical were recorded using KBr pellets. As seen in Figure 3.20, the peak at 1576 cm-1 increased and the peak at 1498 cm-1 declined after reaction with DPPH•. The former peak is assigned to C=C stretching vibration of quinoid (C=C (Q)), while the later peak corresponds to the stretching vibration of benzenoid ring (C=C (B)). The increase of the
81
C=C (Q)/ C=C (B) ratio corresponds to the transformation of C–N to C=N, and indicates that PANI was oxidized during reaction with DPPH• (see Scheme3.6).
Absorbance
C=C (B) C=C (Q)
(a)
(b)
300
600
900
1200
1500
Wavenumber / cm
1800
2100
-1
Figure 3.20 IR spectra of PANI (a) after (b) before reaction with DPPH•.
An elemental analysis of the PANI powders before and after reaction with DPPH radicals by XPS is presented in Table 3.6. The S/N ratio of PANI decreased by 46 %, from 16.6 to 9.1, after reaction with DPPH• for 24 hrs. The lowering of the S/N ratio indicates a loss of C-N+ groups in the PANI powders, since the sulfur (S) is derived mainly from the
82
dopant HSO4-
/
SO42-
162
, associated with C-N+, as has been applied previously to
estimate the level of doping and protonation of PANI
163
. The result provides further
evidence for an oxidation or deprotonation process and movement from C-N+ to C=N.
Table3.6 Elemental analysis of PANI before (A) and after (B) reaction with DPPH free radicals, according to XPS wide spectra. S/N (%)
At % N1s
C1s
O1s
S2p
A
10.28
78.14
9.87
1.71
16.6
B
9.74
80.27
9.11
0.89
9.1
At %: relative atomic concentrations (%)
20 mg of PANI powder was mixed with 200 mL of DPPH• solution (510 µM) for 24 hours. The PANI powders before and after reaction with DPPH free radicals were then investigated using Electron Spin Resonance (ESR) spectrometry. The spectra of these two samples are presented in Figure 3.21, which shows that the PANI powder contained 45 % less free radicals (polarons) after reaction with DPPH•. The diminution of the polaron signal confirms the results obtained from the XPS investigation, indicating a loss of C-N+ species during reaction with DPPH•.
83
Signal Intensity
(a) (b)
328
332
336
340
344
348
Magnetic Field / mT
Figure 3.21 ESR spectra of PANI (a) before (b) after reaction with DPPH•.
The electrochemical properties of the PANI samples before and after reaction with DPPH free radicals were evaluated by cyclic voltammetry as shown in Figure 3.22. At a scan rate of 20 mVs-1, the as-prepared PANI powder showed significant redox activity with the earliest oxidation peak occurring at +121 mV. After reaction with the DPPH radicals, the same level of electroactivity was not obtained on the cast films, and the possibility of
84
further cross-linking reactions resulting from reaction with DPPH• in addition to internal PANI redox processes, needs to be explored further.
40 30
Current / μA
20
(a) 10
(b)
0 -10 -20 -30 -200
0
200
400
600
800
Potential / mV
Figure 3.22 Cyclic voltammograms of PANI powders (a) before (b) after reaction with DPPH•, cast on a glassy carbon working electrode and cycled in 0.1 M HCl at a scan rate of 20 mV s-1.
85
3.6 DPPH• scavenging activity of poly (3,4-ethylenedioxythiophene)
3.6.1 Polymerization of PEDOT PEDOT powders were synthesized chemically by oxidative polymerization of EDOT in an aqueous solution containing various levels of the APS oxidant. The polymerisation mechanism is shown in Scheme 3.7, where the monomers are oxidized to neutral polymers first and further oxidized to polymeric cations doped with anions. Similar to PPy and PANI, bisulfate, HSO4-, and sulfate, SO42-, are expected to be the main dopant anions. The predicted ratio of [HSO4-]/[SO42-] was also determined using the same equation for PPy and PANI (eq. 3.2).
O
O
O
O
1/n
+ 2H+ + 2e-
S
S
S2O82- + 2e -
n
2SO42+
O
O
S
S
1/2S2O 82- + e SO42- + H+
O
O
SO42HSO 4-
Scheme 3.7 Polymerization of PEDOT.
86
+ e-
Table 3.7 showed elemental analysis results and the molar percentage of HSO4-/SO4-2 to EDOT units (Table 3.8), which was calculated based on the molar ratios of carbon to sulfur, with exclusion of the sulfur in the backbone, by adapting the method reported by Chiu et al. 162.
Table 3.7 Elemental analysis results for PEDOT samples prepared with different APS/EDOT ratios. APS/aniline
1.0
1.5
2.0
2.5
C (mass %)
43.05
42.58
42.91
43.38
H (mass %)
3.08
3.11
3.03
3.07
S (mass %)
21.14
20.45
20.45
20.72
O (mass %)
32.73
33.06
33.61
32.83
(HSO4-/SO4-2)/PEDOT
10.5
8.1
7.24
7.47
(mol %)
Table 3.8 Calculation of molar ratio of HSO4-/SO42- to EDOT units for PEDOT1.0 based on the elemental analysis results. Total
(-C6H4O2S-)na
C (mass %)
43.05
43.05
S (mass %)
21.14
19.13
HSO4-/SO4-2
Element
(HSO4-/SO4-2)/PEDOT
2.01 10.5
(mol %)b a PEDOT b calculation based on the molar ratios of carbon to sulfur with exclusion of the sulfur in the backbone
87
The doping level of PEDOT samples was then calculated based on the molar percentage of HSO4-/SO4-2 to EDOT units and the [HSO4-]/[SO42-] ratio at the final pH values of the preparative solution as described in section 3.4.1. As seen in Table 3.9, a higher doping level was obtained as lower APS/aniline ratios were applied during polymerization. It is clear that the doping levels of these polymers depended upon the APS/EDOT ratios, somewhat similar to the trends seen with PPy and PANI.
Table 3.9 pH values of the PEDOT reaction mixture after 24 hours. APS/aniline for preparing PANI
1
1.5
2.0
2.5
pH value
1.44
1.40
1.33
1.27
Doping level
12.8
9.7
8.5
8.7
3.6.2 SEM images The SEM images of the PEDOT powders prepared with various concentrations of APS are shown in Figure 3.23. The polymer particles obtained cluster together and have a mainly granular morphology with diameters of 40 to 150 nm. The morphology of the PEDOT particles are similar to those reported by Zhang et al 164.
88
A
B
C
D
Figure 3.23 Morphology of PEDOT prepared with various ratio of APS/EDOT (A) 1.0 (B) 1.5 (C) 2.0 (D) 2.5.
3.6.3 FTIR spectroscopy In the FTIR spectra of the PEDOT samples (Figure 3.24), vibrations in the region between 1510 and1470 cm-1 are due to ring stretching of the thiophene ring; the vibration at 1340 cm-1 is attributed to C-C and C=C stretching of quinoidal structures; bands at 1188, 1138 and 1080-1060 cm-1 are due to the stretching in the C-O-C bond; and bands at
89
980, 832 and 688 cm-1 are ascribed to the C-S bond in the thiophene ring165, 166. The presence of the band at 1638 cm-1, somewhat more prominent for higher APS/EDOT ratios, reveals the existence of carbonyl groups in the product, which is related to overoxidation.
It can be seen from Fig 3.24, the band at 1510 cm-1 weakens with an increase in the APS level. Diminution of this band was also observed by Seo and Chung167. The absorption has been attributed to the vibration of the double bond in the PEDOT ring. The decline of this band might be due to the incidence of side reactions, such as overoxidation, which leads to destruction of these double bonds.
ν C -C νC =C
Absorbance
1 .2
νC =C
0 .8
0 .4
0 .0
νC =O
800
1000
1200
1400
W avenum ber / cm
1600
1800
-1
Figure 3.24 IR spectra of PEDOT prepared with the ratio of APS to EDOT: from top to bottom are 1, 1.5, 2 and 2.5.
90
3.6.4 DPPH• scavenging activity The DPPH free radical scavenging activity of PEDOT powders is shown in Fig 3.25. After a rapid initial decline within one hour, the absorbance continued to decrease progressively, meaning that the reaction continued more slowly following an initial rapid scavenging. The wide potential range over which CPs can be oxidized means that some parts (eg. the surface) of the polymers can react rapidly with the DPPH free radicals, while other parts (eg. inside the particles) have a slower reducing activity
105, 126
. The
diffusion of free radicals might be an important factor for the reaction rate.
It was found that the polymer prepared with a lower APS to EDOT ratio scavenged more DPPH radicals over a 24-hour period. PEDOT prepared using an APS/EDOT ratio of 1.0 scavenged 16 % of DPPH radicals, while PEDOT formed with an APS/EDOT ratio of 2.5 scavenged only 14 % of DPPH radicals. 1 mg of PEDOT represents 6.7 µmole of EDOT units, while 20.0 mL of 255 µM DPPH• solution represents 0.51 × 10-5 moles of DPPH•. Thus using the largest scavenging values for PEDOT, a ratio of 8 EDOT units per DPPH radical scavenged was obtained.
91
Absorbance at 516nm
2 .6 2 .5 2 .4 2 .3 2 .2 2 .1 2 .0 1 .9 1 .8 1 .7 1 .6 1 .5 1 .4 1 .3
(a ) *
(b ) (c) (d ) (e )
0
5
10
15
20
25
T im e / h r Figure 3.25 DPPH• scavenging capacity of PEDOT synthesized with different APS/EDOT ratios: (a) DPPH• only, (b) 2.5, (c) 2.0, (d) 1.5, (e) 1.0. *: experimental uncertainty.
3.6.5 Conductivity of the PEDOT samples The conductivities of the PEDOT samples are listed in Table 3.10. It is clear that the conductivity of these polymers depended upon the APS/EDOT ratios. The electrical conductivity of PEDOT was lowered from 3.9×10-3 to 5.9×10-4 S/cm as the APS/EDOT moved from 1.0 to 2.0, and was too low to measure at a ratio of 2.5. Previous research has likewise indicated that CPs prepared at a higher concentration of a very powerful
92
oxidant, such as APS, normally present lower conductivities because of overoxidation167. A study reported by Zykwinska et al. also mentions that the overoxidation process possibly decreases the conjugation length of the main chain π-bond and leads to degradation of PEDOT168. Therefore, the decrease of conductivity, and subsequent effects on the DPPH radical scavenging ability of PEDOT samples, are likely due to overoxidation of these polymers, leading to a less regular CP structure and a diminution of the properties of the non-oxidised forms.
Table 3.10 Conductivity values and DPPH• scavenging activity for PEDOT powders. APS/EDOT for preparing PEDOT DPPH scavenged (μmole)
by
Conductivity (mS/cm)
1
PEDOT 0.83
3.9
1.5
2.0
2.5
0.81
0.76
0.73
0.76
0.59
a
a: too low to measure (less than 10-3 mS/cm)
3.7 Conclusions
This study has shown that PPy powders are effective DPPH free radical scavengers, particularly when prepared with a lower APS/pyrrole ratio, and when reduced with hydrazine and when polymer conjugation (and conductivity) is preserved. The optimum initial APS/pyrrole ratio for the synthesis of PPy powders with a superior DPPH free radical scavenging was shown to be around 0.5. The weaker DPPH• scavenging ability found when higher levels of APS were employed to produce the PPy powders can be 93
attributed to defects in the conjugated structure of the polymer due to overoxidation that lowers the reducing power of the polymer. Ammonium persulfate is a particularly strong oxidant, with an oxidation potential of 2.0 V
128
, and when present in excess of that
required to oxidatively polymerise PPy into a fully oxidised state (requiring an APS/pyrrole ratio of about 1.15), can form carbonyl groups on the pyrrole rings, as confirmed in this case by FTIR spectroscopy. The lower conductivities and doping levels of PPy powders formed using higher amounts of the APS oxidant are also likely due to overoxidation, damaging the PPy structure in the process. In addition, the very weak DPPH• scavenging ability of PNMPy indicates the significance of N-H unit in PPy structures, when the conducting polymer acts as an antioxidant.
PANI powders prepared with APS as an oxidant have been found to be efficient DPPH free radical scavengers, especially when prepared with lower level of APS. Using a high ratio of APS/aniline, PANI showed not only low DPPH• scavenging activity but also low conductivity and weak redox properties, which might be due to overoxidation leading to a change of the structure, as confirmed by IR spectra. In order to obtain better electrochemical properties for PANI, it is important to apply a suitable molar ratio of APS to ANI for preparation of PANI powders.
The radical scavenging activity of reduced PANI and dedoped PANI was also evaluated through their reaction with DPPH free radicals. In comparison with as-prepared PANI, the reduced form was the most efficient scavenger (with a greater capacity to be oxidised), followed by as-prepared PANI and dedoped PANI. The reaction of PANI with DPPH
94
radicals leads to the transformation of C–N and C-N+ groups through to more fully oxidized C=N. Spectroscopic evidence from FTIR and ESR showed the change from C-N to C=N and a lowering of the content of C-N+ groups.
The decline of clearly defined oxidation peaks in voltammograms of the PANI powders confirms that structural changes had occurred in the PANI powders during reaction with DPPH radicals.
PEDOT also showed DPPH radical scavenging activity, which was dependent on the molar ratio of APS to monomer used in the chemical polymerization of the polymers. Similar to PPy and PANI, using higher levels of the strong APS oxidant led to both a lowering of DPPH• scavenging ability and the conductivity of the PEDOT powders. The loss of free radical scavenging activity and conductivity are related to overoxidation of the polymers, with evidence for this seen in FTIR spectra.
In summary, the antioxidant ability of PPy, PANI and PEDOT powders is strongly related to the initial ratio of APS to Py, ANI and EDOT monmers using in the polymerization. It has been found that greater DPPH radical scavengers were obtained when a lower level of APS was used for polymerization, and that PPy and PANI showed a much greater free radical scavenging capacity than PEDOT, as summarized below in Figure 3.26. PPy, PANI and PEDOT powders with the optimal antioxidant ability were applied in subsequent studies.
95
DPPH Scavenged / μmole
3 .6
A
3 .2
2 .8
2 .4
0 .4
0 .8
1 .2
1 .6
2 .0
DPPH Scavenged / μmole
A P S /P y
3 .6
B
3 .2 2 .8 2 .4 2 .0 0 .8
1 .2
1 .6
2 .0
2 .4
DPPH Scavenged / μmole
A P S /A N I
0 .8 4
C
0 .8 0
0 .7 6
0 .7 2
0 .8
1 .2
1 .6
2 .0
2 .4
A P S /E D O T
Figure 3.26 Amount of DPPH• scavenged over a 24 hour period for powders formed with different polymerization ratios of (A) APS to Py, (B) APS to ANI and (B) APS to EDOT (n = 2).
96
Chapter 4 ABTS•+ scavenging activity of polypyrrole, polyaniline and poly(3,4-ethylenedioxythiophene)
4.1 Introduction
The ABTS assay has been widely employed for the measurement of the total antioxidant capacity of liquid samples, such as food extracts and beverages169, 170. While the DPPH assay is also designed to evaluate antioxidants in solution, we have successfully developed a modified DPPH assay to use with solid samples171. However, the DPPH free +
radical can only dissolve in organic solvents, such as methanol. The ABTS• radical and its associated assay is applicable to both lipophilic and hydrophilic phases27, and therefore the development of an ABTS assay suitable for solid samples is of interest.
The ABTS assay was originally based upon reaction of ABTS with the ferrylmyoglobin radical, formed through activation of metmyoglobin by hydrogen peroxide, to produce +
the coloured radical cation ABTS• , in the presence or absence of antioxidants26. +
Antioxidants can quench the ABTS•
radical and thereby decolourise the reaction
mixture, as follows: Metmyoglobin + H2O2
ABTS
Ferrylmyoglobin radical
ABTS
97
.+
Antioxidant (AH)
ABTSH +
+
The antioxidant effect is monitored during the production of ABTS• and evaluated by +
measuring the concentration decline of ABTS• at 753 nm using a spectrophotometer.
Conducting polymer powders prepared with ammonium persulfate oxidant to monomer ratios that proved to be the most efficient DPPH scavengers, namely PANI1.5, PPy0.5 +
+
and PEDOT1.0, were used in tests with ABTS• . In this work, the ABTS• and DPPH• scavenging capacity of these conducting polymers have been compared.
4.2 Experimental +
4.2.1 ABTS• radical scavenging activity of PPy0.5, PANI1.5 and PEDOT1.0 +
Sample powders were prepared as described in section 2.3. A stock solution ABTS• was prepared by mixing 7 mM ABTS with 2.45 mM potassium persulfate in water, which +
produced around 70% of ABTS•
radicals (further details are given in section 2.9.2). In
the final procedure, the working solution was freshly prepared by a 11 times dilution of the stock solution with ethanol. 1 mg of each sample powder was added to 5 mL of the working solution and left to stand for various times with agitation using a commercial shaker (KS 125 basic, IKA LABORTECHNIK) with a speed of 500 Mot / min for 15 minutes, before the measurements were taken after 30 and 60 minutes, and for a further +
45 minutes before measurements at 120 and 180 minutes; the absorbance of ABTS• at 753 nm was recorded at these times.
98
4.2.2 Spectroscopic characterization of the conducting polymer powders Structural changes in the conducting polymers were examined using FTIR and X-ray photoelectron spectroscopy (XPS), as described earlier in section 2.10.2.
+
4.2.3 Cyclic voltammetry (CV) and UV- visible characterisation of ABTS• and DPPH• in an ethanolic solution +
An investigation was undertaken of the oxidant strength of the ABTS• and DPPH• radicals through an examination of the electrode potentials at which the species are oxidised and reduced. 0.1 mM of DPPH• or 0.1 mM ABTS diammonium salt were dissolved in ethanol, with 0.1 M of lithium perchlorate (LiClO4) as a supporting electrolyte. Cyclic voltammetric measurements of these two solutions were carried out in a three-electrode cell as shown in Scheme 4.1. An Indium tin oxide (ITO) electrode (0.9 cm × 5.0 cm × 0.05 cm, ABTECH Scientific, Inc., USA) was used as working electrode and a platinum wire was utilized as counter electrode. A silver wire covered with silver chloride (AgCl), which was prepared by dipping the silver wire in Pam’s Brite Bleach for 5 min and then thoroughly rinsed with water, was used as the reference electrode. The difference in potential between this reference electrode and the Ag/AgNO3 ethanolic reference electrode (with 0.1 M of lithium perchlorate (LiClO4) as a supporting electrolyte) was determined to be 0.25V, which was measured using Hewlett Packard 34401A Multimeter.
The CV traces were recorded using a CH Instrument Electrochemical Workstation (650) at a scan rate of 50 mV/s. While the cyclic voltammetric measurement was operating, the 99
optical spectra of the same solution were also recorded in situ at the ITO electrode using a spectrophotometer (Pharma Spec UV-1700, SHIMADZU). The UV-visible spectrum of ABTS solution was recorded in the range from 900 to 350 nm to confirm the generation +
of ABTS• . All of the experiments were performed at room temperature.
Ref erence electrode Pt counter electrode
ITO working electrode
Scheme 4.1 Schematic representation of the cell used for spectroelectrochemistry experiments.
100
4.3 Results and discussion
4.3.1 Development of an ABTS assay for conducting polymer powders Initial experiments were undertaken in which ABTS, myoglobin and PANI powders were mixed, and the reaction was initiated by the addition of hydrogen peroxide, following from established procedures for antioxidants in solution25,
26, 120, 172
. The chemicals
excluding PANI powders were obtained from the Randox Total Antioxidant Status Kit (Dunore Diagnostics, Co. Antrim, United Kingdom). The generation and removal of +
ABTS• in the presence of various amounts of PANI powder are presented in Figure 4.1. For the blank (in absence of PANI powder), the absorbance of the reaction solution increased from 0.11 to peak at 0.18 units after 30 min and then decreased to 0.068 over a +
2 hr period. These results indicate that the production reaction (ABTS → ABTS• ) +
+
dominated for the first 30 minutes, and then the termination reaction (ABTS• + ABTS•
→ nonradical products) became faster3. In the presence of PANI powder, it was notable that the reactions were affected from the time of hydrogen peroxide addition, and the +
conducting polymer seemed to scavenge and remove the ABTS• radical cation in a variable manner. It is possible that the conducting polymer powder also reacted with +
ferrylmyoglobin radical when the generation of ABTS• radical cation was in progress, increasing the complexity of the reactions. For some reason, the addition of the larger +
quantities of conducting polymer (3 and 5 mg) left more ABTS• radicals (a larger 753 nm absorbance) after two hours than the smaller (1 and 2 mg) additions. Therefore, generation of the radical cation prior to the addition of the conducting polymers in the measurement procedure was considered.
101
Absorbance at 753 nm
0.18 0.15 0.12 0.09 (a)
0.06
(b) (c) (d) (e)
0.03 0.00 0
20
40
60
80
100
120
140
Time / min
+
Figure 4.1 Inhibition of production of ABTS• by addition of (a) nil (b) 5.0, (c) 3.0, (d) 1.0 and (e) 2.0 (Δ) mg of PANI powder (prepared using an APS/aniline ratio of 1.5).
+
An improved method for the generation of ABTS• has been reported by Re et al27. Here the blue/green radical cation is produced through the reaction of ABTS and potassium +
persulfate. The antioxidant activity can be evaluated by direct ABTS• radical cation scavenging as follows:
ABTS
K2S2O8
ABTS
.+
Antioxidant (AH)
According to this method, the absorbance of ABTS•
+
ABTSH +
is adjusted to 0.7 with liquid
samples, and the reaction with added antioxidant proceeds for 6 minutes. However, it has
102
reported that 6 min is not enough for some compounds, such as chlorogenic acid and +
caffeic acid, to reach end point when reacted with ABTS• , and a longer reaction time is therefore needed to give more reliable data173, 174. In our case, some modification of the method was also required for use with solid samples. The reaction of various amounts (1 +
to 5 mg) of PANI1.5 and 2 mL of ABTS• with an initial absorbance of 0.7, showed a fast decrease in absorbance to less than 0.1 in 6 minutes in each case. This indicates that the initial absorbance was too low for the radical scavenging power of even 1 mg of the +
conducting polymer. When 1 mg of PANI1.5 powder was reacted with 2 mL of ABTS•
radical cation at initial absorbance readings of 1.0 and 1.9, the absorbance declined sharply to around 0.15 after in 6 minutes; thereafter there was no significant change in +
absorbance for the subsequent 60 minutes. The near complete removal of ABTS• radical cations in a short period of time is not suitable for a correct determination of the +
antioxidant capacity of the samples, indicating that a still higher amount of ABTS• was required for the testing. When the initial absorbance was increased to 2.87, the reading decreased to 1.3 after 6 minutes, to 0.7 after 30 minutes and to a reading of 0.1 after 3 hrs +
+
(for 1 mg of PANI1.5 in 2 mL of ABTS• solution). The volume of ABTS• working solution was finally increased from 2 mL to 5 mL for reaction with 1 mg of conducting polymer powders.
Two control experiments were also undertaken. An excess of ascorbic acid (2.0 mg) was +
added to the working solution and fully reacted with ABTS• radicals. The absorbance reading declined immediately to a value of 0.011 units and remained steady after 3 hours.
103
+
+
This absorbance reading was mostly contributed by the fully reduced ABTS• (ABTSH ) produced in the reaction, and no attempt was made to correct for the small absorbance +
contribution of ABTSH in later experiments. In a further experiment, 1.0 mg of each of the PPy, PANI and PEDOT powder was left in ethanol for 3 hours to see if any soluble PPy forms would develop and contribute to the absorbance reading, but this value only reached 0.008 units for PPy and PEDOT and 0.024 units for PANI after 3 hours. The dissolution of conducting polymer powders into the ethanolic solution is therefore not expected to affect the radical scavenging results.
+
4.3.2 ABTS• scavenging activity of conducting polymer powders +
5 mL of ABTS• working solution was mixed with 1 mg of each sample powder with +
shaking. The absorbance readings of ABTS• working solution at different times in the presence of 1 mg of PPy, PANI and PEDOT powder are presented in Figure 4.2. The conducting polymer powders showed very slow scavenging over the first 30 minutes, then the reaction rate increased, as seen in the decline in the absorbance from 2.87 to 0.32 for PANI, to 0.53 for PPy, and only to 1.51 for PEDOT after 3 hrs. During this time the +
absorbance of a solution of ABTS• radicals with no added conducting polymer declined to 2.68. A biphastic kinetic pattern has been reported by several investigators in this +
procedure, indicating that the reaction of ABTS• with antioxidants can involve both fast and slow steps175, 176. In our case, although a very weak scavenging activity over the first 30 min for these samples is not clear, it is obvious that the both faster and slower effects are involved in the scavenging reactions. Some slower effect might be due to the
104
presence of an intermediate product, produced by the fast initial reaction of the +
conducting polymers with ABTS• , analogous to the effect reported by Walker and Everette that the biphastic kinetics for some aminothiols may be caused by a fast initial +
reaction of the thiol with ABTS• to form the disulfide and then followed by the slow reaction of disulfide with the radicals174.
3.5
Absorbance at 753nm
3.0 (a)
2.5 2.0 1.5
(b)
1.0 (c) (d)
0.5 0.0 0
30
60
90
120
150
180
210
Time / min
+
Figure 4.2 ABTS• scavenging activity of as-prepared conducting polymer powders: (a) ABTS•+ only, (b) PEDOT1.0, (c) PPy0.5 and (d) PANI1.5.
+
The reaction of ABTS• and each of the three as-prepared samples was also carried out +
without shaking. A comparison of ABTS• scavenging activity with and without shaking of the reactant solution is shown in Table 4.1. It can be seen that for all the samples,
105
+
when the reaction proceeded without shaking, fewer ABTS• radicals were quenched than in the procedure undertaken with shaking, with 18 % less scavenging in the case of PPy, 16 % less for PANI and 22 % less for PEDOT over 3 hrs. It is clear that without +
shaking, the transport of ABTS• to the solid conducting polymer powders occurs at a slower rate, indicating that shaking accelerates the reaction rate. The degree of shaking is thus an important factor in establishing the scavenging rate of the test samples and needs to be controlled and undertaken in a consistent fashion. For this reason, the shaking procedure used was standardised to apply a constant speed and same shaking period as described in section 4.2.1.
+
Table 4.1 ABTS• scavenging for PPy, PANI and PEDOT at different times with and without shaking. Shaking
No shaking µ moles
PPy 0.5
1 hr 0.6
2 hr 1.4
3 hr 1.7
1 hr 0.3
2 hr 1.1
3 hr 1.4
PANI 1.5
0.8
1.6
1.9
0.4
1.3
1.6
PEDOT 1.0
0.5
0.7
0.9
0.2
0.5
0.7
+
The PPy, PANI and PEDOT powders were also reduced using hydrazine. The ABTS•
scavenging activity of the reduced CP powders was evaluated using the same procedure +
for as-prepared powders. The decline in absorbance of the ABTS• solutions is shown in Figure 4.3. It can be seen that all of the reduced CP powders showed a quicker
106
scavenging activity over the first 30 minutes, in sharp contrast to the very weak scavenging activity of the as-prepared powders during this initial period. A slower rate of scavenging followed after the initial 30 minute period for the reduced conducting polymers, indicating that there could be different chemical structures, or polymer oxidation states, which vary in their capacity for electron donation (reducing strength). Among these conducing polymers, the reduced PANI and reduced PPy were the most +
effective ABTS• scavengers, with PEDOT showing a much weaker effect even in the reduced state, as seen with the as-prepared CP samples. In the case of PANI, it is possible that the (reduced) benzenoid amine units are more efficient electron and proton donors than the (oxidised) quinoid imine units.
3.0
Absorbance at 753nm
(a) 2.5 2.0 1.5
(b)
1.0 0.5 (c) (d)
0.0 0
25
50
75
100
125
150
175
200
Time / min
+
Figure 4.3 ABTS• scavenging activity of the reduced conducting polymer powders:
107
+
(a) ABTS• only, (b) PEDOT1.0 (c) PPy0.5 (d) PANI1.5. The scavenging capacity of test samples can be calculated as the percentage of free radicals scavenged as follows177, 178:
ABTS • + scavenging (%) = 1 − (
As ) Ac
(eq. 4.1)
where Ac is the initial absorbance (at 753 nm) and As is the absorbance after reacting for time t (after accounting for the absorbance decay of ABTS•
+
itself in the absence of
added conducting polymer).
+
A comparison of the ABTS• (%) scavenging capacity of the as-prepared and reduced +
PPy0.5, PANI1.5 and PEDOT1.0 applying ABTS• scavenged is presented in Table 4.2. As seen in Table 4.2, as-prepared PANI scavenged 82 % of the radical cations, while the reduced form of PANI showed a stronger scavenging strength and removed 91 % of the +
ABTS• radicals. For PPy, the as-prepared and reduced samples scavenged 75 % and 89 % of the radicals respectively. PEDOT was the weakest scavenger, removing only 41 % in the as-prepared state, and 45 % from the reduced state. Compared to the as-prepared +
samples, the conducting polymers reduced using hydrazine scavenged more ABTS• radicals by 9 % for PANI, 14 % for PPy and just 4 % extra for reduced PEDOT.
108
+
Table 4.2. Comparison of ABTS• (%) scavenging capacity for as-prepared and reduced PPy0.5, PANI1.5 and PEDOT1.0 after 3 hours of reaction. +
ABTS• (%) scavenging PPy0.5
PANI1.5
PEDOT1.0
As-prepared
75
82
41
Reduced form
89
91
45
PANI powders were also prepared with the addition of sulfuric acid in the chemical polymerization step. The morphology of the powders is shown in Figure 4.4. During preparation, the molar ratio of APS to ANI was 1.5. Using molar ratios of H2SO4/ANI of 0.5 (PANI15H0.5) and 1 (PANI15H1), all of the powders appeared as amorphous particles with diameters less than 100 nm and no nanotubes were observed in the powders, but there was no significant difference in morphology between the two samples with different acid levels. This is in contrast to the PANI1.5 sample prepared without added acid, which consisted predominantly of high surface area nanotubes (see Figure 3.11B on p.68). The conductivities of PANI15H0.5 and PANI15H1 were also measured and a greater conductivity was obtained for the acidified samples, with 16 mS/cm for PANI15H0.5, 120 mS/cm for PANI15H1, compared to only 0.18 mS/cm for the asprepared PANI1.5.
109
A
B
Figure 4.4. SEM images of (a) PANI15H0.5 (b) PANI15H1.
+
The ABTS• scavenging activity of the PANI samples prepared with and without addition of sulfuric acid are shown in Figure 4.5. It can be observed that although PANI15H0.5 and PANI15H1 were not as efficient as PANI1.5, they still scavenged 69 % 110
+
and 60 % of the ABTS• radicals, respectively, over a 3 hr period. Compared to PANI1.5, the weaker scavenging activity of the PANI15H0.5 and PANI15H1 powders might be due to lack of higher surface area nanostructuring, since the PANI1.5 consisted mainly of nanotubes, which possibly provides a greater surface area (see section 3.5.2). The results are in accordance with those obtained with the DPPH assay mentioned in section 3.5.7, indicating that surface area can be more important factors in the radical scavenging activity of PANI than high conductivity.
Absorbance at 753 nm
3.5 3.0 (a)
2.5 2.0 1.5 1.0
(b) (c)
0.5
(d) 0.0 0
30
60
90
120
150
180
210
Time / min
+
Figure 4.5. ABTS• scavenging activity of conducting polymer powders: +
(a) ABTS• only, (b) PANI15H1 (c) PANI15H0.5 (d) PANI1.5.
111
+
4.3.3 Comparison of DPPH• and ABTS• scavenging capacity of the conducting polymers
+
The ABTS• and DPPH• radical scavenging methods are based upon a decolorization step for determining antioxidant capacities. Both assays are easy to use, have a high sensitivity, and provide a rapid analysis for the antioxidant capacity of the samples being tested. Therefore, it is interesting to compare the results obtained from these two methods to see whether the same samples showed similar trends, and scavenge the test radicals as effectively in both cases.
+
Assuming 70% of the available ABTS was converted to ABTS• radical cations during +
the production reaction, 5 mL of ABTS• working solution would contain 2.3 µmole of +
ABTS• radical cations. According to the elemental analysis results of PPy0.5, PANI1.5 and PEDOT1.0 with deduction of dopants, as given in section 3.4.1, 3.5.1 and 3.6.1, 1.0 mg of PPy0.5 represents 10.8 µmoles of pyrrole units, while 1 mg of PANI1.5 and PEDOT1.0 represent 8.0 µmoles of aniline units and 6.7 µmoles of EDOT units, +
respectively. As calculated based on the percentage of the ABTS• radical scavenged, 1.7 +
µmoles of ABTS• radicals were scavenged by PPy0.5 (a 6 to 1 ratio of pyrrole units to +
+
ABTS• ), 1.9 µmoles were scavenged by PANI (a 4 to 1 ratio of aniline units to ABTS• ) +
and 0.9 µmoles were scavenged by PEDOT (a 7 to 1 ratio of EDOT units to ABTS• ) in the 3 hr reaction period.
112
+
As presented in Table 4.3, the correlation between the ABTS• and DPPH• radical scavenging of these three conducting polymers was high. In comparison in the DPPH• scavenging presented in section 3.4.3, 3.5.7, and 3.6.4, 3 pyrrole units were required to scavenge 1 DPPH• unit, while 2 aniline units and 8 EDOT units were required to react with 1 DPPH• unit, respectively. The results from both ABTS and DPPH assays indicate that PANI has the greatest free radical scavenger among these three conducting polymers, per monomer unit, followed by PPy. PEDOT also showed free radical scavenging activity, however, with a weaker effect than PANI and PPy. The greater free radical scavenging capacity of PPy and PANI might be due to the -N-H groups in their structures, which can donate H-atoms to the free radicals. It has also been reported that intermolecular hydrogen bonding is an important property affecting the antioxidant activity of oligopyrroles, when they operate to stabilize their intermediate radical145. In addition, the low N-H bond dissociation energy (77-80 Kcal/ mol)179-182 may give another explanation +
for higher efficiency of PPy and PANI to react with free radicals, since ABTS• and DPPH• scavenging includes hydrogen atom transfer to the free radicals in addition to electron transfer. The lower efficiency of PEDOT might be due both to a lack of intermolecular hydrogen, available for hydrogen atom transfer, and to the steric hindrance in the structure174 arising from ethylenedioxy group at the 3- and 4- positions, as outlined in section 1.3.
113
+
Table 4.3.Comparison of DPPH• and ABTS• scavenging activity of the conducting polymers. PPy0.5
PANI1.5
PEDOT1.0
(Py : DPPH•/ ABTS•+)
(ANI : DPPH•/ ABTS•+)
(EDOT : DPPH•/ ABTS•+)
ABTS• scavenginga
6:1
4:1
7:1
DPPH• scavengingb
3:1
2:1
8:1
+
a: reaction time is 3 hrs b: reaction time is 24 hrs
+
4.3.4 Spectroscopic characterization of PANI before and after reaction with ABTS• and DPPH• +
The structures of PPy, PANI and PEDOT before and after reaction with ABTS• radicals were investigated by FTIR and the spectra are presented in Figure 4.6. For all these three conducting polymers, there were no significant structural changes observed after reaction +
with the ABTS• radicals, suggesting that the degree of modification of the polymers (an expected oxidation) was not great, or else subsequent reactions of the polymers with solution species could reduce the polymers back to some extent.
114
0.25
A
Absorbance
0.20
(a)
0.15 (b)
0.10
C=O
0.05 0.00 -0.05
600
900
1200
1500
1800
Wavenumber / cm
2100
-1
0.24
B
Absorbance
0.21 0.18 0.15
(a)
0.12 0.09 (b)
0.06 0.03 600
900
1200
1500
Wavenumber / cm
1800
2100
-1
0.35
C
Absorbance
0.30
(a) 0.25 0.20
(b) 0.15 0.10 0.05
600
900
1200
1500
Wavenumber / cm
1800
2100
-1
Figure 4.6 IR spectra of (A) PPy0.5 (B) PANI1.5 (C) PEDOT1.0: (a) before (b) after +
reaction with ABTS• . 115
X-ray photoelectron spectroscopy (XPS) was used to monitor the structural changes of +
PANI when reacted with ABTS• free radicals. The N1 XPS core level spectra for PANI +
1.5 before and after reaction with ABTS• and DPPH• are shown in Figure 4.7. The N1s spectra of PANI can be deconvoluted into three environments: C-N at 399 eV, C=N at 398 eV and C-N+ at 400 eV, which can possibly be split into two components centred at 401 eV and 402 eV122. The PANI sample was polymerized in the absence of a strong acid, such as sulfuric acid. It has been reported that the oxidation of aniline in mild acidic, neutral or alkaline conditions lead to the production of non-conducting oligomers in the early stages of oxidation183, which might lead to a higher C-N content than typical emeraldine salt prepared in the presence of acid (50 % oxidized) (Figure 4.7A). The higher C-N content of the PANI sample prepared under higher pH conditions has been observed and exhibited in our previous report155. It can be seen that the benzenoid amine (C-N) peak was larger (83 %) in the as-prepared PANI powder and decreased to 79 % +
and 66 % after reaction with ABTS• and DPPH· respectively. The positively charged amine (C-N+) also declined from 11 % for the as-prepared PANI to 9.3 % after reaction +
with ABTS• radical cations and to 7.7 % after reaction with DPPH free radicals. However, the quinoid imine (C=N) increased from 6.4 % for the as-prepared PANI to +
11.8 % after reaction with ABTS• radical cations and to 26.5 % after reaction with DPPH free radicals. The results agree with the findings mentioned in section 3.5.8, indicating that PANI was oxidized during reaction with the free radicals, given that benzenoid amine and positively charged amines changed to quinoid imine. On the other hand, no differences in XPS spectra were observed for PPy and PEDOT after reaction +
with ABTS• radical. 116
4000
A
3500
CPS
3000
2500
C-N
2000 +
C-N C=N 1500
1000 394
396
398
400
402
404
406
Binding Energy / eV
1600
B
1400
CPS
1200
C-N 1000
C-N
+
C=N
800
600
Figure 4.7 XPS spectra of
394
396
398
400
402
404
3200
C C-N
2800
CPS
406
Binding Energy / eV
2400
C=N
2000
+
C-N 1600
1200 394
396
398
400
402
Binding Energy / eV
+
and after reaction with (B) ABTS• and
117
404
406
PANI1.5 (A) before,
(C) DPPH•.
+
4.3.5 Comparison of ABTS• and DPPH• oxidant strength using cyclic voltammetry and UV- visible spectrometry +
According to their interaction with antioxidants, both ABTS• and DPPH• can be considered as oxidants. The relative strength of these two free radicals as oxidants can be evaluated using cyclic voltammetry (CV) combined with UV-visible spectrometry of the +
products of electrochemical transformations. As seen in the CV trace of ABTS• in the potential range from –0.1 to 1.1 V (Figure 4.8), there were two reversible waves, as also observed by Scott et al.184. Focusing on the first anodic peak at around 0.5 V, we measured the cyclic voltammogram for 15 cycles and UV-vis spectra at the same time, and the results are shown in Figure 4.9A and B. It can be seen that the absorbance at 753 nm changed cyclically during the cyclic voltammetric experiment, expected to be due to +
the formation and removal of the green/blue ABTS• radical as the potential was cycled. It was also observed in Figure 4.9C that the absorbance increased rapidly when the electrode potential was greater than 0.5 V.
+
The generation of ABTS• was also be confirmed by the oxidation of ABTS with the electrode held at a constant potential of 0.5 V for various times. As seen in Figure 4.9D, the absorbance at 659 nm, 753 nm and 843 nm increased with time as ABTS was oxidized at 0.5 V; the absorbance at these wavelengths has been used to monitor the level
118
+
of ABTS• as it reacts with antioxidants26, 27. The second oxidation peak was then due to +
further oxidation of ABTS• .
20 15
Current / μA
10 5 0 -5 -10 -15 -20 -25 -0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Potential / V
Figure 4.8 Cyclic voltammogram of 0.1 mM ABTS in 0.1 M LiClO4 in ethanol using an Indium tin oxide (ITO) electrode as the working electrode, platinum wire as a counter electrode and silver covered with AgCl as the reference electrode at a scan rate of 50 mV/s.
The CV voltammogram and UV-vis spectra of DPPH• are shown in Figure 4.10, where +
the redox behaviour of DPPH• was recorded between –0.5 and 0.9 V. Similar to ABTS• ,
119
two reversible waves can be seen in Figure 4.10A. It has been found that DPPH• undergoes a reversible one-electron reduction to form a stable anion and a one-electron oxidation to form a stable cation as shown below185: + e-
-
e-
. DPPH
e-
-
-
+e
-
+ DPPH
(eq. 4.2)
DPPH
The anodic peak a in Figure 4.10A was due to the production of DPPH• from the DPPH anion and the anodic peak b was ascribed to the reaction of DPPH• to the DPPH cation, while the cathodic peak b’ was due to the generation of DPPH• through reduction of the DPPH cation and the cathodic peak a’ was ascribed to the reaction of DPPH• to form the DPPH anion, as seen the absorbance changes shown in Figure 4.10C and D, in which case the coloured DPPH• radical is removed at the upper (E > 0.6 V) and lower (E < -0.2 V) potential extremes. Figure 4.10B shows the absorbance changes over the first 10 cycles which further illustrates the cyclic pattern of DPPH• formation and removal.
120
0.030
40 30
0.025
A
0.020
Absorbance
Current / μΑ
20
B
10 0
0.015 0.010
-10 0.005
-20 0.000
-30 -0.2
0.0
0.2
0.4
0.6
0
0.8
100
200
300
400
500
600
Time / s
Potential / V
40
0.10
20 10
-20 0.020
-30
0.015 0.010 0.005 0.000 0.0
0.2
0.4
0.6
0.8
Absorbance
0 -10
-0.2
D
C
Absorbance at 753 nm
Current / μΑ
30
0.05
i a
0.00 400
500
600
700
800
Wavelength / nm
Potential / V
+
Figure 4.9 Generation of ABTS• monitored using cyclic voltammetry and UV-visible spectrometry: (A) CV voltammograms from -0.1 to 0.8 V; (B) associated absorbance changes at 753 nm with time; (C) cyclic representation of absorbance change at 753 nm +
as a function of applied potential (first 3 cycles); and (D) generation of ABTS• at 0.5 V after 0, 10, 120, 240, 360, 480, 600, 720 and 840 seconds (a-i).
121
900
0.964 40
b
A
30 20
a
Absorbance
Current / μA
B
0.960
10 0 -10
b'
-20
0.956
0.952
0.948 -30
a'
-40
0.944 -0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
-100
0
100
200
Time / s
300
400
500
600
700
Time / s
0.963 0.962
C
40
0.946
30
0.945
30
D
20
0.961
0
0.957
-10
0.956 0.955
0.943 0 0.942 0.941
-10
0.940
-20
-20 0.939
0.954
-30
-30 0.938
0.953 -0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
Potential / V
Potential / V
Figure 4.10 Oxidation and reduction of DPPH• monitored using cyclic voltammetry and UV- visible spectrometry: (A) CV voltammogram from -0.5 to 0.9 V, (B) absorbance changes at 753 nm for various times, (C) The CV trace (dash line) and in situ change of absorbance at 753 nm (solid line) with a potential scanning from +0.4 V +0.9 V
-0.5 V
-0.5 V, (D) The CV trace (dash line) and in situ change of absorbance at 753
nm (solid line) with a potential scanning from +0.4 V
122
+0.9 V
-0.5 V
+0.9 V.
0.8
1.0
Current / μΑ
10
0.958
10
Absorbance
0.959
Current / μA
20
0.960
Absorbance
0.944
+
As shown in Figures 4.8 to 4.10, the main peak for the production of ABTS• and DPPH• in the CV traces occurs at 0.47 and 0.05 V, respectively. These results indicate that +
+
ABTS• is a stronger oxidant than DPPH•, since the oxidation potential of ABTS• is higher than that of DPPH•, and it is more difficult to oxidise (and easier to reduce) than DPPH•. When reacting with an antioxidant, such as a conducting polymer, we might +
expect that ABTS• would be more reactive than DPPH•, because the free radicals would be reduced more easily during the reactions. This expectation is consistent with previous +
reports that ABTS• radicals are more reactive than DPPH radicals in tests made with various small molecule antioxidants, including BHT, BHA, α-tocopherol, Trolox and curcumin, which is a phenolic compound obtained from Curcuma longa L.28. However, the fact that changes in FTIR spectra were more pronounced after reaction with DPPH• +
than with ABTS• , the greater number of radicals scavenged per DPPH• unit (Table 4.3, although the DPPH test ran for a longer period of time), and a greater degree of polymer oxidation with DPPH• was indicated from XPS results, indicates that the particular nature of the weak oxidising radical, in addition to its formal electrode potential, will determine the extent of its reaction. On the other hand, unlike DPPH•, which can only dissolve in +
organic solvents, ABTS• can dissolve in both organic and aqueous solutions, thus the ABTS assay is suitable for both lipophilic and hydrophilic systems. This makes the ABTS assay an ideal method to evaluate the free radical scavenging activity of antioxidant samples, including packaging materials, which might otherwise dissolve in an unwanted manner in the organic solvents required for the DPPH assay.
123
4.4 Conclusions +
The modified ABTS assay, where ABTS• radical cation is generated prior to addition of antioxidant samples, is suitable to evaluate the antioxidant capacity of conducting polymers such as PPy, PANI and PEDOT powders. The results of the ABTS assay showed a similar antioxidant ranking of the tested conducing polymer powders to the results obtained from the DPPH assay: PANI1.5 > PPy0.5 > PEDOT1.0. The results also show that the reduced forms of all the three conducting polymers are greater free radical scavengers than the as-prepared samples, which also agrees with the findings for the DPPH assay. The greater scavenging effect of PANI and PPy samples than of PEDOT might be due to the presence of intermolecular hydrogen bonding in PANI and PPy samples, which contain an –NH group in their structures. In a comparison of relative +
oxidant strength, the ABTS• radical was the stronger oxidising agent (it was itself reduced at a higher potential) than DPPH•, with reduction of the radicals on the cathodic +
sweeps occurring from around 0.5 V for ABTS• and from around 0.1 V for DPPH• (versus a silver wire coated with AgCl as the reference electrode). On the other hand, in the case of PANI, a larger increase of oxidised imine (C=N) units was observed after +
reaction with DPPH• relative to reaction with ABTS• , and from the 1 mg PANI samples, +
5.1 µmoles of DPPH• and only 2.3 µmoles for ABTS• were scavenged. Advantages of +
+
using ABTS• radical include testing in aqueous solutions, although when using ABTS•
radicals they need to be generated prior to utilization and after formation the free radicals are normally stable for just two days186.
124
Chapter 5 Antioxidant capacity of a conducting polymer – ethyl cellulose film
5.1 Introduction
Polyaniline (PANI) powders have been demonstrated as efficient free radical scavengers104, 126, 161, and can also be blended with other processable polymers to extend their applications187-195. Ethyl cellulose (EC) is believed to be a suitable material for food packaging because of its stability and non-toxicity towards foodstuffs196. Therefore, it is of great interest to prepare a PANI composite with EC for food packaging applications, which is capable of quenching free radicals and extending the shelf life of food products. Polypyrrole (PPy) powders were also shown to provide high free radical scavenging activity. Therefore, in this work, PANI and PPy EC composites were prepared by a solution-based method and their free radical scavenging activity was evaluated using the Oxygen Radical Absorbance Capacity (ORAC) assay, which is a commonly used antioxidant capacity assay in Food Science2, 121, 197-199.
5.2 Experimental
A PANI EC film, with nanotube forms of PANI produced at high pH, was prepared as described in section 2.6.1; a PPy EC film (with a thickness of 120 ± 12 μm) was also prepared by the same method using PPy powders made with a APS/Pyrrole ratio of 0.5. The corresponding reduced PANI EC film was prepared as described in section 2.6.2. An
125
in situ oxidatively polymerized PANI(OX)EC film was prepared using a synthetic method as described in section 2.6.3.
A PANI PE film was prepared by a melt processing procedure as described in section 2.7. PANI and PPy films were also prepared by electrochemical polymerisation, using a stainless steel sheet (5 × 9 cm2) as a working electrode as described in section 2.8.
The antioxidant capacity of both PANI and PPy containing films was evaluated using the ORAC assay, as described in section 2.9.3, to determine the peroxyl free radical scavenging capacity of various PANI and PPy films. The morphology of the PANI EC films was examined by scanning electron microscopy (SEM) as described in section 2.10.1. A tensile test measurement (ASTM D638) of the PANI EC films was performed as described in section 2.11. XPS was also used to investigate structural changes in the PANI component after reaction with peroxyl free radicals. Before the XPS measurements, 5 mg of PANI powder was soaked in 10 mL of AAPH solution (306 mM) for 3 hrs and then washed with milli-Q water and vacuum dried.
5.3 Results and Discussion
5.3.1 Development of the PANI EC films A PANI EC film was prepared using a solution-based method. Ethyl cellulose (EC) was originally dissolved in dimethyl sulfoxide (DMSO) and then mixed with PANI powders. It was found that the mixture was not able to form a film on a glass surface when cast, and that it took around 6 hrs to remove the solvent. Thus, DMSO seemed unsuitable for
126
making a PANI EC film. Ethanol, which is believed to be safe for contact with foodstuffs, was found to be an appropriate solvent for dissolving EC. PANI EC film was therefore prepared by mixing PANI powder with EC-containing ethanolic solution and then cast on a glass surface. The resulting film showed that the PANI powders did not disperse homogenously in the EC base. The reason for the heterogeneous dispersion was possibly due to low density of the matrix, which was not able to stabilize the dispersion of PANI particles and clusters in a colloidal EC matrix. In order to obtain a homogenous dispersion of PANI particles, the mixture was then concentrated using a vacuum rotator, which also removed bubbles from the matrix at the same time, since air bubbles were readily generated during mixing. After concentration of the matrix and removal of bubbles, a homogenous mixture was obtained. However, the film was difficult to remove from a glass surface. Teflon is known to be a non-stick material, which was therefore chosen for casting PANI EC films because of the easy removal of the film. The thickness of PANI EC films obtained in this way was 120 ± 12 µm.
5.3.2 Morphological study In Figure 5.1, SEM micrographs of a PANI EC film (with 9 % PANI) are shown. A very homogenous dispersion of PANI particles (with some clusters) in the EC matrix was observed. The presence of EC (higher in part І) and PANI powders (higher in part П) in the film was also examined by energy-dispersive X-ray spectrometry (EDXS). The analysis provided molar percentages of O, N and C of the two parts (Table 5.1), and showed a N/C ratio of 0.075 for part I and 0.12 for part II (compared to a theoretical value of 0.17 for pure PANI, and an absence of N in EC). As N was detected in both
127
parts І and П, this showed that PANI was dispersed in the EC matrix to varying degrees. The dispersion of PANI powder in the EC matrix also gives an explanation as to the very low conductivity of the film, below the measurement level of the available instrument (less than 10-6 S cm-1), because the electron transfer between PANI units was blocked by the insulating EC matrix.
A
B
C
Figure 5.1 SEM images of (A) Pure EC, (B) a PANI EC film prepared with 9 % PANI viewed from above, and (C) a cross section of the PANI EC film, where ○ is an area high in EC (part І) and □ is an area high in PANI (part П).
128
Table 5.1 Molar percentage of O, N and C in PANI cluster and EC matrix. Molecular Percentage (At %) Experimental
Theoretical
EC (І)
PANI (П)
EC
PANI
O
10.28
8.49
31.3
0
N
6.29
9.52
0
14.3
C
83.43
81.99
68.7
85.7
N/O
0.61
1.12
0
-
N/C
0.075
0.12
0
0.17
Element
5.3.3 Development of an ORAC assay for PANI-containing films The ORAC-FH assay is commonly applied for the evaluation of water-soluble antioxidants using fluorescein (FH) as a fluorescent probe and AAPH as an oxygen radical generator39, 40, 121, 200. The antioxidant capacity of the samples is determined by the area under the FH decay curve as AAPH oxidizes and removes fluorescein, comparing a blank run with the test sample, to give the net AUC area. In an initial trial, it was found that 20 mg of a PANI EC film (9 %) delayed the decay of fluorescence and led to a net AUC of 17.7 units (Figure 5.2). By contrast, when 18.2 mg of pure EC was tested, a net AUC value of just 3.8 units was obtained. The results indicated that the ORAC-FH method is suitable to examine the free radical scavenging activity of such conducting polymer containing films.
129
After the first trial, various amounts (by weight) of PANI EC film (several small pieces) were tested, and it was found that the net AUC increased when more pieces were used, but not in a very linear manner (Figure 5.3). The low correlation might be due to some parts of some film pieces being covered by the others. The results led to a consideration of applying single PANI EC film pieces for the test of different sizes, from 16 to 100
Relative Fluorescence Intensity
mm2.
1.0 0.8 0.6 0.4
(c)
(a) (b)
0.2 0.0 0
20
40
60
80
Time / min
Figure 5.2 The decline of fluorescence of a fluorescein solution with (a) nil (b) 18.2 mg of EC film and (c) 20.0 mg of PANI EC film, in presence of AAPH at 37 oC.
130
24 22
Net AUC
20 18 16 14 12 4
8
12
16
20
24
28
32
36
Weight / mg
Figure 5.3
Correlation between ORAC area and weight of multiple PANI EC film
pieces.
5.3.4 Peroxyl free radical scavenging activity of PANI EC films The antioxidant activity of the PANI EC film evaluated by ORAC assay is presented in Figure 5.4. These curves show the kinetic behavior of the fluorescein / AAPH system in the absence (control) and in the presence of PANI EC film (9 %). The PANI content of the film neutralized the peroxyl radicals generated in the system and delayed the decay of the fluorescence curve by a certain length of time. The antioxidant capacity was evaluated based on the net AUC; the bigger the area, the greater the antioxidant capacity. 131
Similar to various small molecule antioxidants, when polyaniline acts as a chain-breaking antioxidant (AH), it traps the peroxyl free radical (ROO•) by transferring a hydrogen atom to the radical.
ROO• + AH → ROOH + A•
(eq. 5.1)
The radicals of PANI (A•) are resonance stabilized (Scheme 5.1), and thus will not continue the chain reaction201. However, it is also possible that A• terminates the chain reaction by combining with the peroxyl radical3 or by reaction with another A• unit200:
ROO• + A• → non-radical products
(eq. 5.2)
A• + A• → non-radical products
(eq. 5.3)
132
N
.
H
H
N
..
+
-
e
+
Formation of PANI radical cation
H
H N
N
N
+
H
H
N
.
+
+
H
H
.
+
.
. Resonance of PANI radical cation Scheme 5.1 Formation of PANI radical cations and resonance stabilisation of PANI radical cations.
PANI EC film presented effective peroxyl radical scavenging activity from a 16 mm2 film (containing around 1.6 mg PANI) and the activity increased with an increase in film area. A good linear relationship was obtained between net AUC and the area of the tested film, as shown in Figure 5.4B, which also served to confirmed the generally homogeneity of the PANI EC film.
133
Relative Fluorescence Intensity
1.0 A
0.8 0.6 0.4 0.2
(a) (b) (c)
(e)
(d)
0.0 0
20
40
60
80
100
120
140
160
Time / min
27 24
B
Net AUC
21 18 15 12 9 6 3
0
15
30
45
60
75
Film Area / mm
90
105
2
Figure 5.4 ORAC results of PANI EC films (9 % PANI): (A) fluorescence decay curve of fluorescein induced by peroxyl radicals in presence of various PANI EC films; (a) no film present, (b) 16 mm2 (c) 36 mm2 (d) 64 mm2 and (e) 100 mm2 of PANI EC film; (B) correlation between net AUC and film area (deducted 91% ORAC area of EC, with net AUC of 1.6, 3.6, 6.7 and 8.4 units for film areas of 16 mm2, 36 mm2, 64 mm2 and 100 mm2 respectively). 134
Trolox, a water-soluble α-tocopherol analogue, was used as an antioxidant standard. The Trolox equivalent is commonly applied to measure the antioxidant capacity of test samples. The plot of the net AUC versus Trolox concentration is presented in Figure 5.5. For samples that are soluble in working solution, the relative ORAC value (Trolox equivalents) is calculated as follows40, 41:
Relative ORAC value = [(AUCsample – AUCblank) / (AUCtrolox – AUCblank)] × (molarity of Trolox /molarity of sample)
(eq. 5.4)
In our case, PANI EC film is not soluble in the working solution and films with different areas were used for the test, so that the trolox equivalent of the sample films was expressed as follows:
Relative ORAC value = [(AUCsample – AUCblank) / (AUCtrolox – AUCblank)] × (µ moles of Trolox / area (mm2) of the tested film)
(eq. 5.5)
Using the net AUC of the PANI EC films (Figure 5.4 B) and the Trolox standard curve (Figure 5.5), the antioxidant activity of PANI EC film can also be expressed as a relative ORAC value in Trolox equivalents, for films of different areas, as shown in Figure 5.6.
135
36 32 28
Net AUC
24 20 16 12 8 4 0 0
10
20
30
40
50
Concentration / μ M
-3
Relative ORAC Value (Trolox equivalents, 10 μ mole)
Figure 5.5 Plot of net AUC versus Trolox concentration (µM).
4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0
20
40
60
Film Area / mm
80
100
2
Figure 5.6 Relative ORAC value (Trolox equivalents, µ moles) as a function of film area (mm2) for the PANI EC films (9 % PANI).
136
The Weibull distribution function (eq 5.6) has been used to describe destruction of microorganisms202
and inactivation of enzymes203,
204
under the treatment by high-
intensity pulsed electric fields (HIPEF). The distribution equation was recently also applied by Odriozola-Serrano et al.205 to describe the retention of health related compound, such as vitamin C, under HIPEF. In our case, it was found that the curves obtained by plotting the reduction of fluorescence from the FH- AAPH system against the reaction time were well fitted by using the equation with high values of R2 (Figure 5.7), which has not been reported by other researchers. The kinetic behavior of the system in absence and presence of PANI film could also be described by the Weibull distribution function, which contains the following parameters:
I = I 0e
−(
t
α
)β
(eq. 5.6)
where I is the relative fluorescence intensity, I0 is the initial fluorescence intensity, α is the scale factor (min), β is the shape parameter and t is reaction time (min).
The parameters and R2 for the fitting curves were listed in Table 5.2, which were obtained by fitting equation (5.6) to the experimental data using a non-linear curve fit function in Origin 7.5. With the exclusion of the blank run, the parameter α ranged from 26.7 to 56.1 and increased as the area of the tested film increased. By contrast, the value of β, with a range from 1.6 to 2.3, decreased as the tested film area increased; the smaller the value the longer the tail of the curve. The changes of α and β with the tested film area reflect the in-situ fluorescence intensity, which can be calculated by equation (5.6) and was influenced by the reaction of PANI film and the free radical in the system. A larger α
137
and smaller β obtained from the larger area films pointed to a greater retention of fluorescence level in the reaction system (calculated by eq 5.6), indicating that the films providing more PANI particles to react with the peroxyl free radicals and thus exhibiting a high antioxidant efficiency. Good linear correlations were also obtained between the film area and parameters α, as shown in Figure 5.8, indicating that parameter α of various tested film areas can be predicted using the correlation line, and that their kinetic curves could be estimated by applying equation (5.6). 1/α can be used to represent the reaction rate of the system. In addition, if the kinetic curves are similar between all the tested films, the mathematic function may also be suitable for analyzing curves for PANI EC films with various proportions of PANI or with other polymer composites.
Table 5.2 Regression Coefficients for fitting the Weibull distribution function curve to the ORAC response curves for PANI EC films (9 % PANI). Coefficients
Area of PANI EC film (mm2)
I0
α
β
R2
Nil (blank)
0.997 ± 0.003
18.55 ± 0.05
2.247 ± 0.021
0.99954
16
0.993 ± 0.002
26.69 ± 0.06
2.261 ± 0.016
0.99971
36
0.989 ± 0.003
34.66 ± 0.11
1.980 ± 0.017
0.99948
64
0.985 ± 0.002
47.32 ± 0.12
1.774 ± 0.011
0.99968
100
1.018 ± 0.002
56.05 ± 0.14
1.558 ± 0.008
0.99973
138
Relative Fluorescence Intensity
1.0 0.8 0.6 0.4 0.2
(e)
(d)
(a) (b) (c)
0.0 -20
0
20
40
60
80
100 120 140 160 180
Time / min
Figure 5.7 Fluorescence decay curve of AAPH /FH system in presence of (a) nil (b) 16 mm2 (c) 36 mm2 (d) 64 mm2 and (e) 100 mm2 of PANI EC film. The dots represent experimental data points and the lines are the fitting results.
2.3
56
2.2
52
Parameter β
Parameter α
48 44 40 36
2.1 2.0 1.9 1.8
32
1.7
28
1.6
24
1.5 0
10
20
30
40
50
60
70
Film Area / mm
80
90 100 110
2
0
10
20
30
40
50
60
70
80
90 100 110
2
Film Area / mm
Figure 5.8 Correlation between the tested PANI EC film area and (a) parameter α and (b) parameter β. (a) y = 0.355x + 22.0, R2 = 0.9807 (b) y = -0.008x + 2.33, R2 = 0.9617
139
5.3.5 Characterization of PANI powders by XPS and IR The XPS spectra of a PANI powder (in the absence of added EC) before and after reaction with AAPH peroxyl free radicals is presented in Figure 5.9. The proportions of benzenoid amine (C-N), quinonoid imine (C=N) and positively charged nitrogen (C-N+) in PANI was determined based by curve-fitted the N(1s) core-level spectra, with binding energies (Be) set at 399 eV, 398 eV and 400 eV, respectively. For C-N+ at 400 eV, it is possible that two peaks at 401 eV and 402 eV are involved122. Li et al206 have shown how the change in the [C=N] / [C-N] ratio can be applied to evaluate the oxidation level of PANI. It can be seen that after reaction with peroxyl radicals, the N= content increased from 6.4 % to 18.6 %, while benzenoid amine decreased from 82.5 % to 71.0 %, and positively charged nitrogen changed only from 11.1 % to 10.3 %. Clearly PANI was oxidized by the free radicals, as the [C=N] / [C-N] ratio increased from 0.08 to 0.26. The results support the findings for the application of the DPPH and ABTS assay (see section 4.3.4), which also showed the oxidation of conducting polymers after reaction with free radicals.
The IR spectra of PANI1.5 before and after reaction with peroxyl free radicals are shown in Figure 5.10. It can be seen that there is no significant difference in structural changes between the two tested powders. The difference between the results obtained from XPS and IR spectra might be due to the fact that the oxidation of PANI by peroxyl free radicals readily occurs in the surface layers moreso than in the bulk, since XPS is a surface chemical analysis technique, while IR spectra is obtained by passing a beam of infrared light through the entire tested sample.
140
4000 A
3500
CPS
3000 2500
C-N
2000
+
C-N C=N
1500 1000 394
396
398
400
402
404
406
Binding Energy / eV
4000 B 3500 C-N
CPS
3000 2500 C-N
C=N
+
2000 1500 1000
396
398
400
402
404
406
Binding Energy / eV
Figure 5.9 XPS spectra of PANI (A) before and (B) after reaction with peroxyl free radicals.
141
Absorbance
(a)
(b) 600
800
1000
1200
1400
Wavenumber / cm
1600
1800
-1
Figure 5.10 IR spectra of PANI (A) before and (B) after reaction with peroxyl free radicals.
5.3.6 Peroxyl free radical scavenging capacity of PANI EC films with different PANI contents The peroxyl radicals scavenging activity of PANI EC films prepared with 5 %, 9 % and 17 % of PANI powder was compared and the results are presented in Table 5.3. As can be seen, when a small 16 mm2 film was tested, the peroxyl free radical scavenging capacity of the film with 5 % PANI added was very small, with only 0.6 of net AUC area, while with 9 % PANI, the antioxidant capacity was similar to that containing 17 % PANI, with net AUC areas of 5.4 and 5.3 respectively. However, when larger films were
142
tested, a higher content of PANI consistently led to much greater scavenging capacity, in the order of 17 % > 9 % > 5 %. It is obvious that the difference in peroxyl free radical scavenging between the tested films with various PANI contents is easier to observe when larger films are used. In addition, using greater amounts of samples can minimize the experimental uncertainties. The results pointed out that the peroxyl free radical scavenging capacity of PANI EC film related to the content of PANI in PANI EC film; the higher the content of PANI, the greater the antioxidant capacity. Apart from evaluation of antioxidant capacity, another property of the polymer film, such as mechanical property, was also important to investigate.
Table 5.3 Net AUC of PANI EC films. Film area (mm2)
% of PANI of PANIEC film
16
36
64
100
5
0.6
2.8
4.8
6.6
9
5.4
10.1
18.1
26.0
17
5.3
14.2
25.4
43.7
5.3.7 Mechanical properties of the PANI EC films The mechanical property of the PANI EC films with various PANI contents were obtained by measuring the tensile modulus, between 0.05% and 0.25% strains, and ultimate tensile strength at a crosshead speed of 5 mm/min (Figure 5.11). It can be seen that addition of 5 % and 9 % of PANI powders increased the ultimate tensile strength
143
from 23.1 (blank without PANI) to 24.1 and 28.4 MPa, respectively. However, with addition of 17 % of PANI, the tensile strength decreased to17.6 MPa. The modulus of PANI EC film with 5 % and 9 % of PANI powders decreased from 2.3 (blank) to 1.3 GPa, while with 17 % of PANI powders showed the tensile modulus of 2.1 GPa.
It is clear that the mechanical strength of PANI EC film depends on the proportion of PANI included in the film. The film prepared with 9 % of PANI powder led to an increase in the tensile strength and a decrease in the tensile modulus, compared to the EC only blank, showing good mechanical properties. However, with 17 % of PANI powder, adverse effects were observed. Generally, there was no significant difference in the tensile modulus between the tested films. However, the film with 9% of PANI powders was less brittle than the other tested films, according to its higher tensile strength. With a high Young’s modulus (from 1.3 to 2.1 GPa) and tensile strength (17.6 to 28.4 MPa), the PANI EC films therefore show good mechanical strength, and would be suitable for inclusion in practical applications such as in food packaging.
144
6
28 5 26 24
4
22 3 20 18
2
16 1 14 12
0
4
8
12
16
20
0
Modulus / segment 0.05%-0.25%, GPa
Ultimate Tensile Strength / MPa
30
% of PANI in PANIEC Film
Figure 5.11 Mechanical properties of PANI EC films with varying amounts of PANI added: (Δ) modulus and (●) ultimate tensile strength.
5.3.8 Peroxyl radical scavenging activity of reduced PANI in PANI EC films The antioxidant capacity of the as-prepared PANI EC film was tested when a reduced form of PANI was used, or when the as-prepared PANI film was subsequent exposed to a reducing (hydrazine) solution. As seen in Figure 5.12, the film prepared with the reduced PANI and the reduced as-prepared PANI EC film both showed much a much higher antioxidant effect against peroxyl radicals compared to the (untreated) as-prepared PANI EC film.
145
As is well known, PANI is formed with a variety of oxidation states, which have differing electronic structures. The main structural units of PANI in its base (non-acidied) form, as shown in Scheme 5.2, are leucoemeraldine (totally reduced), emeraldine (half oxidised) and pernigraniline (fully oxidised). The as-prepared PANI powders are expected to be initially in the emeraldine oxidation state. When it reacts with the AAPH peroxyl free radical and acts as an antioxidant, PANI has to be not only an electron donor but also a proton donor. As seen in scheme 5.2, leucoemeraldine (the fully reduced PANI) contains more N-H than emeraldine and pernigraniline, and would be expected to be able to transfer H-atom more easily to the peroxyl free radicals. The structure of reduced PANI, with more N-H, provides an explanation why reduced PANI showed a greater capacity to scavenge peroxyl free radicals than the more oxidized forms.
50
Net AUC
40
30
20
10
0
0
20
40
60
Film Area / mm
80
100
2
Figure 5.12 Comparison of peroxyl free radical scavenging ability of (■) PANI EC film, (●) reduced PANI + EC and (△) reduced PANI EC film. 146
H N
H N
H N
H N
n
Leucoemeraldine Base H N
N
H N
N
n
Emeraldine Base N
N
N
N
n
Pernigraniline Base
Scheme 5.2. The main chemical forms of polyaniline showing different proportions of (reduced) benzenoid and (oxidized) quinoid units.
5.3.9 Peroxyl radical scavenging activity of the in situ oxidatively polymerized PANI(OX)EC film A PANI EC composite was also prepared by polymerizing aniline directly in the EC matrix and exposing this to the APS oxidant. The SEM image of the resulting film is presented in Figure 5.13. Unlike the PANI EC film, which showed PANI clusters dispersed in the EC matrix (see Fig. 5.1), the film showed that PANI was well dispersed 147
within the EC matrix and that no individual PANI particles or clusters could be observed. However, after rinsing with water, the film shrinked and tended to roll parallel to its longer edge on air-drying, which might be due to the removal of the supporting electrolyte (residual APS and its reaction products). A comparison of peroxyl radical scavenging activity of the PANI(OX)EC and the PANI EC films prepared using preformed PANI powders (from above) is shown in Figure 5.14. It was found that the antioxidant activity was higher for the PANI(OX)EC film, as seen by the slope of the linear regression being 1.9-fold higher for PANI(OX)EC film. The stronger activity of the PANI(OX)EC film may be owing to more uncovered PANI particles being homogenously located on the surface of the film, which is evidenced by the SEM image.
Figure 5.13 SEM image of PANI(OX)EC film (cross section) prepared by synthesizing PANI in the matrix of EC and casting the mixture onto a Teflon sheet.
148
80
a
Net AUC
60
40
b
20
0 0
20
40
60
F ilm A re a / m m
80
100
2
Figure 5.14 Net AUC of (a) PANI(OX)EC film prepared in matrix of EC (17 % PANI), and (b) PANI EC as-prepared films (17 % PANI). a) y = 0.859x-1.05, R2 = 0.9850 b) y = 0.446x-2.39, R2 = 0.9968.
5.3.10 Peroxyl free radical scavenging of pure PANI and of PPy films prepared electrochemically on a stainless steel working electrode A PANI film was prepared electrochemically using a stainless steel working electrode (5
× 9 cm2) at 1.1 V (versus an Ag/AgCl (+207 mV vs. SHE) reference electrode) with a thickness of 110 ± 8 μm, and a PPy film was prepared using the same procedure but at 0.8 V with a thickness of 110 ± 5 μm. The growth of the polymer films during electropolymerization was monitored in their cyclic voltammograms, as shown in Figure 5.15. It can be seen that the current on the first cycle increased sharply at around 0.8V for PPy and around 1.05 V for PANI, at which the polymerization of PPy and PANI began to take place.
149
The Net AUC of electropolymerised PANI and PPy films is listed in Table 5.4, in comparison with results for PANI EC films, and further composite PPy EC films formed by blending PPy powders with EC prior to casting a composite film. It can be seen that PANI EC film and PANI film formed electrochemically on the stainless steel electrode showed a greater scavenging capacity for peroxyl free radicals than the PPy EC and pure PPy films which instead displayed quite low net AUC responses. The results indicate that the weak activity of PPy was not due simply to the nature of its incorporation in the EC matrix. According to the results obtained from the DPPH and ABTS assays, PPy showed excellent DPPH• and ABTS·+ scavenging activity, comparable and only a little weaker than that of PANI (see section 3.4.3 and 4.3.2). The reasons for the very weak reaction of PPy with peroxyl radicals in the ORAC assay is not clear and needs to be investigated in the future work. Further the pure PANI film exhibitied a higher AUC result than the PANI EC film, but not several times more as might be expected given than the PANI EC film contained only 9% PANI. However, the greater thickness of the PANI EC films combined with some movement of the radical containing solution into the film interior, plus differences in surface roughness, could contribute to the result obtained.
Table 5.4 Net AUC of PANI EC and PPy EC cast films, and of PANI and PPy film prepared electrochemically on a stainless steel working electrode.
PANI EC film
5.3
Film area (mm2) 36 64 9.8 17.3
PANI film
5.7
15.7
28.4
35.1
PPy EC film
1.8
2.9
4.2
5.6
PPy film
1.3
1.9
3.5
3.5
16
150
100 25.1
9
10
6
8
3
6
0
4
-0.2
0.0
0.2
0.4
0.6
0.8
A
Current / mA
Current / mA
12
1.0
Potential / V
2 0 -2 -4 -0.2
0.0
0.2
0.4
0.6
0.8
1.0
Potential / V
20
3
16
2
Current / mA
1
12 8
0 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2
Current / mA
4
B
-1
Potential / V
4 0 -4 -0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Potential / V
Figure 5.15 Cyclic voltammograms for the polymerization of (A) PPy in 0.1M NaHSO4, and (B) PANI in 0.1 M H2SO4. Inserts: the first three cycles of PPy and PANI. 151
5.3.11 Peroxyl free radical scavenging activity of PANI PE film Polyethylene (PE) is commonly used in packaging, including foodstuffs 15, and therefore a composite of PE with PANI is of considerable interest. The PANI PE film was prepared using a melt processing method, where a high temperature up to 180oC was used for the process. The peroxyl radical scavenging activity of polyaniline polyethylene (PANI PE) film was then determined using the ORAC assay and the results are shown in Figure 5.16.
It was noted that this film presented remarkable peroxyl radical scavenging activity with a large net AUT, which increased with a large area of the film exposed to the test solution, and similar to the PANI EC films. The efficient antioxidant activity makes the PANI PE film a promising choice for consideration in food packaging. However, the complicated procedure, including pretreatment of the PANI sample as described in section 2.7, and the high temperature needed for the process, still needs to be taken into account.
152
Relative Fluorescence Intensity
1.0
A
0.8 0.6 0.4 0.2
(a) (b) (c)
(d)
(e)
0.0 0
20
40
60
80
100
120
140
160
Time / min
40
B
Net AUC
30
20
10
0 0
20
40
60
Film Area / mm
80
100
2
Figure 5.16 ORAC results of PANI PE film (10 % PANI): (A) relative fluorescence versus time; and (B) ORAC area versus PANI PE film area.
153
5.4 Conclusions
A PANI EC film was prepared using a solution method and PANI particles and clusters were homogenously dispersed in EC matrix, as shown by SEM images. The antioxidant capacity of the PANI EC films was examined using the ORAC assay giving the AUC area as the output. The results showed that the PANI EC films presented efficient peroxyl free radical scavenging activity even using an area as small as 16 mm2 and the activity increased when more film area was exposed to the test solution. A good correlation between net AUC and area of tested film was obtained, confirming the homogenous dispersion of active PANI powder across the film.
Using XPS, the structural change of PANI after reaction with peroxyl free radicals was observed, with benzenoid amine (C-N) converting to quinonoid imine (C=N) after the reaction with free radicals. A PANI EC film with higher proportion of PANI showed higher effect of free radical scavenging. However, the mechanical property of the highest PANI content film (17 % PANI) showed a little higher modulus but much lower tensile strength than the lower PANI content films (5 to 9 %), meaning that with 17 % of PANI, the film was more brittle. When prepared with reduced PANI or after reduction of the asprepared PANI EC film with hydrazine, the PANI EC films showed much more efficient radical scavenging, indicating that a higher amine content of PANI is more effective for H-atom donation.
The PANI-EC film can also be prepared by chemically polymerzising aniline in an EC matrix prior to casting the mixture on a Teflon sheet. The SEM image of this film showed
154
a homogenous mixing of PANI and EC. When reacted with peroxyl free radicals, this film showed greater effect than the as-prepared PANI EC film, which might be due to have more PANI concentrated at the film surface. Although the film exhibited excellent antioxidant ability, some shrinking of the film after rinsing with water was a problem that remains to be solved.
PANI PE prepared using a melt processing method also showed excellent peroxyl free radical scavenging ability. However, the procedure for making the film is more complicated than that for solution-based method and higher temperature of up to 180oC was needed during the operation, which may limit the application of this approach.
155
Chapter 6 The application of the conducting polymer films in food packaging
6.1 Introduction
Fish oils are rich sources of polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)207, 208, which are highly unsaturated fatty acids and are easily attacked by oxygen-containing free radicals. The free radical chain reaction is widely known as a common mechanism of lipid oxidation, which leads to deterioration and off-flavours in oils209. The PANI EC film has been demonstrated to be an effective free radical scavenger. Therefore, it was expected that the conducting polymer film could be applied for the inhibition of oxidation of fish oil.
6.2 Experimental
6.2.1 Measurement of peroxide value (PV) An aliquot of 3.5 g of fish oil, with or without PANI film (2×2 cm2), was placed in a 25 mL conical flask and incubated at 60 oC exposed to the air for various times. 3.0 g of the fish oil was then removed for peroxide value measurement using the AOCS Official Method Cd 8-53 as described in section 2.12. Butylated hydroxytoluene (BHT) was dissolved in absolute ethanol to a concentration of 0.1 %.
156
6.2.2 Determination of DHA and EPA in fish oil using gas chromatography The ω-3 PUFA content of Ropufa oil, with and without addition of PANI EC film (2×2 cm2), and incubated at 60oC for 5 and 8 days, was determined by gas chromatography (GC) using tridecanoic acid (C:13) as internal standard as described in section 2.13. Data analysis and peak integration were performed using GC ChemStation software (rev. A.06.03 [509]).
6.2.3 Statistical Analysis Student’s t-test was used to determine the significant difference between the mean peroxide values of the tested oils at the level of p < 0.05.
6.3 Results and discussion
6.3.1 Oxidation of lipid Lipid oxidation normally begins with the formation of free lipid radicals, which may be initiated by singlet oxygen or other sources (eq. 6.1). The peroxyl radical is produced through the reaction of lipid radicals with oxygen, which is then converted into lipid hydroperoxide by abstraction of a hydrogen atom (eq. 6.2 and eq. 6.3); the process can be repeated many times (i.e. a chain reaction). The oxidation reactions are presented as follows (X• = singlet oxygen or other sources; LH = lipid; AH = antioxidant)4:
X• + LH → L•+ H•
(eq. 6.1)
L• + O2 → LOO•
(eq. 6.2)
LOO• + LH → LOOH + L•
(eq. 6.3)
157
The extent of oxidation of the lipid can be expressed using the peroxide value (PV) and determined by reducing hydroperoxides to hydroxyl derivatives through reaction of hydroperoxides with iodide ions, which are oxidized to free iodine (I2) simultaneously (eq. 6.4). The iodine is then titrated with a solution of sodium thiosulfate and reduced to iodides, while sodium thiosulfate is oxidized to tetrathionate (eq. 6.5)4. The PV is expressed as milliequivalents of peroxide / kg of oil (mEq O2/kg oil); the larger the PV, the greater the level of oxidation of the tested oil.
LOOH + 2I- + 2H+- → LOH + I2 + H2O
(eq. 6.4)
I2 + 2S2O32- → S4O62- + 2I-
(eq. 6.5)
Ropufa oil was placed under accelerated storage in an oven at 60oC. Oxidative changes of the fish oil were measured by the PV from 2 to 14 days. As seen in Figure 6.1, the PV increased from 0 to 380 after incubation for 10 days and decreasse to 274 after incubation for a further 4 days. Such oxidative changes, measured by PV, were also observed by Ogwok et al.208. Generally, hydroperoxides present in food lipids are unsaturated and can be oxidized to secondary oxidation products, such as aldehydes. The secondary oxidation products are also unstable and can be further oxidized to tertiary products, such as compounds with a shorter chain length formed through the cleavage of the secondary oxidation products. The lowering of the PV might be due to further oxidation of some peroxides to secondary and tertiary oxidation products. Because of this subsequent decline in PV, the test was limited to 8 days of accelerated aging.
158
PV / mEq peroxides/Kg oil
400 350 300 250 200 150 100 50 0 2
4
6
8
10
12
14
Day
Figure 6.1 PV of Ropufa oil under accelerated storage conditions (60oC).
6.3.2 The influence of PANI EC film on oxidation of Ropufa oil The influence of the PANI EC film on oxidation of the Ropufa oil is presented in Figure 6.2, with a steady increase in PV again seen over the 8 day trial period. The presence or absence of the PANI EC film (9 %) did not change the PV value significantly after storage for 2 days. However, the PV was lower in the presence of PANI EC by 30 %, 11 % and 8 % respectively after storage for 4, 6 and 8 days at 60oC, compared to that of the oil maintained without a conducting polymer film exposed to the oil. In the presence of
159
the PANI EC film (17 %), the PV was lower by 29 %, 18 % and 12 % respectively after storage for 4, 6 and 8 days, a little more in the final stages than the PANI EC (9 %) films., The results showed that there was a significant difference in PV between Ropufa oil in the presence and absence of a PANI EC film (9 %) (p < 0.05), and between Ropufa oil with and without a PANI EC film (17 %) (p < 0.05). However, there was no significant difference (p > 0.05) between Ropufa oil results in presence of PANI EC films containing 9 % versus 17 % PANI powders. In addition, the results showed that PANIEC film is able to delay the oxidation of fish oil and that inhibition of oxidation is proportionally more effective in the early stages, as evidenced by the lower PV obtained.
Antioxidants, such as BHT, have been reported to inhibit lipid oxidation of fish muscle and oil13, 15. The inhibition effect is due to free radical trapping by transfer of hydrogen atoms from the antioxidant to free radicals and/or by reaction of the antioxidant radicals with lipid radicals as shown below3, 210.
L• + AH → LH + A•
(eq. 6.6)
L• + A• → LA
(eq. 6.7)
PANI powders have been demonstrated to possess free radical scavenging activity as described in chapter 3, 4 and 5, which provides an explanation to their inhibitory effects on the oxidation of fish oil.
160
300 (a) (b) (c)
PV / mEq peroxides/kg oil
250 200 150 100 50 0 1
2
3
4
5
6
7
8
9
Day
Figure 6.2 Peroxide value (PV) of Ropufa oil at 60oC with (a) no film, (b) PANIEC film (9 %), (c) PANIEC film (17 %) (n = 3).
The PV of 3.0 g of Ropufa oil in presence of various amount of BHT, a commonly used antioxidant for prevention of oxidative rancidity of fat13, 15, under accelerated storage condition (60oC) for 8 days is shown in Figure 6.3. Based upon the lowering of PV in the presence of PANI films and BHT under these test conditions, as shown in Figures 6.2 and 6.3, 2×2 cm2 of PANI EC film with 9 % of PANI powders presents a similar antioxidant effect as 1.8 μmoles of BHT, while the film with 17 % of PANI powders presents a similar effect as 2.8 μmoles of BHT.
161
PV / mEq peroxides/kg oil
260
240
220
200
180
160 0
2
4
6
8
10
12
14
BHT / μmole
Figure 6.3 Peroxide value of Ropufa oil in the presence of various amounts of the antioxidant BHT after 8 days of storage at 60oC. y = 243 – 7.21x, R2 = 0.9567
The inhibition of the PANI EC film (9 %) on oxidation of Ropufa oil was also investigated at 40 oC, and the results are presented in Figure 6.4. It can be seen that after incubation for 8 days, the PV is still lower than 20, indicating that a very slow rate of oxidation proceeded at this temperature. It was also observed that there was no significant difference in PV between Ropufa oil in the presence of and absence of conducting polymer films after incubation for 8 days (p > 0.05). The result might be due to the fact that the oxidation of the oil is too slow and only very small amount of hydroperoxides were produced at this temperature.
162
PV / mEq peroxides/kg oil
14 12 10 8 6 4 2 0 0
2
4
6
8
Day
Figure 6.4 Peroxide value (PV) of Ropufa oil at 40oC with (▲) no film, (•) PANIEC film (9 %) (n = 3).
6.3.3 Influence of α-tocopherol on the oxidation of Ropufa oil The effect of α-tocopherol on the oxidation of Ropufa oil is shown in Figure 6.5. It can be seen that PV increased with the addition of α-tocopherol, contrary to the expectation that it will act as an antioxidant and lower the PV of the tested oil. The acceleration of oxidation of Ropufa oil might be due to the production of α-tocopherol radicals generated at the higher temperature (60oC). The radical may act as a prooxidant, when it is not inactivated211. Moreover, α-tocopherol has been reported to show prooxidative effects at
163
high concentrations212, 213. The high level of α-tocopherol might be another reason for the more rapid oxidation of the Ropufa oil.
400
PV / mEq peroxides/kg oil
350 300 250 200 150 100 50 0 -50 0
2
4
6
8
Day Figure 6.5 Peroxide value (PV) of 3.0 g of Ropufa oil with (•) nil and (▲) 4.5 mg of αtocopherol added (n = 3).
6.3.4 The influence of PANI EC film on oxidation of avocado oil The PV of avocado oil stored at 60oC for up to 8 days was evaluated and the results are presented in Figure 6.6. As is shown in this figure, the PV was very low after incubation for 8 days. While the PV was lower in the avocado oils in the presence of the PANI EC film, the difference was not statistically significant (p > 0.05). The results might be due to the very slow oxidation of the avocado oil. Rich in fatty acids, especially oleic acid, a mono unsaturated fatty acid214, avocado oil seems to be more stable than Ropufa oil (fish
164
oil). It has been reported that it is easier to abstract a hydrogen atom from unsaturated fatty acid molecules, especially di- and tri-unsaturated acids, which are then changed into free radical, than for saturated fatty acids4,
215
. Lack of EDA and PHA might be the
reason why avocado oil is more stable to high temperatures than Ropufa oil.
PV / mEq peroxides/Kg oil
14 12 10 8 6 4 2 0
0
2
4
6
8
Day
Figure 6.6 Peroxide value (PV) of avocado oil at 60oC in presence of (▲) nil and (•) PANI EC film (9 %) (n = 3).
6.3.5 Fatty acid composition of Ropufa oil Ropufa oil was incubated at 60oC for various times in the absence and in the presence of PANI EC film (9 %), to evaluate the influence of the conducting polymer film on the
165
fatty acid content of fish oil after accelerated oxidation. The composition and fatty acid content of the Ropufa oil before and after accelerated oxidation are listed in Table 6.1. The concentration of each fatty acid was calculated by applying the relative response factor (RRF) to internal standard, as follows216: RRFi =
Cf =
( Ai / C i ) ( As / C s )
Af RRFi
×
(eq. 6.8)
Cs As
(eq. 6.9)
where Ai is the area of the identified fatty acid peak using known standards, Ci is the concentration of the identified fatty acid standard, As is the area of the internal standard,
Cs is the concentration of the internal standard, Af is the area of identified fatty acid peak in the tested sample, and Cf is the concentration of the identified fatty acid in the tested sample.
As seen in Table 6.1, the fish oil contained a considerable amount of fatty acids with high levels of palmate acid (C16:0), elaidic acid (C18:1n9t) and two long chain ω-3 fatty acids, cis-11,14,17-eicosapentaenoic acid
(C20:5n3) (EPA) and cis-4,7,10,13,16,19-
docosahexaenoic acid (C22:6n3) (DHA), which are believed to be beneficial for human health217. After incubation at 60oC, some decline in the content of EPA and DHA was observed by 6-8 % after 8 days, with smaller changes in the remaining fatty acids. However, no significant difference in the fatty acid content of the oils was seen in the presence or absence of the PANI EC film. Thus the small lowering of the rate of increase of peroxide value noted above in the presence of the PANI EC film did not carry through to a noticeable difference in the concentration of the polyunsaturated fatty acid content.
166
Table 6.1 Fatty acid composition in wt % of Ropufa oil determined by GC (n = 2). 5 days at 60 oC
8 days at 60 oC
Fatty acid oil at RT
oil
oil + film
oil
oil + film
8 Myristic acid (C14:0)
6.0 ± 0.3
6.1 ± 0.2
6.1 ± 0.1
5.9 ± 0.2
5.75 ± 0.01
12 Palmitic acid (C16:0)
16.3 ± 0.9
16.4 ± 0.4
16.4 ± 0.3
16.2 ± 0.5
15.8 ± 0.1
13 Palmitoleic acid (C16:1)
6.0 ± 0.3
6.1 ± 0.1
6.1 ± 0.1
6.0 ± 0.2
5.86 ± 0.03
16 Stearic acid (C18:0)
3.4 ± 0.2
3.5 ± 0.1
3.5 ± 0.1
3.5 ± 0.1
3.39 ± 0.02
17 Elaidic acid (C18:1n9t)
10.5 ± 0.6
10.6 ± 0.2
10.6 ± 0.2
10.6 ± 0.3
10.27 ± 0.05
18 Oleic acid (C18:1n9c)
2.3 ± 0.1
2.33 ± 0.04
2.34 ± 0.04
2.3 ± 0.1
2.27 ± 0.01
20 Linoleic acid (C18:2n6c)
1.9 ± 0.1
1.85 ± 0.03
1.87 ± 0.03
1.9 ± 0.1
1.80 ± 0.01
22 Linolenic acid (C18:3n3c)
1.3 ± 0.1
1.30 ± 0.01
1.31 ± 0.02
1.29 ± 0.04
1.25 ± 0.01
12.5 ± 0.8
12.0 ± 0.2
12.1 ± 0.2
11.8 ± 0.4
11.6 ± 0.1
18.0 ± 1.1
17.0 ± 0.3
17.2 ±0.3
16.7 ± 0.6
16.4 ± 0.2
30 Cis-11,14,17Eicosapentaenoic acid (C20:5n3) (EPA) 35 Cis-4,7,10,13,16,19Docosahexaenoic acid (C22:6n3) (DHA)
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6.4 Conclusions
Ropufa oil is rich source of fatty acids, especially EPA and DHA, which are well known as long chain omega-3 polyunsaturated fatty acids. After incubation at 60oC for 5 and 8 days, no significant difference in the fatty acid content between the oil with and without added PANI EC film was observed. However, it is obvious that the PV of the Ropufa oil in the presence of the PANI EC film is lower than in absence of the film, demonstrating an inhibitory effect of the conducting polymer film on oxidation of fish oil. The PV of Ropufa oil incubated at 40oC was very low, and no significant difference in PV was observed between the oil tested with and without an added PANI EC film. Compared to Ropufa oil, avocado oil was more stable to high temperature (60oC) accelerated oxidation, which might be due to its richness in monounsaturated fatty acid but lack of di- and triunsaturated acids. α-tocopherol, a commonly used antioxidant, was surprisingly found to accelerate the oxidation of fish oil at 60oC. It is possible that α-tocopherol acts as both antioxidant and prooxidant under these conditions.
168
Chapter 7 Conclusions and future work
7.1 General conclusions
This study has presented the development of antioxidant conducting polymers (CPs) for packaging applications, with a focus on the free radical scavenging effects of the materials. Polpyrrole (PPy), polyaniline (PANI) and poly(3,4-ethylenedioxythiophene) (PEDOT) powders were prepared by chemical polymerization, using various concentrations of ammonium persulfate (APS), which is a strong oxidant. It was found that PPy, PANI and PEDT powders are effective DPPH free radical scavengers, particularly when prepared using a lower APS/monomer ratio, as a high level of APS led to overoxidation of the CPs, as detected using IR and Raman spectroscopy. In the case of PPy, a clear C=O absorption in the IR spectra and a blue shift of the C=C peak in the Raman spectra were observed when PPy was prepared at a high level of APS. Furthermore, the conductivity and doping level of the CP powders formed using higher concentrations of the APS oxidant were lower than those prepared using low levels of APS, again likely also due to overoxidation, damaging the CP structures during the preparation processes.
In this study, two methods for evaluating antioxidant properties of solid samples, modified forms of the DPPH and ABTS assays, have been developed. Initially, the antioxidant effects of PPy, PANI and PEDOT powders prepared using various ratios of APS to Py, ANI and EDOT were evaluated using the DPPH assay. The results showed
169
that the optimum initial APS/pyrrole ratio for the synthesis of PPy powders, as superior DPPH radical scavengers, was around 0.5, while the optimum initial APS/ANI ratio for PANI was 1.5 and with APS/ EDOT the optimum ratio for PEDOT was 1.0. In each case weaker DPPH scavenging abilities were found when higher levels of APS were employed to produce the CP powders, which can be attributed to overoxidation. PPy and PANI were found to show a much greater free radical scavenging capacity than PEDOT. The free radical scavenging activities of reduced PPy and PANI were also tested using the DPPH assay, and the results showed that the reduced forms of PPy and PANI exhibited better DPPH radical scavenging abilities than the as-prepared powders, indicating that the reduced CPs are greater electron and/or hydrogen donors compared to the as-prepared CPs.
+
The ABTS• radical scavenging activities of PPy 0.5, PANI 1.5 and PEDOT 1.0 (the optimum APS to monomer forms as determined from the DPPH assay) were examined using a modified ABTS assay. Similar to the results obtained from the DPPH assay, PANI 1.5 was the best ABTS radical scavenger among the three tested CP powders, followed by PPy 0.5, while PEDOT 1.0 presented a much weaker scavenging effect. The results also showed that the reduced forms of all the three conducting polymers were more effective ABTS free radical scavengers than the as-prepared samples, which confirms the findings of the DPPH assay. In the case of PANI, powders formed from an initial high pH preparative solution (i.e. no added strong acid), which contained PANI nanotubes, exhibited stronger free radical scavenging activity, compared with the regular granular PANI formed under more acidic conditions, evidenced by the results obtained
170
from both the DPPH and ABTS assays. It is clear that the higher surface area nanostructured forms (even though these had a lower electronic conductivity) enhance the efficiency of CPs, when they act as free radical scavengers.
Using spectroscopic methods, it was found that reaction of the CPs with the free radicals +
DPPH• and ABTS• led to structural changes in the CPs. In the case of PANI, using corelevel N1s XPS spectra, the [C=N] / [C-N] ratio increased after reaction with DPPH• and +
ABTS• . An increase in imine (C=N) units was also observed in the IR spectra after PANI reacted with DPPH•. The oxidation of PANI indicates that the conducting polymer acted as an antioxidant, with reducing power, when it reacted with the free radicals.
Both the DPPH and ABTS assays are commonly used for the evaluation of antioxidant capacity in Food Science, because they are simple, rapid, sensitive and reproducible. DPPH• is a commercially available stable free radical and is easy to use, with no requirement of pre-treatment, but is only suitable for hydrophobic systems. Compared to DPPH•, the advantage of ABTS•
+
is that it is suitable for both hydrophilic and +
hydrophobic systems. However, the ABTS• radical cations need to be generated prior to utilization and are normally stable for just two days. Although there are a few limitations in the application of theses two methods, the testing results still indicate the suitability of the modified DPPH and ABTS assays for evaluating the antioxidant capacity of CP powders.
171
A PANI-ethyl cellulose (EC) film was successfully prepared using a solution-based method, where PANI particles and clusters were homogenously dispersed in an EC matrix. The resulting PANI EC films showed good mechanical properties. The process for the preparation of the PANI EC film is simple and low-cost, and is also suitable for the preparation of various CP EC films using different CP powders and CP containing materials, when the appropriate solvents are applied.
A modified ORAC antioxidant assay was also developed, which is suitable for evaluating the antioxidant capacity of CP containing films; films soluble in methanol cannot be evaluated using the DPPH assay. The antioxidant capacities of the tested samples can be quantitatively determined using their net AUC area and using fluorescein as the fluorescent probe. The ORAC assay has been widely used to assess the free radical antioxidant activity of pure compounds, fruits and vegetable extracts, wine and biological fluids19, 41, 218. To our knowledge, this is the first study of the ORAC kinetic behavior for conducting polymer composites. The methodology can also be used for other polymer films.
The results show that the PANI EC films present very efficient peroxyl free radical scavenging activity even using an area as small as 16 mm2 and the activity increased when more film area was exposed to the test solution. The results also showed that a PANI EC film with a higher proportion of PANI presented a higher efficiency for free radical scavenging, and that the PANI EC films exhibited much more efficient radical scavenging when prepared with reduced PANI, or alternatively after reduction of the as-
172
prepared PANI EC film with hydrazine, confirming that a higher amine content in the PANI portion is more effective for free radical scavenging. The ORAC method is particularly important when CP composites are prepared for bioapplications, because the peroxyl free radical is one of the dominant radicals in biological systems and in oxidized lipids. The marked peroxyl radical scavenging abilities of PANI composites showed their suitability for bioapplications, including food packaging.
Structural changes of PANI powders, after reaction with peroxyl radicals, were detected using XPS. The results indicate that benzenoid amine (C-N) converted to quinonoid imine (C=N) after reaction with peroxyl free radicals, which is in agreement with the findings obtained with the DPPH and ABTS assays. From the results indicated above, XPS is a very useful technique for tracking structural changes of CPs after reaction with free radicals.
+
PPy was found to be a strong free radical scavenger for both DPPH• and ABTS• , but only displayed a weak scavenging effect with respect to peroxyl radicals. The fact that small molecule antioxidants shows different scavenging activities to different free radicals has been reported by Tabart et al. (2009)219, where antioxidants such as kaempferol-O-glucoside (a flavonol), hesperidin and naringenin (flavonons), exhibited little to no antioxidant activity in the ABTS and DPPH assays, but strong activity in the ORAC assay. It is possible that the free radical scavenging activity of PPy is mainly based on electron donation, not on direct transfer of hydrogen atoms to the substrate.
173
A PANI film prepared by chemically polymerizing aniline within an EC matrix prior to casting the mixture on a Teflon sheet (PANI(OX)EC), and a PANI-polyethylene (PE) film prepared using a melt processing method at 190 oC, also showed excellent peroxyl free radical scavenging ability. However, the poor texture of the PANI(OX)EC film and the complicated process used to prepare the PANI PE film needs to be improved in future work, but the PANI PE system is of particular interest given the widespread use of polyethylene in packaging industries.
The oxidation of unsaturated lipids is generally caused by free radical chain reactions. Therefore, fish oil, which is rich in unsaturated fatty acids, was used to evaluate chain reaction terminating effects of the PANI EC film. The oxidation of lipids with and without added PANI EC film was determined by measurement of the peroxide value (PV). After incubation at 60oC for various days, it was found that the presence of the PANI EC film retarded the oxidation of Ropufa oil to a certain extent, as seen in the lowering of the PV. The results demonstrate an inhibitory effect of the conducting polymer film on the oxidation of fish oils.
Although numerous studies have focused on the fabrication of CP composites and their applications, little research has been undertaken on the antioxidant effect of CP composites.
The PANI EC film materials are expected to be used as free radical
scavengers not only for food products, but also in pharmaceutical and cosmetic packaging, to protect the product from the undesirable influence of free radicals.
174
7.2 Future research
PANI has been demonstrated to present free radical scavenging effects with DPPH, ABTS and peroxyl free radicals. It will be interesting to investigate the free radical quenching effects of derivatives of PANI, such as poly(o-anisidine), which is a methoxysubstituted polyaniline and has been reported to show excellent antioxidant properties for protecting rubber against oxidation102,
103
. PANI composites containing reduced CP
powders show greater peroxyl free radical scavenging activity than those containing asprepared powders. Their influence on the oxidation of foodstuffs needs to be investigated further.
Both of the PANI(OX)EC and PANI PE films showed significant peroxyl radical quenching activity, but the poor texture of the PANI(OX)EC film and the complicated process used to prepare the PANI PE film, as described in section 2.7, may limit their application. The addition of surfactants to improve the texture of PANI(OX)EC films and preparing PANI PE films with dedoped PANI for simpler processing can be examined.
Since PANI EC films have shown inhibitory effects with regard to the oxidation of fish oils, the influence of CP containing films on the oxidation other foods and beverages, along with pharmaceutical and cosmetic products susceptible to free radical damage, can be investigated. In addition, the safety and reliability of CP films for use as packaging materials in the above fields also needs to be investigated, particularly to develop forms in which the leaching of any polymeric material into the food product is excluded, since one of the main concerns with new packaging developments is in the migration of
175
packaging materials into the consumable products. Therefore, leaking of CP from the packaging materials to the packaged stuffs has to be monitored and the examination of CP in packaged products needs to be carried out. It will be important to ensure that in forming and processing the conducting polymers, that all small molecule oligomers and side reaction products are effectively removed prior to blending with the food grade plastic, to ensure that only longer polymer chains remain that will not leach into the packaged product. Liquid chromatography-mass spectroscopy is suitable for the examination, because the technique combines the physical separation of liquid chromatography and the mass analysis of mass spectrometry, which allows the undesirable components, such as the small oligomers of the CPs, to be separated and identified.
176
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