The Development of Smart Sensors for Aquatic Water Quality

The Development of Smart Sensors for

Aquatic Water Quality Monitoring

A thesis submitted to the University of Manchester for the degree of

Doctor of Philosophy in the Faculty of Engineering and Physical

Sciences

2014

CRAIG ALEXANDER

SCHOOL OF CHEMICAL ENGINEERING AND ANALYTICAL

SCIENCE

Craig Alexander

2

Contents

List of Figures ..........................................................................................................................................9

List of Tables..........................................................................................................................................16

Abstract ..................................................................................................................................................18

Declaration .............................................................................................................................................19

Copyright Statement ...............................................................................................................................19

Acknowledgements ................................................................................................................................20

List of Abbreviations ..............................................................................................................................21

Project Overview .................................................................................................................................. 24

1 Introduction ................................................................................................................................. 25

1.1 Aquarium Chemistry ................................................................................................................... 25

1.1.1 Analytes from the Nitrogen Cycle ................................................................................................ 26

1.1.1.1. Ammonia .................................................................................................................... 26

1.1.1.2. Nitrite .......................................................................................................................... 28

1.1.1.3. Nitrate ......................................................................................................................... 28

1.1.2 Aquarium pH ............................................................................................................................... 30

1.1.3 Chlorine .................................................................................................................................... 30

1.1.4 Dissolved Oxygen ....................................................................................................................... 31

1.1.5 Phosphate .................................................................................................................................... 32

1.1.6 Water Hardness ........................................................................................................................... 32

1.1.6.1. General Hardness ........................................................................................................ 32

1.1.6.2. Carbonate Hardness ..................................................................................................... 33

1.1.7 Summary of Parameters ............................................................................................................... 34

1.2 Current Methods of Analysis ....................................................................................................... 35

1.2.1 Test Kits .................................................................................................................................... 35

1.2.1.1. pH ............................................................................................................................... 36

1.2.1.2. Ammonia .................................................................................................................... 37

1.2.1.2.1. Nessler’s Reagent Method ........................................................................................... 37

1.2.1.2.2. Salicylate Method ........................................................................................................ 37

1.2.1.3. Nitrite .......................................................................................................................... 38

Craig Alexander

3

1.2.1.4. Nitrate ......................................................................................................................... 39

1.2.2 Commercial Sensors .................................................................................................................... 40

1.2.3 Benefits of the Proposed Device .................................................................................................. 40

1.3 Introduction to Chemical Sensors ................................................................................................ 42

1.3.1 Electrochemical Sensors .............................................................................................................. 43

1.3.1.1. Potentiometry .............................................................................................................. 43

1.3.1.2. Reference Electrodes ................................................................................................... 45

1.4 Ion-Selective Electrodes .............................................................................................................. 48

1.4.1 Selective Polymeric Membranes .................................................................................................. 48

1.4.2 Selectivity of ISEs ....................................................................................................................... 50

1.4.3 Polymer Membrane Electrodes .................................................................................................... 55

1.4.4 Miniaturisation of ISEs ................................................................................................................ 56

1.4.4.1. Coated-Wire Electrodes ............................................................................................... 56

1.4.4.2. Screen-Printed ISEs ..................................................................................................... 57

1.4.5 Sol-gels .................................................................................................................................... 59

1.4.5.1. Sol-gel Chemistry ........................................................................................................ 59

1.4.5.2. Sol-gel Ion-Selective Membranes ................................................................................ 61

1.4.6 ISEs for Aquarium-Significant Analytes ...................................................................................... 63

1.4.6.1. pH ............................................................................................................................... 63

1.4.6.2. Ammonia/Ammonium ................................................................................................. 65

1.4.6.3. Nitrite .......................................................................................................................... 67

1.4.6.4. Nitrate ......................................................................................................................... 69

1.5 Impedimetric Interdigitated Electrode Chemical Sensors ............................................................. 72

1.5.1 Electrical Impedance ................................................................................................................... 72

1.5.2 Interdigitated Electrodes .............................................................................................................. 74

1.5.3 IDEs for Chemical Sensing Applications ..................................................................................... 75

1.5.3.1. Ion-Selective Conductometric Microsensors ................................................................ 75

1.5.4 IDE Fabrication Techniques ........................................................................................................ 76

1.5.4.1. Photolithography ......................................................................................................... 77

1.5.4.2. Screen-Printing............................................................................................................ 79

Craig Alexander

4

1.5.5 Membrane Coating Techniques.................................................................................................... 80

1.5.5.1. Spin-Coating ............................................................................................................... 80

1.5.5.2. Dip-Coating ................................................................................................................ 81

1.5.5.3. Drop-Coating .............................................................................................................. 81

1.5.5.4. Screen-Printing............................................................................................................ 81

1.6 Aims and Objectives .................................................................................................................... 82

2 Materials and Methods................................................................................................................. 83

2.1 Preparation of Ion-Selective Membranes...................................................................................... 83

2.1.1 Materials .................................................................................................................................... 83

2.1.2 Polyvinyl Chloride Membranes.................................................................................................... 84

2.1.3 Sol-Gel Membranes ..................................................................................................................... 85

2.1.3.1. MTES ......................................................................................................................... 85

2.1.3.2. DEDMS/TEOS............................................................................................................ 85

2.1.4 Dielectric Screen-Printing Paste Membrane ................................................................................. 86

2.2 Construction of Ion-Selective Electrodes ..................................................................................... 87

2.2.1 Materials .................................................................................................................................... 87

2.2.2 Polymer Membrane Electrodes .................................................................................................... 87

2.2.3 Coated-Wire Electrodes ............................................................................................................... 88

2.3 Potentiometric Measurements ...................................................................................................... 89

2.3.1 Measurement Instrumentation ...................................................................................................... 89

2.3.2 Sensor Testing ............................................................................................................................. 89

2.3.2.1. Materials ..................................................................................................................... 89

2.3.2.2. Nitrate ISEs ................................................................................................................. 90

2.3.2.2.1. Stock Solutions ........................................................................................................... 90

2.3.2.2.2. Calibrations ................................................................................................................. 90

2.3.2.2.3. Selectivity Determination ............................................................................................ 91

2.3.2.3. Ammonium ISEs ......................................................................................................... 92

2.3.2.3.1. Stock Solutions ........................................................................................................... 92

2.3.2.3.2. Calibrations ................................................................................................................. 92


Craig Alexander

5

2.3.2.4. pH ISEs ....................................................................................................................... 94

2.3.2.4.1. Stock Solutions ........................................................................................................... 94

2.3.2.4.2. Calibrations ................................................................................................................. 94


2.3.2.5. Nitrite ISEs ................................................................................................................. 95

2.4 Fabrication of Interdigitated Electrodes ....................................................................................... 96

2.4.1 Lift-off Photolithography ............................................................................................................. 96

2.4.1.1. Materials ..................................................................................................................... 96

2.4.1.2. Sensor Design ............................................................................................................. 97

2.4.1.3. Fabrication Process ..................................................................................................... 98

2.4.1.4. Reduced Geometry IDEs ........................................................................................... 100

2.4.2 Screen-Printing .......................................................................................................................... 101

2.4.2.1. Materials ................................................................................................................... 101

2.4.2.2. Sensor Design ........................................................................................................... 102

2.4.2.3. Fabrication Process ................................................................................................... 104

2.4.2.3.1. Attempt One .............................................................................................................. 104

2.4.2.3.2. SP IDE Design 1 ....................................................................................................... 104

2.5 Construction of Ion-Selective Impedimetric Microsensors ......................................................... 106

2.5.1 Photolithographically-prepared IDE Sensors .............................................................................. 106

2.5.1.1. IDE Design 1............................................................................................................. 106

2.5.1.2. IDE Design 2............................................................................................................. 107

2.5.2 Screen-Printed IDEs .................................................................................................................. 107

2.5.2.1. SP IDE Design 1 ....................................................................................................... 107

2.6 Impedance Measurements .......................................................................................................... 108

2.6.1 Measurement Instrumentation .................................................................................................... 108

2.6.2 Sensor Testing ........................................................................................................................... 109

2.7 Ion-Selective Impedimetric Microsensors Methodology ............................................................ 110

2.7.1 Materials .................................................................................................................................. 110

2.7.2 Nitrate Sensors .......................................................................................................................... 110

2.7.2.1. Stock Solutions ......................................................................................................... 110

Craig Alexander

6

2.7.2.2. Pure Water Calibrations............................................................................................. 111

2.7.2.3. Background Effects ................................................................................................... 112

2.7.2.4. Selectivity ................................................................................................................. 112

2.7.2.5. Reproducibility.......................................................................................................... 114

Ammonium Sensors............................................................................................................................ 114

2.7.2.6. Stock Solutions ......................................................................................................... 114

2.7.2.7. Measurements in Water ............................................................................................. 114

2.7.2.8. Selectivity ................................................................................................................. 114

2.7.3 pH Sensors ................................................................................................................................ 115

2.7.3.1. Stock Solutions ......................................................................................................... 115

2.7.3.2. Calibrations ............................................................................................................... 116

2.7.4 Nitrite Sensors ........................................................................................................................... 117

2.7.4.1. Stock Solutions ......................................................................................................... 117

2.7.4.2. Measurements in Water ............................................................................................. 117

2.7.4.3. Selectivity ................................................................................................................. 117

3 Potentiometric Characterisation of Ionophores ........................................................................... 119

3.1 Nitrate-Selective Electrodes ....................................................................................................... 119

3.1.1 Nitrate PMEs ............................................................................................................................. 119

3.1.2 Nitrate CWEs ............................................................................................................................ 124

3.2 Ammonium-Selective Electrodes ............................................................................................... 129

3.3 Hydrogen Ion-Selective Electrodes ............................................................................................ 133

3.4 Conclusions for Chapter 3.......................................................................................................... 136

4 Nitrate Sensing using IDE Design 1 .......................................................................................... 140

4.1 Sensor Calibration ..................................................................................................................... 140

4.2 PVC Membrane Sensors ............................................................................................................ 147

4.2.1 Calibrations ............................................................................................................................... 147

4.2.2 Selectivity.................................................................................................................................. 155

4.3 Sol-gel Membrane Sensors ........................................................................................................ 157

4.3.1.1. DEDMS/TEOS Membrane ........................................................................................ 157

4.3.1.1.1. Calibrations ............................................................................................................... 157

Craig Alexander

7

4.3.1.1.2. Selectivity ................................................................................................................. 167


5 Nitrate Sensing using IDE Design 2 ........................................................................................... 174

5.1 Sensor Calibration ..................................................................................................................... 174

5.2 PVC Membrane Sensors ............................................................................................................ 177

5.2.1.1. Spin-coated Membranes ............................................................................................ 177

5.2.1.1.1. Calibrations ............................................................................................................... 177

5.2.1.1.2. Selectivity ................................................................................................................. 182

5.2.1.2. Drop-coated Membranes ........................................................................................... 183

5.2.1.2.1. Calibrations ............................................................................................................... 184

5.2.1.2.2. Selectivity ................................................................................................................. 202


6 Nitrate Sensing using SP IDE Design 1...................................................................................... 211

6.1 Issues Experienced with Initial Sensor Designs .......................................................................... 211

6.2 SP IDE Design 1 Calibration ..................................................................................................... 212

6.3 SP IDE Design 1 Selectivity ...................................................................................................... 224


7 pH, Ammonium and Nitrite Sensing using IDE Design 2 ........................................................... 230

7.1 Ammonium Sensors .................................................................................................................. 230

7.2 Nitrite Sensors ........................................................................................................................... 234

7.3 pH Sensors ................................................................................................................................ 238


8 Conclusions and Suggestions for Further Work ......................................................................... 246

8.1 Conclusions ............................................................................................................................... 246

8.2 Future Work .............................................................................................................................. 250

8.2.1 Individual Sensor Testing .......................................................................................................... 250

8.2.2 Membrane Characterisation ....................................................................................................... 252

8.2.3 Membrane Materials .................................................................................................................. 252

8.2.4 IDE Geometry ........................................................................................................................... 253

8.2.5 Screen-Printed Sensors .............................................................................................................. 254

Craig Alexander

8

8.2.6 Considerations for a Prototype Device ....................................................................................... 254

8.2.7 Sensor Longevity ....................................................................................................................... 255

8.2.8 Data Display .............................................................................................................................. 256

9 References ................................................................................................................................. 257

10 Appendices ................................................................................................................................ 265

10.1 Molar Equivalent Concentrations of Ions ................................................................................... 265

10.2 Details of Commercial Ionophores Used .................................................................................... 266

10.3 Potentiometric Data Acquisition System .................................................................................... 267

Final word count: 56,647

Craig Alexander

9

List of Figures

1.1 Algae accumulation caused by high levels of nitrate in an aquarium ........................................... 29

1.2 Structure of the two forms of phenol red which result in the observed colour change ................. .36

1.3 Structure of three forms of bromothymol blue which result in a colour change at varying pH

values…... ................................................................................................................................... 36

1.4 Structure of indosalicylate. .......................................................................................................... 38

1.5 Schematic representation of a double-junction Ag/AgCl reference electrode with

1 M LiCH3COO as outer filling solution. ..................................................................................... 47

1.6 (A) Potassium tetrakis-[3,5,-bis-(trifluoromethyl)phenyl]borate – used as an ionic additive in

cation-selective membranes; (B) tridodecylmethyl ammonium chloride (TDMACl)– used as an

ionic additive in anion-selective membranes ................................................................................ 49

1.7 Structures of three commonly-used plasticisers within ISEs (A) dioctyl sebacate (DOS),

(B) o-nitrophenyloctyl ether (NPOE), (C) dioctyl phthalate (DOP).. ............................................ 50

1.8 Example calibration curve for calculating the selectivity coefficent using the FIM ....................... 52

1.9 Schematic diagram of a typical PME set-up, showing the cell potential of an ISE being

measured against a SCE reference ............................................................................................... 55

1.10 Structures of three typical silicon alkoxide sol-gel precursors – (A) methyltriethoxysilane (MTES);

(B) tetraethoxysilane (TEOS); (C) diethoxydimethylsilane (DEDMS) ......................................... 60

1.11 Selection of the Hofmeister series showing the order of anion hydrophobicity ............................. 67

1.12 A typical AC sine wave voltage and current. ................................................................................ 72

1.13 Schematic diagram of a typical IDE design. ................................................................................. 74

1.14 Cross-section schematic representation of the electric field penetration depth from a

typical IDE when an AC voltage is applied. ................................................................................. 75

1.15 Schematic representation of an ISCOM ....................................................................................... 76

1.16 Photolithographic preparation of thin-film electrodes using (a) chemical etching and

(b) lift-off procedures .................................................................................................................. 78

1.17 Schematic diagram of screen-printing for the fabrication of thick-film electrochemical sensors. .. 79

2.1 Schematic representation of IDE Design 1 ................................................................................... 98

2.2 Microscope image (4x) of the digits of IDE Design 1, fabricated in-house using a mask-less,

lift-off photolithographic technique followed by e-beam deposition of gold metal ..................... 100

2.3 IDE Design 2 ............................................................................................................................. 101

2.4 Schematic representation of the screens used to produce 6 different IDE designs. ...................... 103

2.5 Schematic representation of SP IDE Design 1 ............................................................................ 104

2.6 Outline of the screen-printing fabrication process of SP IDE Design 1 ....................................... 105

2.7 Microscope image (4x) of the electrode digits of SP IDE Design 1 ............................................ 106

Craig Alexander

10

2.8 Front panel of the LabVIEW program used to control the Agilent E4980A LCR meter .............. 108

2.9 Schematic diagram of the experimental set-up for testing IDE ion sensors using the Agilent

E4980A LCR meter. .................................................................................................................. 110

3.1 Calibration graph of two nitrate-selective PMEs prepared using a PVC membrane containing

1% w/w TDAN as the ionophore. .............................................................................................. 119

3.2 Calibration graph of two nitrate-selective PMEs prepared using a PVC membrane containing

1% w/w NO3V as the ionophore ................................................................................................ 121

3.3 Selectivity determination of TDAN PME 1 using the FIM in 1000 ppm Cl- and

1000 ppm NO2-. ........................................................................................................................ .122

3.4 Selectivity determination of NO3V PME 1 using the FIM in 1000 ppm Cl- and

1000 ppm NO2-. ......................................................................................................................... 123

3.5 Calibration graph of two nitrate-selective CWEs prepared by dip-coating the electrode into a

PVC membrane ‘cocktail’ containing 1% w/w TDAN as the ionophore ..................................... 124

3.6 Calibration graph of four nitrate-selective sol-gel CWEs (two MTES and two DEDMS)

containing TDAN as the ionophore ............................................................................................ 125

3.7 Selectivity determination of TDAN PVC CWE 1 using the FIM in 1000 ppm Cl- and

1000 ppm NO2- .......................................................................................................................... 126

3.8 Selectivity determination of TDAN MTES CWE 1 using the FIM in 1000 ppm Cl- and

1000 ppm NO2- .......................................................................................................................... 127

3.9 Selectivity determination of TDAN DEDMS CWE 1 using the FIM in 1000 ppm Cl- and

1000 ppm NO2-. ......................................................................................................................... 128

3.10 Calibration graph of two ammonium-selective PMEs prepared using a PVC membrane

containing 1% w/w NH4I as the ionophore ................................................................................ 129

3.11 Selectivity determination of NH4I PME 1 using the FIM in 1000 ppm of the interfering cations

sodium, potassium, magnesium and calcium .............................................................................. 130

3.12 Calibration graph of two ammonium-selective PMEs prepared using a PVC membrane

containing 1% w/w NH4I as the ionophore, with the ionic additive omitted ............................... 131

3.13 Selectivity determination of NH4I PME 1 (ionic additive omitted) using the FIM in 1000 ppm

of the interfering cations sodium, potassium, magnesium and calcium.. ..................................... 132

3.14 Calibration graph of two hydrogen ion-selective PMEs prepared using a PVC membrane

containing 1% w/w HIII as the ionophore.. ................................................................................ 133

3.15 Calibration graph of two hydrogen ion-selective PMEs prepared using a PVC membrane

containing 1% w/w HV as the ionophore. .................................................................................. 134

3.16 Selectivity determination of HIII PME 1 using the FIM in 0.1 M of the interfering cations

sodium, potassium and magnesium.. .......................................................................................... 135

3.17 Selectivity determination of HV PME 1 using the FIM in 0.1 M of the interfering cations

sodium, potassium and magnesium.. .......................................................................................... 136

Craig Alexander

11

4.1 Conductance (A) and capacitance (B) spectra for the addition of NO3- to an uncoated

IDE Design 1 (1 Vrms input amplitude) ....................................................................................... 140

4.2 Conductance response of uncoated IDE Design 1 upon addition of 1–100 ppm nitrate at

six frequencies.. ......................................................................................................................... 141

4.3 Capacitance response of uncoated IDE Design 1 upon addition of 1–100 ppm nitrate at

six frequencies.. ......................................................................................................................... 142

4.4 Deionised water baseline-subtracted conductance (Gm – G0) against log NO3- concentration for

uncoated IDE Design 1 at six frequencies.. ................................................................................ 143

4.5 Deionised water baseline-subtracted capacitance (Cm – C0) against log NO3- concentration for

uncoated IDE Design 1 at six frequencies.. ................................................................................ 144

4.6 Logarithm of the change in conductance (Gm – G0) against the logarithm of nitrate concentration

for uncoated IDE Design 1 at six frequencies.. ........................................................................... 145

4.7 Logarithm of the change in capacitance (Cm – C0) against the logarithm of nitrate concentration

for uncoated IDE Design 1 at three frequencies ......................................................................... 146

4.8 Conductance (A) and capacitance (B) spectra for the addition of NO3- to TDAN PVC spin-coated

IDE Design 1 (1 Vrms input amplitude).. ..................................................................................... 147

4.9 Conductance response of TDAN PVC spin-coated IDE Design 1 upon addition of

1–100 ppm nitrate at six frequencies.. ........................................................................................ 148

4.10 Capacitance response of TDAN PVC spin-coated IDE Design 1 upon addition of


4.11 Deionised water baseline-subtracted conductance (Gm – G0) against log NO3- concentration

for TDAN PVC spin-coated IDE Design 1 at 2 MHz. ................................................................ 150

4.12 Logarithm of the change in conductance (Gm – G0) against the logarithm of nitrate

concentration for TDAN PVC spin-coated IDE Design 1 at 2 MHz. .......................................... 150

4.13 Deionised water baseline-subtracted conductance (Gm – G0) against log NO3-

concentration for TDAN PVC spin-coated IDE Design 1 at 2 MHz.. ......................................... 151

4.14 Measured baseline conductance value (G0) of TDAN PVC spin-coated IDE Design 1 in six

separate sample matrices at 2 MHz, prior to the addition of nitrate............................................. 153

4.15 Measured conductance response (Gm) of TDAN PVC spin-coated IDE Design 1 against

log NO3- concentration in six separate sample matrices at 2 MHz .............................................. 154

4.16 Baseline-subtracted conductance (Gm – G0) of TDAN PVC spin-coated IDE Design 1 against

log NO3- concentration at 2 MHz, in six separate sample matrices. ............................................ 154

4.17 Deionised water baseline-subtracted conductance (Gm – G0) of three TDAN PVC spin-coated IDE

Design 1 devices against log NO3- concentration at 2 MHz, for reproducibility determination.... 155

4.18 Selectivity determination of TDAN PVC spin-coated IDE Design 1.. ......................................... 156

4.19 Conductance (A) and capacitance (B) spectra for the addition of NO3- to TDAN DEDMS

spin-coated IDE Design 1 (1 Vrms input amplitude). ................................................................... 158

4.20 Conductance response of TDAN DEDMS spin-coated IDE Design 1 upon addition of

1–100 ppm nitrate at six frequencies. ......................................................................................... 159

Craig Alexander

12

4.21 Capacitance response of TDAN DEDMS spin-coated IDE Design 1 upon addition of



for TDAN DEDMS spin-coated IDE Design 1.. ......................................................................... 161


for TDAN DEDMS spin-coated IDE Design 1 ........................................................................... 161


TDAN DEDMS spin-coated IDE Design 1 ................................................................................ 162


TDAN DEDMS spin-coated IDE Design 1 ................................................................................ 163

4.26 Measured baseline conductance value (G0) of TDAN DEDMS spin-coated IDE Design 1 in six

separate sample matrices prior to the addition of nitrate. ............................................................ 165

4.27 Measured baseline capacitance value (C0) of TDAN DEDMS spin-coated IDE Design 1 in six

separate sample matrices prior to the addition of nitrate. ............................................................ 166

4.28 Selectivity determination of TDAN DEDMS spin-coated IDE Design 1 (conductive response).. 168

4.29 Selectivity determination of TDAN DEDMS spin-coated IDE Design 1 (capacitive response).. . 169

5.1 Conductance (A) and capacitance (B) spectra for the addition of NO3- to an uncoated

IDE Design 2 (1 Vrms input amplitude) ....................................................................................... 174


uncoated IDE Design 2 at six frequencies and varying input amplitudes. ................................... 175


uncoated IDE Design 2 at six frequencies and varying input amplitudes .................................... 176

5.4 Conductance (A) and capacitance (B) spectra for the addition of NO3- to TDAN PVC spin-coated

IDE Design 2 (10 mVrms input amplitude). ................................................................................. 178

5.5 Conductance response of TDAN PVC spin-coated IDE Design 2 upon addition of 1–100 ppm

nitrate at six frequencies. ........................................................................................................... 179

5.6 Capacitance response of TDAN PVC spin-coated IDE Design 2 upon addition of 1–100 ppm

nitrate at six frequencies. ........................................................................................................... 180


for TDAN PVC spin-coated IDE Design 2 at 2 MHz. ................................................................ 181

5.8 Deionised water baseline-subtracted capacitance (Cm – C0) of three TDAN PVC spin-coated IDE

Design 2 devices against log NO3- concentration, for reproducibility determination. .................. 181

5.9 Selectivity determination of TDAN PVC spin-coated IDE Design 2. ............................................ 182

5.10 Conductance response versus time of TDAN PVC drop-coated IDE Design 2 (10% w/v) upon

addition of 1–100 ppm nitrate at five frequencies. ...................................................................... 184

5.11 Capacitance response versus time of TDAN PVC drop-coated IDE Design 2 (10% w/v) upon

addition of 1–100 ppm nitrate at five frequencies. ...................................................................... 185

5.12 Conductance deionised water baseline settling response of a dry TDAN PVC drop-coated IDE

Design 2 (10% w/v) at five frequencies. ..................................................................................... 186

Craig Alexander

13

5.13 Capacitance deionised water baseline settling response of a dry TDAN PVC drop-coated IDE

Design 2 (10% w/v) at five frequencies. ..................................................................................... 187

5.14 Conductance response of TDAN PVC drop-coated IDE Design 2 (10% w/v) upon the addition

of 50 ppm NO3- to a deionised water background at five frequencies. ........................................ 188

5.15 Capacitance response of TDAN PVC drop-coated IDE Design 2 (10% w/v) upon the addition










5.20 Conductance response versus time of TDAN PVC drop-coated IDE Design 2 (2% w/v) upon

addition of 1–100 ppm nitrate at four frequencies. ..................................................................... 196

5.21 Capacitance response versus time of TDAN PVC drop-coated IDE Design 2 (2% w/v) upon



for TDAN PVC drop-coated IDE Design 2 (2% w/v) at two frequencies.................................... 198

5.23 Logarithm of the change in capacitance (Cm – C0) against the logarithm of nitrate concentration for

TDAN PVC drop-coated IDE Design 2 (2% w/v) at 100 kHz. ................................................... 198

5.24 Conductance response versus time of NO3V PVC drop-coated IDE Design 2 (2% w/v) upon


5.25 Capacitance response versus time of NO3V PVC drop-coated IDE Design 2 (2% w/v) upon



for NO3V PVC drop-coated IDE Design 2 (2% w/v) at three frequencies. ................................. 201

5.27 Logarithm of the change in capacitance (Cm – C0) against the logarithm of nitrate concentration for

NO3V PVC drop-coated IDE Design 2 (2% w/v) at two frequencies. ........................................ 201

5.28 Selectivity determination of TDAN PVC drop-coated IDE Design 2 (2% w/v) using the FIM

(conductive response) ................................................................................................................ 202

5.29 Selectivity determination of TDAN PVC drop-coated IDE Design 2 (2% w/v) at 100 kHz using

the FIM (capacitive response) .................................................................................................... 203

5.30 Selectivity determination of NO3V PVC drop-coated IDE Design 2 (2% w/v) using the FIM

(conductive response). ............................................................................................................... 204

5.31 Selectivity determination of NO3V PVC drop-coated IDE Design 2 (2% w/v) using the FIM

(capacitive response). ................................................................................................................ 205

6.1 Microscope image (4x) of the carbon screen-printed IDE sensors produced at the University of

Bedfordshire.. ............................................................................................................................... 211

Craig Alexander

14

6.2 Conductance (A) and capacitance (B) spectra for the addition of NO3- to an uncoated SP IDE

Design 1 (1 Vrms input amplitude) ............................................................................................. .213


uncoated SP IDE Design 1 at six frequencies and varying input amplitudes. .............................. 214


uncoated SP IDE Design 1 at six frequencies and varying input amplitudes ............................... 215

6.5 Conductance (A) and capacitance (B) spectra for the addition of NO3- to SP IDE Design 1 with

a layer of plasticised dielectric paste containing 1% w/w TDAN as the ionophore

(10 mVrms input amplitude). ....................................................................................................... 216

6.6 Comparison of the conductance response of SP IDE Design 1 when a membrane containing

1% w/w TDAN was printed over the sensing area with a ‘blank’ membrane which did not

contain any ionophore. ............................................................................................................... 217

6.7 Comparison of the conductance response of SP IDE Design 1 coated with a ‘blank’ membrane

containing no ionophore when zero, one or two ‘build-up’ layers of non-modified dielectric

paste were printed prior to the modified layer containing NPOE as the plasticiser. ..................... 218

6.8 Conductance spectrum for the addition of NO3- to TDAN SP IDE Design 1

(10 mVrms input amplitude).. ...................................................................................................... 219

6.9 Conductance response versus time of TDAN SP IDE Design 1 upon addition of 1–100 ppm

nitrate at four frequencies. ......................................................................................................... 219


for TDAN SP IDE Design 1.. ..................................................................................................... 220


TDAN SP IDE Design 1.. .......................................................................................................... 221

6.12 Measured baseline conductance value (G0) of TDAN SP IDE Design 1 in four separate sample

matrices prior to the addition of nitrate. ..................................................................................... 222

6.13 Deionised water baseline-subtracted conductance (Gm – G0) of TDAN SP IDE Design 1 against

log NO3- concentration in four separate sample matrices. ........................................................... 223

6.14 Baseline-subtracted conductance (Gm – G0) of three TDAN SP IDE Design 1 devices against log

NO3- concentration, for reproducibility determination. ............................................................... 224

6.15 Selectivity determination of TDAN SP IDE Design 1 using the FIM.. ........................................ 225

7.1 Conductance response versus time of NH4I PVC drop-coated IDE Design 2 (2% w/v) upon the

addition of 50 ppm NH4+ to a deionised water background at five frequencies. .......................... 231

7.2 Capacitance response versus time of NH4I PVC drop-coated IDE Design 2 (2% w/v) upon the

addition of 50 ppm NH4+ to a deionised water background at five frequencies. .......................... 232

7.3 Conductance response versus time of NO2I PVC drop-coated IDE Design 2 (2% w/v) upon the

addition of 5 ppm NO2- to a deionised water background at five frequencies. ............................. 235

7.4 Capacitance response versus time of NO2I PVC drop-coated IDE Design 2 (2% w/v) upon the

addition of 5 ppm NO2- to a deionised water background at five frequencies. ............................. 236

7.5 Conductance response versus time of HIII PVC drop-coated IDE Design 2 (2% w/v) between

pH 9.04–6.24 at five frequencies. ............................................................................................... 240

Craig Alexander

15

7.6 Capacitance response versus time of HIII PVC drop-coated IDE Design 2 (2% w/v) between


7.7 Conductance response versus time of HV PVC drop-coated IDE Design 2 (2% w/v) between


7.8 Capacitance response versus time of HV PVC drop-coated IDE Design 2 (2% w/v) between


10.1 Calibration graph from a pH electrode obtained using a PHTX-22 pH/ORP preamplifier which was

powered using a linear lab supply at 10 V. .......................................................................................... 268

Craig Alexander

16

List of Tables

1.1 dH scale for water hardness ......................................................................................................... 33

1.2 Summary of the acceptable criteria of each of the most important analytes identified for

acceptable water quality within freshwater aquaria ...................................................................... 34

1.3 Comparison of the features of the proposed sensor device with the options currently available .... 41

1.4 Summary of some of the performance characteristics of H+-selective ionophores used within

pH-ISEs that have been described in the literature ....................................................................... 64

1.5 Summary of some of the performance characteristics of NH4+-selective ionophores..................... 66

1.6 Summary of some of the performance characteristics of NO2--selective ionophores ..................... 68

1.7 Summary of some of the performance characteristics of NO3--selective ionophores ..................... 70

2.1 Composition of each component required to produce PVC ion-selective membranes containing

1% w/w ionophore ....................................................................................................................... 84

2.2 Details of internal filling solutions used within PMEs for each target ion ..................................... 88

2.3 Volume of respective stock solutions required to produce 500 ml nitrate calibration solutions

with fixed 1000 ppm interfering ions, for selectivity coefficient determination ............................ 91

2.4 Volume of respective stock solutions required to produce 500 ml ammonium calibration solutions

with fixed 1000 ppm interfering ions, for selectivity coefficient determination ............................ 93

2.5 Volume of 0.1 M HCl required to add to 50 ml stock solution to produce 250 ml pH buffer

calibration solutions ..................................................................................................................... 94

2.6 Chemical composition of Megaposit SPR-220 positive photoresist .............................................. 96

2.7 Chemical composition of Megaposit MF-26A developer solution ................................................ 96

2.8 Geometric parameters of each IDE design shown in Figure 2.4 .................................................. 103

2.9 Composition of each phosphate buffer stock solution required to produce 0.1 M solutions of

each buffer at the required pH .................................................................................................... 116

3.1 Potentiometric selectivity coefficients of NH4I PME 1 .............................................................. 130

3.2 Potentiometric selectivity coefficients of NH4I PME 1 (ionic additive omitted) ......................... 132

3.3 Potentiometric selectivity coefficients of HIII PME 1 ................................................................ 135

3.4 Potentiometric selectivity coefficients of HV PME 1 ................................................................. 136

7.1 Selectivity determination of NH4I PVC drop-coated IDE Design 2 (2% w/v)

(conductive response) ................................................................................................................ 233

7.2 Selectivity determination of NH4I PVC drop-coated IDE Design 2 (2% w/v)

(capacitive response) ...................................................................................................................................... 234

7.3 Selectivity determination of NO2I PVC drop-coated IDE Design 2 (2% w/v)

(conductive response) ....................................................................................................... 237

Craig Alexander

17

7.4 Selectivity determination of NO2I PVC drop-coated IDE Design 2 (2% w/v)

(capacitive response) ................................................................................................................. 238

7.5 Final pH and TDS concentration of the diluted buffer solutions used to test the response of the

impedimetric pH sensors ........................................................................................................... 239

8.1 The resulting concentration, in ppm, of unionized ammonia present following additions of an

ammonium stock solution to a test sample at various pH values ................................................. 251

10.1 Concentration values of species stated in ppm throughout the thesis converted to the

molar equivalent ....................................................................................................................... 265

10.2 Details of the commercial ionophores used in the experimental work described throughout

this thesis .................................................................................................................................. 266

Craig Alexander

18

Abstract

Name of the University: The University of Manchester

Author: Craig Alexander

Degree Title: Doctor of Philosophy (PhD)

Thesis Title: The Development of Smart Sensors for Aquatic Water Quality Monitoring

Date: September 2014

The focus of this project was to investigate the use of interdigitated electrodes (IDEs) as

impedimetric ion-selective chemical sensors for the determination of several important

analytes found within a freshwater aquarium. The overall aim of this research was to

work towards a prototype sensing device that could eventually be developed into a

commercial product for sale to aquarium owners.

Polyvinyl chloride and sol-gels containing commercially-available ionophores for four

aquarium-significant ions (NH4+, NO2

-, NO3

- and pH) were prepared and investigated

for use within polymeric ion-selective membranes. Three separate IDE transducers were

produced using either photolithography or screen-printing microfabrication techniques.

A sinusoidal voltage was applied to the IDEs and an LCR meter was used to measure

changes in the conductance and capacitance of the ion-selective membrane layer

deposited over the electrode digits.

Each ionophore, when tested within potentiometric ion-selective electrodes (ISEs), was

found to be suitable for further investigation within IDE devices. Sol-gels were

investigated as a potential membrane material for a coated wire electrode; however,

poor response characteristics were observed. An IDE sensor fabricated in-house using

lift-off photolithography and spin-coated with a polymeric membrane was found to

produce non-selective responses caused by changes in the conductivity of the test

solution. IDE devices with reduced geometric parameters were purchased and coated

with a selective polymeric membrane. When the membrane was spin-coated,

non-selective responses were observed; therefore, drop-coating of the membrane

material was investigated. This initially resulted in an unacceptably long response time;

however, this effect was reduced by decreasing the membrane solution viscosity prior to

drop-coating. A fully-screen printed carbon IDE device was fabricated by incorporating

the ionophore into a support matrix based on a commercial dielectric paste. Matrix

interferences to the sensor response were reduced by printing ‘build-up’ layers over the

sensing area prior to the ion-selective membrane.

Two novel routes for monitoring the water quality of an aquarium, using IDE sensors

fabricated by either photolithography or screen-printing, have been demonstrated. Due

to the commercial aspect of this project, it is important to consider the final cost of

producing these sensors. Both of the techniques used to produce ion-selective sensors

require further experimentation to optimise the sensor response, prior to integration

within a multi-analyte sensing prototype.

Craig Alexander

19

Declaration

I declare that no portion of the work referred to in the thesis has been submitted in

support of an application for another degree or qualification of this or any other

university or other institute of learning.

Copyright Statement

i. The author of this thesis (including any appendices and/or schedules to this thesis)

owns certain copyright or related rights in it (the “Copyright”) and s/he has given The

University of Manchester certain rights to use such Copyright, including for

administrative purposes.

ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic

copy, may be made only in accordance with the Copyright, Designs and Patents Act

1988 (as amended) and regulations issued under it or, where appropriate, in accordance

with licensing agreements which the University has from time to time. This page must

form part of any such copies made.

iii. The ownership of certain Copyright, patents, designs, trademarks and other

intellectual property (the “Intellectual Property”) and any reproductions of copyright

works in the thesis, for example graphs and tables (“Reproductions”), which may be

described in this thesis, may not be owned by the author and may be owned by third

parties. Such Intellectual Property and Reproductions cannot and must not be made

available for use without the prior written permission of the owner(s) of the relevant

Intellectual Property and/or Reproductions.

iv. Further information on the conditions under which disclosure, publication and

commercialisation of this thesis, the Copyright and any Intellectual Property and/or

Reproductions described in it may take place is available in the University IP Policy

(see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=487), in any relevant

Thesis restriction declarations deposited in the University Library, The University

Library’s regulations (see http://www.manchester.ac.uk/library/aboutus/regulations) and

in The University’s policy on Presentation of Theses.

Craig Alexander

20

Acknowledgements

I would like to take this opportunity to thank my supervisor Professor Peter Fielden for

all of his advice and continued support throughout this project, and also Professor Nick

Goddard for his role as co-supervisor. I would also like to thank Dr. Sara Baldock for

all of the invaluable help she has provided throughout my PhD. Thank you to all of the

other post-docs in the miniaturisation group, past and present, especially Dr. Bernard

Treves Brown, who spent many hours writing LabVIEW code and helping me to set up

equipment, Dr. Behnam Bastani, who advised in the preparation of sol-gels and using

the e-beam, and Dr. Jeremy Hawkes, who helped me to set up several pieces of

instrumentation. My thanks to Dr. Sung Quek for allowing me to use his LabVIEW

program to control the LCR meter.

I would like to thank Dr. Tim Harvey for all of his help as my industrial co-supervisor

during the early stages of the project, and Barry Jones from Compact Instruments who

has supported the project financially throughout.

I would also like to thank Dr. Barry Haggett from the University of Bedfordshire, and

Dr. Craig Banks and Dr. Jonathan Metters from Manchester Metropolitan

University/Kanichi Research, for the many hours they spent providing specialist help

producing screen-printed electrodes for this project.

Finally, I would like to thank my family, friends and girlfriend for all of their continued

support.

This project was supported by funding from the Engineering and Physical Sciences

Research Council (EPSRC).

Craig Alexander

21

List of Abbreviations

%RSD Relative standard deviation

µ-TAS Micro-total analysis system

AC Alternating current

AOB Ammonia-oxidising bacteria

APPA American Pet Products Association

AU Auxiliary Electrode

BHT Butylated hydroxytoluene

CAS Chemical Abstracts Service

CP Conducting polymers

CWE Coated-wire electrode

DEDMS Diethoxydimethylsilane

dH Degrees of hardness

DO Dissolved oxygen

EIS Electrochemical impedance spectroscopy

FIM Fixed interference method

FPM Fixed primary ion method

GH General hardness

HIII Hydrogen Ionophore III

HV Hydrogen Ionophore V

IDE Interdigitated electrode

ISAB Ionic strength adjustment buffer

ISCOM Ion-selective conductometric microsensor

ISE Ion-selective electrode

ISFET Ion-selective field effect transistor

IUPAC International Union of Pure and Applied Chemistry

KH Carbonate hardness

Craig Alexander

22

LDL Lower detection limit

MPM Matched potential method

MSM Mixed-solution method

MTES Methyltriethoxysilane

N-E Nicolskii-Eisenman

NH4I Ammonium Ionophore I

NO2I Nitrite Ionophore I

NO3V Nitrate Ionophore V

NOB Nitrite-oxidising bacteria

NPOE o-nitrophenyl octyl ether

NTC Negative temperature coefficient

OATA Ornamental Aquatic Trade Association

PEG Polyethylene glycol

PFMA Pet Food Manufacturers Association

PID Proportional-integral-derivative

PME Polymer membrane electrode

ppm Parts per million

PVC Polyvinyl chloride

RE Reference Electrode

rpm Revolutions per minute

SCE Saturated calomel electrode

SHE Standard hydrogen electrode

SP-ISE Screen-printed ion-selective electrode

SSM Separate-solution method

TAN Total ammonia nitrogen

TDAN Tetradodecylammonium nitrate

TDMACl Tridodecylmethylammonium chloride

Craig Alexander

23

TDS Total dissolved salts

TEOS Tetraethoxysilane

THF Tetrahydrofuran

UDL Upper detection limit

UV Ultraviolet

WE Working Electrode

Craig Alexander

24

Project Overview

The main objective of this PhD project was to develop ion-selective chemical sensors,

with the specific application of monitoring key ions within a tropical freshwater

aquarium. Both technical and financial assistance to the project was provided by

Compact Instruments Ltd., who sought to exploit the findings of this project to develop

a prototype multi-analyte sensing device, with the eventual outcome of producing a

commercial product for sale to home aquarium owners.

This thesis seeks to address the feasibility of producing such a device by establishing

which chemical species are the most important for aquatic water quality monitoring. It

will also discuss some of the approaches for testing that aquarium hobbyists currently

have available. The limitations of these existing methods will be discussed along with

potential technologies that could be utilised to produce an easy-to-use sensing device.

The benefits that this would offer over current methodologies will also be evaluated.

As the primary motivation of this project was to produce a device to be sold to

household consumers, this research focuses on low-cost technologies which could be

easily implemented on a commercial scale. The eventual product would also be used by

non-specialists; therefore, it was also of great importance to consider the final design of

the proposed sensor, to ensure that it could be operated by the user in a low maintenance

fashion without any prerequisite chemical knowledge.

Craig Alexander

25

1 Introduction

The keeping of fish as pets within the home is a longstanding and popular hobby in

many countries all over the world. Recent data from the Ornamental Aquatic Trade

Association (OATA) indicated that there are currently around 3–3.5 million households

in the UK who own either an ornamental aquarium or a pond, making fish the third

most popular pet category after dogs and cats1. The Pet Food Manufacturers Association

(PFMA) suggests that around 9% of UK households own a fish tank2 and there are

almost 15 million aquaria throughout Europe3. A survey conducted by the American Pet

Products Association (APPA) states that in 2013, 14.3 million homes in the USA kept

freshwater fish, with a further 1.8 million keeping saltwater fish4. Furthermore, it is

estimated that the fishkeeping industry is worth more than 1 billion US dollars each

year.

1.1 Aquarium Chemistry

Owning an aquarium can be a very rewarding pastime, providing that adequate care and

maintenance of the artificial habitat takes place regularly. Generally, a home aquarium

set up for the keeping of fish will fall into one of two categories: freshwater, which can

either be cold-water or tropical, or marine (saltwater). Each of these artificial

environments provides a complex and challenging matrix for the analysis of many

chemical species. Such species are very important for maintaining the health of the fish,

which can be sensitive to even slight changes in various physical or chemical properties

within the environment.

The purpose of this section is to discuss the key chemical constituents within a tropical

freshwater aquarium; often described collectively as its ‘water quality’. Various ionic

Craig Alexander

26

species dissolved within the aquatic habitat provide a delicate environment which must

be suitably monitored and controlled. A decline in the water quality of an aquarium can

easily spoil the enjoyment of an aquarium hobbyist; as even small changes can quickly

lead to issues such as murky water; stress, illness or even death of often expensive

aquatic organisms. Marine water has other specific considerations that will not be

addressed in this thesis.

Throughout this thesis, analyte concentrations will be quoted in units of parts per

million (ppm), equivalent to 1 mg of solute dissolved in a 1 litre solution, as this is

standard fishkeeping nomenclature. A table stating the molar equivalent concentrations

can be found in Appendix 10.1.

1.1.1 Analytes from the Nitrogen Cycle

Ammonia, nitrite and nitrate are three primary nitrogenous species that exist within an

aquarium; each of these has its own specific effects on water quality. Ammonia is

converted in a two-step oxidation to nitrite and then nitrate in a process called

nitrification, commonly referred to as the biological filtration of an aquarium5.

1.1.1.1. Ammonia

Ammonia is produced in an aquarium via protein metabolism in fish and is expelled

from the gills. A smaller quantity also comes from ammonification of organic nitrogen

which arises from faeces and uneaten food6.

When dissolved in an aqueous environment, ammonia exists in two forms, unionized

ammonia (NH3) and the ammonium ion (NH4+). These species are collectively referred

Craig Alexander

27

to as the total ammonia nitrogen (TAN) concentration6. The equilibrium between the

two forms of ammonia dissolved in water is shown in Equation 1.1.

𝐇𝟑𝐎+(𝒂𝒒) + 𝐍𝐇𝟑 (𝒂𝒒)

𝐇𝟐𝐎(𝒂𝒒) + 𝐍𝐇𝟒+

(𝒂𝒒) Equation 1.1

This equilibrium is heavily dependent on the pH and temperature of the solution in

which the ammonia is dissolved7. The pKa of the ammonium ion is 9.26

8; hence

dissolved unionized ammonia only becomes thermodynamically favourable in alkaline

conditions. If the TAN concentration, pH and temperature are known, it is possible to

calculate the free unionized NH3 concentration, as described by Thurston et al7.

Equation 1.2 shows the expression for calculating the fraction of unionized ammonia, f,

at a particular pH. This value is then multiplied by the TAN concentration to give the

concentration of unionized ammonia within the sample.

𝐟 = 𝟏 (𝟏𝟎𝐩𝐊𝐚−𝐩𝐇 + 𝟏)⁄ Equation 1.2

As the equilibrium is also temperature dependent, Thurston et al. produced an empirical

mathematical expression to relate the equilibrium constant with temperature, which can

simply be inserted into Equation 1.2 as the pKa term7. This is shown in Equation 1.3,

where T is the temperature (K).

𝐩𝐊𝐚 = 𝟎. 𝟎𝟗𝟎𝟏𝟖𝟐𝟏 + 𝟐𝟕𝟗𝟐. 𝟗𝟐 𝐓⁄ Equation 1.3

This expression can be used in the temperature range of 0–50 °C and the pH range of

6.0–10.0.

It is of upmost importance to monitor the concentration of ammonia within a freshwater

aquarium as it is one of the most common water quality problems, due to its toxicity to

fish9. This toxicity is predominantly caused by the unionized form of ammonia, whilst

Craig Alexander

28

NH4+ is effectively non-toxic within the aquatic environment

10, due to increased

permeability of NH3 across biological membranes11

.

Different species of fish exhibit varied tolerances to ammonia; however, it is generally

accepted that its levels in freshwater aquaria should be maintained as close to zero as

possible. OATA guidelines state that the unionized ammonia concentration should not

exceed 0.02 ppm12

. This value will be treated as the upper limit for NH3 throughout this

thesis.

The first step of nitrification is carried out by Nitrosomonas and other ammonia-

oxidizing bacteria (AOB), which break down ammonia to form nitrite, NO2- 9

.

1.1.1.2. Nitrite

Nitrite (NO2-) is the intermediate species formed during the nitrification process. The

anion is absorbed by the gills and diffuses into the bloodstream, where its toxic action is

believed to arise due to oxidation of haemoglobin, resulting in respiratory problems and

eventually death13

.

OATA guidelines state that the nitrite concentration within a freshwater aquarium

should not exceed 0.2 ppm.

Nitrobacter and other nitrite-oxidizing bacteria (NOB) facilitate the final stage of the

nitrification process, the oxidation of nitrite to form nitrate, (NO3-) 9

.

1.1.1.3. Nitrate

Nitrate, NO3-, is the final and least toxic product formed during the oxidation of

ammonia. Nitrate permeates poorly through the gills which corresponds to its relatively

low toxicity when compared with the two previously discussed nitrogenous species. At

Craig Alexander

29

toxic concentrations, nitrate transports through the fish’s gills and diffuses into the

bloodstream, where it has a similar toxic action to nitrite, affecting the oxygen-carrying

ability of haemoglobin, eventually resulting in death14

.

Although nitrate can be tolerated at much higher levels than either ammonia or nitrite

before there are any toxic effects on fish its presence can cause algae to grow, which

leads to oxygen depletion within an aquarium9. Figure 1.1 shows an algal bloom within

an aquarium caused by high nitrate levels.

OATA recommends that nitrate levels do not exceed 50 ppm in a freshwater aquarium.

Figure 1.1: Algae accumulation caused by high levels of nitrate in an aquarium, taken from

Roberts et al.9

As mentioned previously, the two-step oxidation of ammonia to form nitrite and then

nitrate is often referred to as biological filtration. When a new aquarium is first set-up a

‘conditioning’ period is required before any fish are added. During this period the levels

of AOB and NOB increase on surfaces within the tank, which increases the efficiency

of nitrification and reduces the ammonia and nitrite levels. Regular water changes are

Craig Alexander

30

required to eliminate nitrate from an aquarium as it will continue to accumulate as the

other two species are oxidized5. It is therefore important to ensure that all three of the

nitrogenous species are adequately monitored to prevent them from reaching dangerous

levels within the closed aquarium system.

1.1.2 Aquarium pH

pH is a measure of the activity, although in practical terms it is a measure of the molar

concentration of hydrogen cations in an aqueous solution, and is defined by

Equation 1.4.

𝐩𝐇 = −𝐥𝐨𝐠 [𝐇+] Equation 1.4

As mentioned is section 1.1.1.1, the pH of the aquarium has effects on other water

quality factors, therefore it is very important that it is monitored. pH is a much more

species-specific indicator of water quality as some tropical fish prefer a more acidic, or

alkaline, environment than others. Generally, a near-neutral pH is favourable in a

tropical freshwater aquarium with most species preferring water with a pH of 5.5–8.5 9.

However, rapid fluctuations in pH are more likely to cause harm to fish, however, rather

than a specific pH value.

1.1.3 Chlorine

Chlorine is routinely added to tap water around the world as a disinfectant to kill

waterborne pathogens. As tap water is commonly used to fill aquaria, the presence of

chlorine must be considered as this species is highly toxic to fish. When Cl2 is dissolved

in water it forms hypochlorous acid (HOCl) which equilibrates with hypochlorite anions

(ClO-) above pH 5.0, which damages the gill tissue of fish at very low concentrations

5.

Craig Alexander

31

No values are published for safe levels of chlorine within an aquarium. It is generally

accepted that it should be completely removed from the water before it is added to the

aquarium9.

Aquarium owners will generally treat tap water with a sodium thiosulfate-based

dechlorinating agent5 when setting up an aquarium and performing water changes,

making regular chlorine monitoring less critical than some of the other water quality

indicators.

The chloride anion (Cl-) is generally well tolerated by freshwater fish in the expected

range found in most tap water samples, up to a maximum of 250 ppm, so there is little

requirement for this to be regularly tested in an aquarium.

1.1.4 Dissolved Oxygen

A supply of dissolved oxygen (DO) within an aquarium is required not only for fish

respiration but also for respiration in bacteria and plants15

. The saturation of DO is

dependent on the physical and chemical properties of the aqueous environment. For

example, less oxygen can be dissolved as temperature or salinity increases. At a typical

aquarium temperature of 25 °C, 100% saturation of DO corresponds to 8.3 ppm16

. The

average saturation in an aquarium is around 70% (5.8 ppm at 25 °C)17

. OATA

guidelines recommend a DO concentration of at least 6 ppm is maintained within a

freshwater aquarium.

By ensuring than an aquarium is not overstocked with fish, and providing a source of

agitation such as an airstone, DO levels can be sustained at near saturation. Therefore it

is not critical for this water quality parameter to be regularly monitored.

Craig Alexander

32

1.1.5 Phosphate

The orthophosphate anion (PO43-

) has similar effects within an aquarium to nitrate as it

is not considered directly harmful to aquatic organisms in the levels likely to be

observed in an aquarium. PO43-

, however, acts as a fertiliser that can cause algal blooms.

This can occur at low PO43-

concentrations therefore, most literature states that the

concentration should not exceed 0.05 ppm18

. Aquarists should be particularly mindful of

the phosphate concentration within their aquarium when using phosphate-based buffers

to stablise aquarium pH; although most pH buffers intended for aquarium use no longer

contain phosphate5.

1.1.6 Water Hardness

Water hardness is a broad term that relates to the amount of dissolved minerals in the

water. Fishkeeping literature generally deals with water hardness as two separate

parameters; general hardness (GH), usually defined as the total concentration of

magnesium (Mg2+

) and calcium (Ca2+

) cations19

, and carbonate hardness (KH), which is

concerned with the amount of free carbonate (CO32-

) and bicarbonate (HCO3-) anions

within the aquarium.

1.1.6.1. General Hardness

Although other multivalent cations such as iron (Fe3+

), aluminium (Al3+

) and manganese

(Mn2+

) may be present in aquarium water, the main species that lead to ‘hard’ water are

Ca2+

and Mg2+

predominantly from carbonate salts (~80%) with the remainder from

sulfate and chloride salts5. For fishkeeping purposes, GH is commonly expressed in

units of ppm with respect to CaCO3, or more simply, using the degrees of hardness (dH)

scale, where 1 dH is equivalent to 17.8 ppm CaCO3. This method provides aquarists

Craig Alexander

33

with a simple and manageable way of monitoring water hardness within their aquarium

as opposed to using scientific nomenclature relating to concentrations of individual

ions. The definitions of what category of hardness a particular dH value belongs to vary

between different fishkeeping advice publications. Table 1.1 shows a typical dH scale

along with its corresponding equivalent CaCO3 concentration and hardness category.

dH Approximate equivalent

concentration of CaCO3/ppm Hardness category

0–4 0–70 Very soft

4–8 70–140 Soft

8–12 140–210 Medium-hard

12–20 210–350 Hard

>20 >350 Very hard

Table 1.1: dH scale for water hardness

Suitable levels of GH for most ornamental freshwater fish usually lie between

~6–18 dH, although it is very much a species-dependent parameter.

1.1.6.2. Carbonate Hardness

The KH (from the German spelling of carbonate, karbonat) of aquarium water provides

a pH buffering capacity, allowing for a more stable pH value. It is sometimes referred to

in fishkeeping literature as alkalinity, due to the ability of a solution with a high KH to

maintain an alkaline pH, although this is scientifically inaccurate. Aquaria with a low

KH will likely have a low pH, and also be susceptible to rapid drops or fluctuations in

pH due to a limited buffering capacity provided from the equilibrium between carbonate

and bicarbonate anions. KH is usually expressed in the same units as GH, where the

total concentration of CO32-

and HCO3- are converted to an equivalent value with respect

to CaCO3. Table 1.1 is therefore applicable for both GH and KH. KH values should not

drop below approximately 4 dH15

, particularly when live plants are kept. Low KH

Craig Alexander

34

affects the ability of aquarium water to dissolve carbon dioxide, which is required for

photosynthesis5.

1.1.7 Summary of Parameters

Table 1.2 provides a summary of each of the water quality factors discussed in this

section, along with the respective acceptable limits in a freshwater aquarium.

Analyte Acceptable concentration

within a freshwater aquarium

Ammonia <0.02 ppm (as NH3)

Nitrite <0.2 ppm

Nitrate <50 ppm

pH pH 5.5–8.5

(species dependent)

Chlorine/Chloramine Not detectable

Chloride Drinking water levels

(<250 ppm)

Phosphate <0.05 ppm (as PO43-

)

Dissolved Oxygen >6 ppm

General Hardness

6–18 dH

(100–300 ppm as CaCO3)

(species dependent)

Carbonate Hardness >4 dH

(>70 ppm as CaCO3)

Table 1.2: Summary of the acceptable criteria of each of the most important analytes identified for

acceptable water quality within freshwater aquaria

After reviewing both scientific and fishkeeping literature which discuss the key

chemical components present within a tropical freshwater aquarium, the focus of the

experimental work in this thesis will be on the determination of the aquarium pH and

the analysis of the three nitrogenous species as these appear to be the most crucial for

fish health. An aquarium hobbyist can be confident in the water quality of their

aquarium if these four components are known to be at acceptable levels. It is beneficial,

Craig Alexander

35

rather than essential, that others such as phosphate and water hardness are known;

therefore they are deemed outside the immediate scope of this work.

1.2 Current Methods of Analysis

As mentioned previously, the monitoring of aquarium water quality is very important to

fish health, and therefore various methods of analysis have been developed that are

available to aquarists. The purpose of this section is to discuss some of the options

currently available for water quality monitoring within aquaria, along with their

limitations, which are to be addressed throughout this thesis.

1.2.1 Test Kits

Currently, most fish keepers will carry out routine colourimetric chemical tests to check

the levels of various key analytes within their aquarium, usually in the form of test

strips or indicator solutions. These are often sold as packs containing multiple tests for

several ions; the most basic will typically test the pH, ammonia, nitrite and nitrate levels

as these are seen as the most critical. More expensive and specialist test kits can be

purchased for other analytes such as water hardness and phosphate.

The process for checking water quality parameters using the indicator solutions

provided with test kits usually involves removing a sample of water from the aquarium

and placing it into a small vial, adding the relevant indicator solution(s), and waiting to

observe a colour change. The coloured solution within the vial is held against a colour

chart which corresponds to an estimation of the concentration of the target species. This

value can then be checked against a given range from the literature to inform the user if

the levels within their tank are safe, or if any further action needs to be taken.

Craig Alexander

36

1.2.1.1. pH

pH indicators are weak acids or bases that are protonated and deprotonated at different

pH values, with each form existing as a different colour. The observed colour change

occurs gradually over a range of concentrations of hydrogen ions and therefore a

reasonable estimation can be made towards the pH of the tested sample.

Various pH indicators are used in aquarium test kits and each has more favourable

properties depending on whether the pH is being tested over a broad or narrow range, or

if marine water or freshwater is the test sample. Three common indicators include:

bromothymol blue (pH range 6.0–7.6) and phenol red (pH range 6.4–8.2), used for low

and high range freshwater pH testing, respectively; and m-cresol purple

(pH range 7.6–9.2), used in saltwater pH test kits. Figure 1.2 shows the two coloured

forms of phenol red observed upon addition of either OH- or H

+, and Figure 1.3 shows

the three species involved in the colour change of bromothymol blue.

Figure 1.2: Structure of the two forms of phenol red which result in the observed colour change

20

Figure 1.3: Structure of three forms of bromothymol blue which result in a colour change at

varying pH values21

Craig Alexander

37

1.2.1.2. Ammonia

Colourimetric testing for dissolved ammonia is usually based on either the Nessler or

Salicylate reactions, both of which are long-standing and well researched methods.

1.2.1.2.1. Nessler’s Reagent Method

The reaction of the Nessler reagent, an alkaline solution of mercury (II) iodide and

potassium iodide with the formula K2HgI4, was first suggested for the direct, qualitative

analysis of aqueous ammonia in 1856 by Julius Nessler. Nichols and Willets further

investigated the reaction scheme followed by this process, outlining any intermediates

formed along with the observed colour changes22

. In the presence of ammonia, a

yellow-coloured species is produced with an intensity that is proportional to the

concentration of ammonia in the sample. The reaction is shown in Equation 1.5.

𝟐𝐊𝟐𝐇𝐠𝐈𝟒 + 𝐍𝐇𝟑 + 𝟑𝐊𝐎𝐇 → 𝐇𝐠𝟐𝐎𝐈𝐍𝐇𝟐 + 𝟕𝐊𝐈 + 𝟐𝐇𝟐𝐎 Equation 1.5

1.2.1.2.2. Salicylate Method

The other colourimetric method for ammonia determination which is often used in

aquarium test kits is known as the salicylate method. This method is based on the

reaction between phenol and hypochlorite to form a blue-coloured indophenol. This is a

multi-step process, outlined below in Equations 1.6 and 1.7, which first requires the

formation of monochloramine via the reaction of ammonia with hypochlorite

(Equation 1.6). Monochloramine then reacts with salicylate to form 5-aminosalicylate

(Equation 1.7), which is oxidized in the presence of an iron catalyst to form

indosalicylate, shown in Figure 1.4.

Craig Alexander

38

𝐍𝐇𝟑 + 𝐎𝐂𝐥− → 𝐍𝐇𝟐𝐂𝐥 + 𝐎𝐇− Equation 1.6

Equation 1.7

Figure 1.4: Structure of indosalicylate

In the presence of an excess of the yellow-coloured catalyst, the blue indosalicylate

appears green. The intensity of the green colour observed is directly proportional to the

amount of ammonia present in the sample23

.

1.2.1.3. Nitrite

The most common nitrite indicators are based on the Griess reaction24

, a diazotisation

reaction which utilises any NO2- within the tank to form an azo-dye compound. The

colour of the azo-dye intensifies depending on the amount of free NO2- that is available

to react. The reaction scheme for this is outlined in Equation 1.8 and 1.9 25

. This two-

step mechanism proceeds via the reaction of nitrite with sulfanilamide (Equation 1.8)

followed by the addition of N-1-naphthylethylenediamine dihydrochloride (NED) to

form the azo-dye (Equation 1.9).

Craig Alexander

39

Equation 1.8

Equation 1.9

1.2.1.4. Nitrate

The determination of aquarium nitrate levels usually involves the reduction to nitrite

with cadmium metal prior to testing using the Griess reaction as above24

. Therefore one

limitation of this method is that the total concentration of both nitrate and nitrite is

observed.

Performing wet chemical tests to indicate the concentration of the target species offers

many sources of error, particularly for non-specialist users. The interpretation of the

results by comparing against colour charts is a subjective process, which could lead the

user to believe that their test is reading higher or lower than in actuality. Users may also

cause erroneous results due to poor adherence to a multi-step testing process, inefficient

shaking of the sample vial or not allowing sufficient time for the observed colour

Craig Alexander

40

change to occur. Finally, some of the chemicals involved are potentially harmful if

incorrectly handled.

1.2.2 Commercial Sensors

An alternative to using colourimetric test kits for aquarium water quality monitoring is

to use chemical sensors. Commercial electrochemical sensors, in the form of

potentiometric ion-selective electrodes (described later in sections 1.3 and 1.4), are

available to purchase for each of the most important target ions found within a home

aquarium (pH, NH4+, NO2

-, NO3

-). These are generally in the form of a handheld probe

that is submerged into the sample to obtain a reading, rather than an in situ device

offering continual monitoring. Sensors such as these are normally intended for

laboratory, rather than home, use and as such are very expensive, often costing several

hundreds of pounds for one probe. This prohibitive cost makes them a non-viable option

for most aquarium hobbyists. Such devices also need to be calibrated prior to use which

would require the aquarium owner to keep standard solutions of each target ion at

known concentrations; thus potentially adding user error to the analysis.

1.2.3 Benefits of the Proposed Device

The availability of a high quality, reliable and affordable sensor array for in situ

determination of the key aquarium water quality factors would eliminate the problems

associated with test kits, described in section 1.2.1, whilst providing a more viable

alternative for aquarium hobbyists to the expensive commercial probes that are

currently available.

A device providing continual monitoring with a suitable output would alert users

immediately if their aquarium required attention, rather than relying on routine

Craig Alexander

41

intermittent testing. Such a device would also provide an instantaneous output of

multiple parameters, as opposed to trial-and-error testing to deduce the cause of an

existing problem. The subjectivity of chemical testing could be removed by providing

the user with a quantitative or semi-quantitative result. Rather than interpreting the

results from a colour chart, the concentration of the species of interest would be

conveyed directly as a value, or by way of a ‘traffic-light’ system where the user is

alerted when the analyte of interest approaches levels where attention is required. By

incorporating a suitable transmitter and receiver unit into the device the results could

also be output to electronic equipment such as a standalone screen, computer or mobile

phone.

Another important benefit that the proposed device would offer is the complete removal

for the requirement of any toxic or harmful chemicals. Batch calibrations of the device

could be performed during manufacture, which would eliminate the need to provide

calibrant solutions. Table 1.3 summarises the benefits that the proposed sensor would

offer over the testing methods that are currently available.

Proposed sensor Test kits Commercial sensors

Continual monitoring

Free from chemicals

Inexpensive

Use without pre-calibrating

Quantitative, or semi

quantitative result Results outputted to a screen,

PC or mobile device Table 1.3: Comparison of the features of the proposed sensor device with the options

currently available

Craig Alexander

42

1.3 Introduction to Chemical Sensors

Chemical sensors have been defined as “a device which responds to a particular analyte

in a selective way through a chemical reaction and can be used for the qualitative or

quantitative determination of the analyte”26

. The primary function of a chemical sensor

is to provide real-time information about a test sample relating to the concentration of a

particular target analyte, or group of analytes27

. The purpose of this section is to

introduce some of the important features of chemical, specifically electrochemical,

sensors, which will form the main focus of the experimental work in this thesis.

Over the past few decades, much research has been carried out to attempt to miniaturise

traditional ‘bench-top’ laboratory techniques, including using chemical sensors in place

of conventional analytical processes. Using sensors offers several advantages for many

analytical applications compared with established macro-scale laboratory techniques,

such as the removal of complicated sample preparation or pre-treatment stages.

Chemical sensors also offer a more feasible route for on-site and in situ analysis whilst

also facilitating integration within a miniaturised device for single or multi-analyte

sensing28

.

A chemical sensor requires two main components: a recognition element, which imparts

the selectivity of the sensor enabling it respond to a particular analyte with minimal

matrix interferences; and a transducer, which converts a change in analyte concentration

into a measureable signal29

. A biosensor is a specific type of chemical sensor that

utilises a biological receptor, such as an enzyme or antibody, to facilitate a

highly-selective response to a particular target analyte.

Craig Alexander

43

Many different transducers for chemical sensors exist, including optical, piezo-electic

and thermal; however, for the purpose of this thesis the focus will be on

electrochemical transducers.

Chemical sensors must perform acceptably across a number of performance

characteristics for them to be successfully used for their desired application. These

include: dynamic range, precision, response time, operational lifetime, sensitivity, limit

of detection and selectivity against interfering species27, 29

.

1.3.1 Electrochemical Sensors

Three electrochemical processes that are often utilised in transducers for chemical

sensors are: potentiometry, voltammetry and electrical impedance29

. Voltammetric

sensors will not be considered in this thesis and impedimetric devices will be described

in detail throughout section 1.5.

1.3.1.1. Potentiometry

Potentiometric sensors involve the measurement of the potential difference between an

indicator electrode (EIND) and a suitable reference electrode (EREF) at zero current, as

shown in Equation 1.10 29

. Reference electrodes will be described in further detail in

section 1.3.1.2.

∆𝐄 = 𝐄𝐜𝐞𝐥𝐥 = 𝐄𝐈𝐍𝐃 − 𝐄𝐑𝐄𝐅 Equation 1.10

The Nernst equation defines the logarithmic relationship between the observed potential

and the ionic activities of the species in an electrochemical cell. Equation 1.11 shows a

typical half-cell reaction and Equation 1.12 shows the corresponding Nernst equation,

where E is the measured electrode potential (V); E° is the standard electrode potential

Craig Alexander

44

for the half-cell (V); R is the universal gas constant (8.3145 J mol-1

K-1

); T is the

temperature (K); n is the stoichiometry of the electrons involved, F is the Faraday

constant (96,485 C mol-1

); and aox and aR are the activities of the respective species.

𝐎𝐱 + 𝐧𝐞− 𝐑 Equation 1.11

𝐄 = 𝐄° +

𝐑𝐓

𝐧𝐅 𝐥𝐧 (

𝒂𝑶𝒙

𝒂𝑹)

Equation 1.12

For dilute solutions, the activities can be assumed to be equal to the molar

concentration29

. The activity of a species, X, is related to the molar concentration by the

activity coefficient, γ, as shown in Equation 1.13.

𝐚𝐗 = γ𝐗[𝐗] Equation 1.13

Assuming ax=[X], at room temperature (298 K) Equation 1.12 becomes:

𝐄 = 𝐄° +

𝟐. 𝟓𝟔𝟖 × 𝟏𝟎−𝟐

𝐧 𝐥𝐧 (

[𝐎𝐱]

[𝑹])

Equation 1.14

As conventional molar concentrations are usually expressed in powers of 10, Equation

1.14 can be multiplied by 2.303 to convert from a natural logarithm to a base 10

logarithm:

𝐄 = 𝐄° +

𝟎. 𝟎𝟓𝟗𝟐

𝐧 𝐥𝐨𝐠 (

[𝐎𝐱]

[𝑹]) Equation 1.15

[R] is generally a metal with a fixed concentration and therefore Equation 1.15 can be

further simplified:

Craig Alexander

45

𝐄 = 𝐄° +

𝟎. 𝟎𝟓𝟗𝟐

𝐧 𝐥𝐨𝐠([𝐎𝐱]) Equation 1.16

For experimental purposes where E° is not known and RT/F may differ from the

theoretical value, a more practical form of the Nernst equation can be written. This is

shown in Equation 1.17, where [C] is the concentration of the species of interest.

𝐄 = 𝐊 +

𝐒

𝐧 𝐥𝐨𝐠[𝐂] Equation 1.17

When a graph of E against log [C] is plotted, a straight line with a slope (S) and

intercept (K) is obtained. The values obtained experimentally can be compared with the

theoretical values, where K=E° and S=0.0592/n at 298 K 29

. A potentiometric sensor

can be deemed ‘Nernstian’ if the measured slope fits closely with the theoretical value,

i.e. for a monovalent cation at 298 K there would be a 59.2 mV increase in potential per

decade of change in ion concentration.

The construction of the indicator electrode can be prepared in such a way so that its

response to one particular ion is increased whilst minimising the interference observed

in the measured potential from any other ions present in the solution. These

potentiometric sensors are called ion-selective electrodes (ISEs) and will be discussed in

section 1.4.

1.3.1.2. Reference Electrodes

As mentioned previously, potentiometric sensors require a suitable reference electrode

against which the potential can be measured. It is not possible to measure the absolute

potential of a half-cell directly; instead it is the difference between the potential of the

half-cell of interest (the sensing, or indicator electrode) and the potential of a reference

Craig Alexander

46

electrode that is measured. An ideal reference electrode must therefore provide an

accurate and stable potential that is not influenced by the test solution30

.

The standard hydrogen electrode (SHE) involves the redox reaction of hydrogen gas at a

platinum electrode in an acidic solution and is used to compare the potential of

half-cells, allowing the compilation of a list of standard electrode potentials at known

temperatures. The SHE half-cell is shown in Equation 1.18. By convention, the SHE is

assigned a potential value of zero under standard conditions (298 K, 1 atm, 1 M H+).

𝟐𝐇+ + 𝟐𝐞− 𝐇𝟐(𝒈) Equation 1.18

Although the SHE has a great importance, it is not the most well-suited reference

electrode in practical situations. The two most common and commercially-available

electrochemical reference electrodes are the silver-silver chloride electrode (Ag/AgCl)

and the saturated-calomel electrode (SCE).

Ag/AgCl reference electrodes are produced by coating a layer of AgCl onto a piece of

Ag wire, which is submersed into a saturated or near-saturated (3 M) solution of KCl.

The electrode potential at 25 °C against the SHE is +0.223 V. The half-cell reaction is

shown in Equation 1.19 31

.

𝐀𝐠𝐂𝐥(𝒔) + 𝐞− 𝐀𝐠(𝒔) + 𝐂𝐥(𝒂𝒒)− Equation 1.19

The SCE is produced by placing mercury in contact with a paste consisting of

mercury (I) chloride and potassium chloride in a solution of saturated KCl. The half-cell

has a potential of +0.244 V against the SHE and proceeds according to Equation 1.20.

𝐇𝐠𝟐𝐂𝐥𝟐(𝒔) + 𝟐𝐞− 𝟐𝐇𝐠(𝒍) + 𝟐𝐂𝐥(𝒂𝒒)− Equation 1.20

Craig Alexander

47

The most practical reference electrode for use when obtaining potentiometric

measurements is a double-junction Ag/AgCl reference electrode. In many cases, a

simple single-junction reference is unsuitable for use, particularly when Cl- and/or K

+

ions are present in the sample solution. A double-junction reference electrode consists

of two compartments, one inside the other. The inner compartment contains the

Ag/AgCl electrode and is filled with a suitable electrolyte, such as saturated KCl. The

outer compartment is filled with an electrolyte that contains ions different from those

present in the sample solution, ensuring that any leakage from the outer compartment

does not affect the measurement. Lithium acetate is seen as a ‘universal’ outer

compartment filling solution within double-junction reference electrodes and is suitable

for use for the measurement of most ions. A schematic diagram of a double-junction

reference electrode is shown in Figure 1.5.

Figure 1.5: Schematic representation of a double-junction Ag/AgCl reference electrode with 1 M

LiCH3COO as outer filling solution32

Craig Alexander

48

1.4 Ion-Selective Electrodes

ISEs are potentiometric sensors which allow the selective determination of the activity,

and hence concentration, of one ionic species in the presence of others. The selective

ionic recognition is achieved using a membrane as the sensor surface, and the chemical

equilibrium which takes place across the membrane produces an electrode potential that

can be related to the concentration of the target ion by the Nernst equation33

.

1.4.1 Selective Polymeric Membranes

Various membrane materials have been used in potentiometric sensors to afford a

selective response, including modified glass, as used in pH-sensitive electrodes, and

crystalline materials such as LaF3 doped with EuF2, used within fluoride-selective

electrodes. Whilst both of these have their uses, they are limited in terms of the number

of ions that can be selectively determined using either of these methods34

. The

development of ISEs that incorporate a polymeric membrane containing an immobilised

ion-binding receptor has led to a vast amount of research in this field. In the literature

terms such as ion-exchanger or ion-carrier are used if the receptor is electrically charged

and neutral receptors are termed ionophores27

, for the purpose of this thesis, however,

‘ionophore’ will be used to refer to the receptor regardless of its charge.

Although others have been reported35,36,37

, the most widely used polymer material for

the preparation of ion-selective membranes is polyvinyl chloride (PVC)38

. The

selective-membrane ‘cocktail’ is prepared by dissolving PVC, along with an appropriate

plasticiser and the desired ionophore, into a suitable solvent, such as tetrahydrofuran

(THF)39

. When a neutral ionophore is used as the selective receptor, it is also necessary

to add a lipophilic ionic additive with a charge opposite to that of the target analyte to

Craig Alexander

49

prevent co-extraction of the counter-ion into the membrane40

. For cation-selective

membranes, alkali salts of tetraphenylborate derivatives (Figure 1.6A) are commonly

used as the ionic additive, whereas anion-selective membranes will generally include a

quaternary ammonium salt additive38

(Figure 1.6B).

A typical PVC membrane composition will consist of approximately 30–33% w/w

PVC, 60–66% w/w plasticiser and 1–10 % w/w ionophore41

with the ionic additive (if

required) present in a molar ratio of around 1:2, with respect to the ionophore34

.

Figure 1.6: (A) Potassium tetrakis-[3,5,-bis-(trifluoromethyl)phenyl]borate – used as an ionic

additive in cation-selective membranes, (B) tridodecylmethyl ammonium chloride (TDMACl) –

used as an ionic additive in anion-selective membranes27

The role of the plasticiser within an ion-selective membrane is not only to provide

mechanical plasticity to the membrane, but also to function as a solvent for the

membrane components; thus ensuring mobility of the ionophore within the membrane38

.

Plasticised polymers are essentially viscous liquids; and are commonly referred to in the

literature as liquid membranes. A plasticiser must exhibit good lipophilicity and be

compatible with the other membrane materials and the chosen solvent42

. The most

common plasticisers used in ISEs include phthalates, sebacates and

ortho-nitrophenyloctyl ether (NPOE)43

. The structures of some of these are shown in

Figure 1.7. Choosing the correct plasticiser for use within a particular ion-selective

membrane has been shown to improve both the selectivity and the detection limits

of an ISE44

.

Craig Alexander

50

Figure 1.7: Structures of three commonly-used plasticisers within ISEs (A) dioctyl sebacate (DOS),

(B) o-nitrophenyloctyl ether (NPOE), (C) dioctyl phthalate (DOP). Adapted from Buhlmann et al.34

1.4.2 Selectivity of ISEs

The selectivity of any chemical sensor is arguably its most important analytical

characteristic, as understanding how the sensor performs in the presence of other

species helps to ascertain whether the device is suitable for use in the intended

target sample45

.

An ideal ISE would respond only to the target ion within a solution that could contain

any number of potential interfering ions. In reality, this situation is highly unlikely,

especially where the sample matrix contains species with similar characteristics to the

target ion, such as size and charge, and hence the term ion-selective rather than

‘ion-specific’ is quoted. The potentiometric selectivity coefficient (kA,Bpot

) is used to

quantitatively evaluate the contribution that interfering ions have to the measured

potential46

. An additional term is inserted into the Nernst equation to account for the

effect of an interfering ion, to give the Nicolskii-Eisenman (N-E) equation, shown in

Equation 1.21, where aA and aB are the ionic activities and nA and nB are the charge

numbers of the target and interfering ions, respectively.

Craig Alexander

51

𝐄 = 𝐄° +

𝟐. 𝟑𝟎𝟑𝐑𝐓

𝐧𝐅 𝐥𝐨𝐠(𝒂𝐀 + 𝐤𝐀,𝐁

𝐩𝐨𝐭(𝒂𝐁)𝐧𝐀 𝐧𝐁⁄ ) Equation 1.21

The smaller the value of kA,Bpot

, the greater the ability of the ISE to distinguish the target

ion from the interfering ion. Its logarithm is generally taken to convert it to a more

convenient number. Like the Nernst equation, the N-E equation is also based on

activities of the ions, rather than the molar concentration. An ionic strength adjustment

buffer (ISAB) can be added to each sample solution used to adjust the total ionic

strength to a similar value, thus making the activity coefficient equal for each target ion

concentration33

. Therefore aA=CA and aB=CB, where CA and CB are the molar

concentrations of the target and interfering ions, respectively.

Several methods have been described for the experimental determination of kA,Bpot

. The

International Union of Pure and Applied Chemistry (IUPAC) recommended guidelines

outlining these methods were first published by Guilbault et al. in 197647

. These were

then revised in 1995 by Umezawa et al. to include the limitations of the N-E equation48

.

These procedures can generally be placed into two categories: mixed solution methods

(MSMs), where the electrode is placed into a test solution containing both primary and

interfering ions, and separate solution methods (SSMs), where the effect of CA and CB

are investigated independently.

Two common MSMs are the fixed interference method (FIM) and the fixed primary ion

method (FPM). The FIM involves measuring the potential of a solution containing fixed

CB with varying CA, whereas the FPM measures varying CB at fixed CA. The obtained

potential values are plotted against the log of the concentration of the target ion in the

case of the FIM, or the interfering ion for the FPM. The selectivity coefficient is

calculated by extrapolating the linear portion of the resulting plot to the intersection

Craig Alexander

52

which gives the value (CA for the FIM, CB for the FPM) to be entered into

Equation 1.22 48,49

; the FIM is illustrated graphically in Figure 1.8.

𝐤𝐀,𝐁

𝐩𝐨𝐭=

𝐂𝐀

(𝐂B)𝐧𝐀 𝐧𝐁⁄ Equation 1.22

Figure 1.8: Example calibration curve for calculating the selectivity coefficient using the FIM,

modified from Mikhelson et al.32

Another MSM is referred to as the two solution method (TSM). The potential of one

solution containing only the ion of interest at a known concentration is measured to give

EA, and the potential of a separate solution containing both the target and interfering

ions is also measured to give EA+B. The value of the potential difference between these,

ΔE = EA+B – EA, is entered into Equation 1.23 to calculate kA,Bpot

.

𝐤𝐀,𝐁𝐩𝐨𝐭

= 𝐂𝐀 (𝐞(𝚫𝐄𝐧𝐀𝐅 𝐑𝐓⁄ ) − 𝟏) (𝐂𝐁)⁄𝐧𝐀 𝐧𝐁⁄

Equation 1.23

The main benefit of using a MSM to calculate kA,Bpot

is that it simulates the actual

conditions in which the sensor is to be used, which gives a direct indication of the

Craig Alexander

53

performance of the sensor in the test sample, and in the case of the FIM and FPM there

is no risk of carry over contamination between measurements50

. However, there are also

SSMs that are accredited by IUPAC that are often the preferred method for determining

kA,Bpot

as they are generally simpler and allow the user to understand the behaviour of the

electrode when the target ion is not present.

The first SSM involves measuring one solution containing the target ion (A) at a known

concentration, and a separate solution containing the interfering ion (B) at the same

concentration as that of the target ion. The recorded potentials, EA and EB are entered

into Equation 1.24 to obtain kA,Bpot

.

𝐥𝐨𝐠 𝐤𝐀,𝐁

𝐩𝐨𝐭=

(𝐄𝐁 − 𝐄𝐀)

𝟐. 𝟑𝟎𝟑 𝐑𝐓 𝐧𝐀𝐅⁄+ (𝟏 − 𝐧𝐀 𝐧𝐁⁄ )𝐥𝐨𝐠 𝐂𝐀 Equation 1.24

A second SSM involves producing calibration plots of both the target and non-target

species. The concentrations of each ion that produce the same measured electrode

potential are entered into Equation 1.22 to obtain a value for the selectivity coefficient.

The limitations of using N-E equation-based methods for selectivity determination of

ISEs were critically evaluated after several papers reported discrepancies between

obtained kA,Bpot

values when different experimental conditions were used48

. The N-E

equation requires the ISE to exhibit a Nernstian response to both the target and

interfering ions, whilst it is also not particularly well suited when comparing ions of

different charge51

. As such, more recent research has focused around exploring methods

for obtaining selectivity information from ISEs that are not based on the N-E equation.

A matched potential method (MPM) for determining kA,Bpot

independent of the N-E

equation was first described by Gadzekpo et al52

. In this method the selectivity

Craig Alexander

54

coefficient is defined as the ratio of the activities (concentrations) of each species that

give rise to the same change in potential under the same conditions. The potential of a

background solution containing a known concentration of the primary ion (CA) is

measured followed by the addition of a further known concentration of the primary ion

(CA’); the resulting change in potential is then recorded. A solution of the interfering ion

(CB) is added to an identical background solution until the same change in the measured

potential is observed. kA,Bpot

is calculated according to Equation 1.25.

𝐤𝐀,𝐁

𝐩𝐨𝐭=

(𝐂𝐀 , − 𝐂𝐀)

𝐜𝐁 Equation 1.25

Despite numerous publications spanning almost 40 years addressing the topic of

potentiometric selectivity coefficient determination, it appears that there has yet to be a

fully definitive method that can be used in all cases. The revised IUPAC guidelines state

that methods based on the N-E equation are acceptable when both the target and

interfering species are of equal charge and produce Nernstian responses, whereas the

MPM is recommended if either or both of these criteria are not fulfilled48

. Attempts at

producing alternative methods have continued since the production of these

recommendations50,53,54

. When developing ion-selective sensors, the analyst must

choose a method that is the most appropriate for determining whether the sensor

provides adequate selectivity against the likely interfering ions found in the test sample

so it can be used with confidence for its intended purpose.

Craig Alexander

55

1.4.3 Polymer Membrane Electrodes

The conventional design of an ISE is often referred to as a polymer membrane electrode

(PME). In this design, an internal Ag/AgCl reference electrode is immersed into an

aqueous electrolyte, referred to as the inner filling solution, which is separated from the

sample by the ion-selective membrane55

. To produce a polymeric membrane that is

suitable for such a construction, the plasticised PVC-based ‘cocktail’ described in

section 1.4.1 is cast onto a flat surface, such as a glass petri-dish, to allow evaporation

of the solvent56

. The membrane that is produced is robust and dry to touch, but is

effectively a liquid due to the excess of added plasticiser. Once dry, an

appropriately-sized disc can simply be cut out and attached to one end of an inert

electrode body. The inner filling solution will generally contain the ion of interest at a

fixed dilute concentration, along with a Cl- salt to maintain a stable potential

39. This

sensing electrode and the chosen external reference electrode, as described in section

1.3.1.2, are connected to a suitable voltmeter and submerged into the test solution. A

diagram of the typical experimental configuration is shown in Figure 1.9.

Figure 1.9: Schematic diagram of a typical PME set-up, showing the cell potential of an ISE being

measured against a SCE reference57

Craig Alexander

56

1.4.4 Miniaturisation of ISEs

Due to aspects of its design, such as the requirement for an inner filling solution, the

standard PME is limited in terms of further miniaturisation. Miniaturisation of ISEs is

seen as beneficial where the intended use of the sensor is in a flow cell device or

micro-total analysis system (µ-TAS) for single or multi-ion sensing58

. Attempts at

miniaturisation have mainly been focused around replacing the liquid inner filling

solution/membrane interface with a solid-contact.

1.4.4.1. Coated-Wire Electrodes

One of the first attempts at miniaturising traditional ISEs was the development of the

coated-wire electrode (CWE). CWEs are produced by depositing the selective

polymeric material directly over the surface of the indicator electrode, thus eliminating

the need for an inner filling solution. These were first described by Freiser et al59,60

. The

membrane material is usually the same composition as is used in PMEs; however the

polymer is cast onto the surface of the electrode via dip-coating of the viscous solution.

The solvent evaporates from the surface, leaving behind a thin, polymeric ion-selective

film61

.

Various conductive materials have been used as the internal wire electrode, including

platinum, copper, silver and graphite62

. Numerous publications have described PVC

CWEs for the determination of a variety of ionic species, including cations such as

copper63

, silver64

, lead65

and palladium66

, as well as anions such as nitrate67

.

As well as being much easier to miniaturise than traditional PMEs, CWEs provide a

simpler and inexpensive route to producing ISEs. Although they have been shown to

Craig Alexander

57

perform comparatively well in terms of analytical characteristics, they do have

limitations with regards to drift and reproducibility33

.

1.4.4.2. Screen-Printed ISEs

Screen-printing technology provides a manufacturing route for the mass-production of

cost-effective, highly reproducible chemical sensors68

. The fabrication technique of

screen-printing for the production of thick-film chemical microsensors will be discussed

later in section 1.5.4.2. This section will focus on some of the research that has been

published on the development of screen-printed ISEs (SP-ISEs) as a means for

producing miniaturised, all-solid-state, potentiometric ion-selective devices.

Several SP-ISEs have been described in the literature. Some of these utilise a

screen-printed transducer coupled to a traditional ion-selective membrane, whereas

others have attempted to produce fully screen-printed devices that also incorporate a

screen-printed membrane layer. For a screen-printable membrane to be produced, the

selective components of the membrane (ionophore, plasticiser, additive) must be

incorporated into a suitable support matrix as conventional ion-selective membrane

solvents are not compatible with this technique due to their volatility69

.

Meyerhoff et al. examined the use of alternative membrane materials with improved

adhesion to silicon substrates for use within planar, all-solid-state ISEs. These were a

polyurethane (PU)-based matrix and a silicone rubber-based matrix, both of which

offered greater adhesion to the substrate than PVC without compromising the

electrochemical performance70

. This research eventually led to the production of a

selective polymer membrane matrix that was suitable for screen-printing by using

alternative, high boiling-point solvents as opposed to THF69,71

. SP-ISEs for K+, Ca

2+,

Craig Alexander

58

NH4+, Na

+, pH

71, and Cl

- 69 were prepared using this method, all of which showed

Nernstian responses with high reproducibility. The main disadvantage of this method is

the slightly more complicated membrane fabrication procedure involved. THF is still

required during the preparation of the membrane ‘cocktail’ to dissolve the selective

components, then the solvent which is suitable for screen-printing is added, before the

THF is allowed to evaporate.

Screen-printing of miniaturised, potentiometric transducers followed by manual

deposition of the polymeric, ion-selective membrane have been described by Walsh et

al.72

, Zielinska et al.73

and Musa et al.74

.

Koncki et al. produced fully screen-printed ISEs with excellent response characteristics

by incorporating the selective components into a commercially-available dielectric

screen-printing paste to produce the ion-selective membrane. In this case, the use of low

temperature inks also ensured that there is no degradation of any of the membrane

materials during the printing process. SP-ISEs for NH4+, K

+ and NO3

- were developed

using this method75,76

.

As miniaturised SP-ISEs still require the use of a suitable reference electrode to

measure the potential against, screen-printing technology has been utilised to produce

miniaturised reference electrodes. Koncki et al. also developed an all-solid-state, fully

screen-printed Ag/AgCl reference electrode77

. This could easily be combined with a

SP-ISE to produce a fully screen-printed potentiometric cell for use within a particular

analysis.

As screen-printing is potentially a low-cost, mass-production fabrication technique, it is

particularly suitable when the intended use of the developed product is for the

commercial market68

. Sensor designs can be printed onto inexpensive, flexible polymer

Craig Alexander

59

substrates which allows them to be used disposably76

; the sensing element could simply

be replaced if any degradation in the response was observed. This is a particularly

attractive feature for an aquarium sensor that is to be operated by a non-specialist user.

The sensors would not require an exceptionally long working lifetime, and the effects of

biofouling would effectively be negated. Therefore, the user would not be required to

carry out any regular cleaning, maintenance or calibration of the sensing device but

simply to remove and replace the disposable sensors at regular intervals.

Other attempts at miniaturising potentiometric ISEs have included using conducting

polymers (CP)78,79

and ion-selective field effect transistors (ISFETs)80

.

1.4.5 Sol-gels

Although it is still the most commonly used material, PVC does have several drawbacks

for use within polymeric selective membranes containing an ionophore. The operational

lifetime of a PVC-based ISE is often dictated by leaching of the plasticisers and

ionophores from within the membrane matrix into the test sample, causing the sensor to

lose its selectivity81,82

. Some of the plasticisers often used in PVC membranes also have

a highly toxic effect on aquatic organisms that would render their use within an

aquarium sensor particularly unsuitable83

. As such, alternative membrane materials have

been investigated, including sol-gels.

1.4.5.1. Sol-gel Chemistry

The sol-gel process provides a simple route to preparing hybrid inorganic-organic

materials with a wide range of potential applications84

. Sol-gels are prepared through

the hydrolysis and then condensation of metal alkoxides. One of the most common

examples is the preparation of silica gels from the sol-gel polymerisation of silicon

Craig Alexander

60

alkoxides85

. The typical reaction scheme proceeds according to Equation 1.26

(hydrolysis) and Equation 1.27 (water or alcohol condensation). The reaction takes

place in a mutual solvent such as ethanol in the presence of either an acid or base as the

catalyst84

.

𝐒𝐢(𝐎𝐑)𝟒 + 𝐧𝐇𝟐𝐎 → 𝐒𝐢(𝐎𝐑)𝟒−𝐧(𝐎𝐇)𝐧 + 𝐧𝐑𝐎𝐇 Equation 1.26

𝐒𝐢(𝐎𝐑)𝟒−𝐧(𝐎𝐇)𝐧 + 𝐒𝐢(𝐎𝐑)𝟒−𝐧(𝐎𝐇)𝐧 →

𝐒𝐢(𝐎𝐑)𝟒−𝐧(𝐎𝐇)𝐧−𝟏𝐎𝐒𝐢(𝐎𝐇)𝐧−𝟏(𝐎𝐑)𝟒−𝐧 + 𝐇𝟐𝐎 𝐨𝐫 𝐑𝐎𝐇 Equation 1.27

The viscosity of the resulting solution increases gradually as the sol-gel forms creating a

rigid, porous network. The sol-gel can be fully dried through evaporation of the

remaining alcohol and water to form a xerogel, allowing the simple preparation of

glass-like thin films86

. Numerous silicon alkoxide precursors can be used to produce

silica gels with different properties, some examples are shown in Figure 1.10.

Figure 1.10: Structures of three typical silicon alkoxide sol-gel precursors (A) methyltriethoxysilane

(MTES), (B) tetraethoxysilane (TEOS), (C) diethoxydimethylsilane (DEDMS)

Craig Alexander

61

The relatively mild reaction conditions required to produce sol-gels allows for the

incorporation of selective molecules into the matrix. This can be done either through

physical entrapment, by simply mixing them with the sol prior to its gelation, or by

covalently binding the receptor to functional groups within the silicate matrix to prevent

leaching into the sample86

. This has resulted in research being carried out into the use of

sol-gels for several chemical analysis applications, including as ion-selective

membranes within chemical sensors.

1.4.5.2. Sol-gel Ion-Selective Membranes

Kimura et al. first described utilising a sol-gel as an ion-selective membrane, by

incorporating the respective ionophores into a sol-gel glass based on a 3:1 ratio of the

precursors DEDMS and TEOS, for use within ISFETs for potassium and sodium

monitoring. Initially, just TEOS was used but this was found to produce a brittle

membrane; however, an increased amount of DEDMS in the mixture afforded a more

stable membrane which showed a highly selective response to both cations87-89

. These

membranes were further developed by covalently attaching the receptor molecules

within the sol-gel to improve the lifetime of a sensor90

. A similar membrane

composition was shown to be suitable for use within a conventional PME when an

appropriate support at the tip of a commercial ISE body was employed91

. Kimura et al.

have since used similar sol-gel compositions to produce chloride92

and calcium93

ISFETs, and modified the surface of a conventional pH glass electrode with a selective

sol-gel for Na+ and Cl

- sensing

94. A different research group employed a

DEDMS/TEOS-based membrane to produce a sulfate ISFET95

. Mixtures of TEOS and

DEDMS have become the most popular precursors for producing ion-selective sol-gel

membranes. This composition has since been used to prepare ISEs for copper96

, lead97

,

Craig Alexander

62

vanadyl98

, strontium99

and lanthanum100

ions where the viscous sol-gel solution was

coated over the surface of a graphite electrode.

Santos et al. tested three different sol-gel prescursors: MTES, TEOS and

tetramethoxysilane (TMOS), for their suitability as ion-selective membrane materials. It

was found that TEOS and TMOS produced brittle membranes, however MTES, when

mixed with a small quantity of polyethylene glycol (PEG) 6000, afforded a suitable

membrane that could be simply dip-coated onto the surface of a graphite electrode101

.

This sol-gel membrane was successfully employed for use within valproate-101

and

ampicillinate-102

selective electrodes for pharmaceutical applications.

Alternative sol-gel ion-selective membranes have been prepared by using

methacryloxypropyltrimethoxysilane (MAPTMS) and TMOS with immobilised

crown-ether ionophores103

, and by reacting either 1,4-butanediol or ethylene glycol with

(3-isocyanatopropyl)triethoxysilane to produce a sol-gel that has been used within

chloride-104, 105

, carbonate-106

and nitrite-107

selective electrodes. These membranes were

cast onto a flat surface to produce a membrane suitable for use within PMEs.

Craig Alexander

63

1.4.6 ISEs for Aquarium-Significant Analytes

A vast amount of research has been carried out over the past several decades into

producing ISEs with optimised performance characteristics for a wide range of ions in

various analytical situations. This includes numerous publications discussing sensors

utilising ionophore-doped polymeric membranes for the determination of the four

previously discussed key ionic analytes in freshwater aquaria. The purpose of this

section is to identify some of the ionophores for these four ions that have been quoted in

the literature, and hence could be used in the development of an ionophore-based

sensing device for use within freshwater aquaria.

1.4.6.1. pH

Arguably the most established and widely-used chemical sensor is the glass pH

electrode. Due to several reasons, such as its fragility and unsuitability for

miniaturisation, research has been carried out into producing pH sensors based on

polymeric membranes containing H+-selective ionophores

108. Examples of ionophores

for pH determination include, amongst others; tridodecylamine109

,

methyldioctadecylamine110

dioctylaniline111

, azobenzene derivatives112

, phenoxazine

derivatives113, 114

, alkyldibenzylamines115

, calix[4]arenes108, 116, 117

,

tetrabenzylalkylenediamines118

and metalloporphyrins119

. Table 1.4 provides a summary

of some of the key response characteristics from some of these pH ISEs.

Craig Alexander

64

log

𝐤𝐇

+,𝐂

𝐚𝟐

+𝐩

𝐨𝐭

-11.1

(FIM

)

-10.6

(FIM

)

-11.7

(FIM

)

-11.2

(FIM

)

-11.3

(FIM

)

Not

stat

ed

-11.1

(FIM

)

log

𝐤𝐇

+,𝐍

𝐚+

𝐩𝐨

𝐭

-10.4

(FIM

)

-10.3

(FIM

)

-12.3

(FIM

)

-10.9

(FIM

)

-9.8

(FIM

)

-10.3

(FIM

)

-11.1

(FIM

)

log

𝐤

𝐇+

,𝐊+

𝐩𝐨

𝐭

-9.8

(FIM

)

-10.0

(FIM

)

-10.8

(FIM

)

-10.5

(FIM

)

-9.9

(FIM

)

-8.3

(FIM

)

-10.4

(FIM

)

Res

po

nse

tim

e (s

)

0.4

0.5

0.8

< 1

0

50–60

15

50–60

Slo

pe

(mV

/pH

)

57.8

58.4

57.4

58.2

56.5

54.2

57.3

pH

ran

ge

4.5

–11.0

3.0

–11.0

1.7

–13.2

4.0

–12.0

2.0

–10.0

2.0

–11.0

1.5

–9.0

Ele

ctro

de

typ

e PM

E

PM

E

PM

E

PM

E

PM

E

PM

E

PM

E

Mem

bra

ne

ma

teri

al

PV

C

PV

C

PV

C

PV

C

PV

C

PV

C

PV

C

Ion

op

ho

re

Tri

dod

ecyla

min

e109

Met

hy

ldio

ctad

ecyla

min

e110

4,4

’-bis

[(N

,N-

dio

cty

lam

ino

)met

hyl]

azob

enze

ne

112

9-(

die

thyla

min

o)-

5-

oct

adec

ano

yli

min

o-5

H-

ben

zo[a

]ph

eno

xaz

ine

(ET

H 5

29

4)1

14

Oct

yld

iben

zyla

min

e115

p-t

ert-

buty

lcal

ix[4

]are

ne-

ox

acro

wn

-4108

N,N,N’,N’-

tetr

aben

zylm

ethy

len

edia

min

e118

Table 1.4: Summary of some of the performance characteristics of H+-selective ionophores used

within pH-ISEs that have been described in the literature

Craig Alexander

65

1.4.6.2. Ammonia/Ammonium

The biological and environmental importance of dissolved ammonia in various samples

has led to significant research being carried out into its real-time determination. Air-gap

electrodes for the direct analysis of dissolved NH3 have been developed, which involve

measuring the change in pH of an internal electrolyte caused by the diffusion of NH3

through a gas-permeable membrane120

, as have ionophore-based sensors for

NH4+ sensing.

The natural antibiotic nonactin is one of the earliest examples of a neutral ionophore for

use within ISEs, and provides an excellent response to ammonium121

. Although very

useful for NH4+ determination, nonactin has shown poor selectivity, particularly against

interfering K+ and Na

+ ions

122. As such, numerous publications have attempted to

produce alternative synthetic ionophores with improved selectivity; these include

19-crown-6 derivatives123, 124

, thiazole-containing benzo-crown ethers125

,

trisphenoxy-2,4,6-triethylbenzenes126

, THF-containing crown ethers127

and a

15-crown-5-functionalised carbosilane dendrimer128

. Some of the key performance

characteristics of these ionophores are summarised in Table 1.5.

Craig Alexander

66

log

𝐤𝐍

𝐇𝟒

+,𝐂

𝐚𝟐

+𝐩

𝐨𝐭

-2.6

2

(FIM

)

~ -

4.5

(SS

M)

-2.6

(SS

M)

-5.2

(MP

M)

~ -

2.5

(SS

M)

-2.7

3

(FIM

)

log

𝐤𝐍

𝐇𝟒

+,𝐌

𝐠𝟐

+𝐩

𝐨𝐭

-4.2

(FIM

)

~ -

3.8

(SS

M)

-4.5

(SS

M)

-4.7

(MP

M)

~ -

1.9

(SS

M)

-2.9

(FIM

)

log

𝐤𝐍

𝐇𝟒

+,𝐍

𝐚+

𝐩𝐨

𝐭

-2.1

4

(FIM

)

-3.5

2

(SS

M)

-3.7

(SS

M)

-2.8

(MP

M)

~ -

0.8

(SS

M)

-2.8

5

(FIM

)

log

𝐤𝐍

𝐇𝟒

+,𝐊

+𝐩

𝐨𝐭

-0.3

8

(FIM

-1.0

0

(SS

M)

-1.3

(SS

M)

-1.0

(MP

M)

-1.8

4

(SS

M)

-2.8

(FIM

)

Res

po

nse

tim

e (s

)

0.2

Not

stat

ed

Not

stat

ed

Not

stat

ed

30

6

Slo

pe

(mV

/dec

)

57.0

58.1

59.4

55.1

53.9

53.3

LO

D

(mo

l d

m-3

)

5 x

10

-6

5 x

10

-6

3 x

10

-6

2.5

x 1

0-6

1.5

8 x

10

-5

3.9

x 1

0-6

Ele

ctro

de

typ

e PM

E

PM

E

PM

E

PM

E

PM

E

PM

E

Mem

bra

ne

ma

teri

al

PV

C

PV

C

PV

C

PV

C

PV

C

PV

C

Ion

op

ho

re

Non

acti

n1

21

TD

19

C6

123

TD

B18

C6

125

1,3

,5-

Tri

s(b

enzy

lox

yp

hen

ox

ym

ethy

l)

-2,4

,6,-

trie

thylb

enze

ne1

26

1,4

,6,9

,11

,14

,16

,19

-

tetr

aoxo

cycl

oei

cosa

ne

127

15

-cro

wn

-5-f

un

ctio

nal

ised

carb

osi

lan

e d

end

rim

er128

Table 1.5: Summary of some of the performance characteristics of NH4+-selective ionophores

Craig Alexander

67

1.4.6.3. Nitrite

The development of receptors for use within ISEs has generally been more problematic

for anions than for cations. This is due to several factors, for example, anions are

commonly larger than cations, have more geometrical forms and are more susceptible to

changes in pH129

. When using a hydrophobic polymeric membrane as the recognition

element, it is much easier to sense some anions than others due to their relative

lipophilicity, i.e. more lypophilic anions will diffuse into the membrane phase more

readily than less liphophilic anions. The selectivity of anion-selective electrodes could

therefore be predicted according to the Hofmeister series, some of which is shown in

Figure 1.11.

𝐂𝐥𝐎𝟒− > 𝐈− > 𝐒𝐂𝐍− > 𝐍𝐎𝟑

− > 𝐂𝐥𝐎𝟑− > 𝐁𝐫− > 𝐍𝐎𝟐

− > 𝐂𝐥−

Figure 1.11: Selection of the Hofmeister series showing the order of anion hydrophobicity130

Research into producing anion-selective electrodes has therefore focused around

producing anti-Hofmeister responses, by producing ionophores that shift the target

anion further up the series, shown above, whilst discriminating between anions with

similar characteristics.

Several different ionophores with excellent nitrite selectivity have been reported. These

have mostly been cobalt complexes, including derivatives of Vitamin B12131-133

,

tetraphenylporphyrinato cobalt (III) acetate134

, Co(II)-salen135

and Co(II)-salophen136

.

Alternative nitrite ionophores have been described utilising indium porphyrins130

,

uranyl salophen derivatives137

and rhodium porphyrin and salophen complexes138

. A

summary of the performance characteristics of some of these ionophores is provided in

Table 1.6.

Craig Alexander

68

log

𝐤𝐍

𝐎𝟐

−,𝐂

𝐥−𝐩

𝐨𝐭

-4.6

(FIM

)

-3.9

-3.6

(MP

M)

-2.1

(SS

M)

Not

stat

ed

-4.4

6

(MP

M)

Not

stat

ed

-2.0

log

𝐤𝐍

𝐎𝟐

−,𝐍

𝐎𝟑−

𝐩𝐨

𝐭

-4.2

(FIM

-4.6

-4.3

(MP

M)

-2.9

(SS

M)

-2.4

(FIM

)

-4.0

(MP

M)

-3.4

1

(MP

M)

-2.1

Res

po

nse

tim

e (s

)

3.9

15

30

Not

stat

ed

Not

stat

ed

10

10

Not

stat

ed

Slo

pe

(mV

/dec

)

-57.3

-60.3

-60.3

-51.0

-56.2

-58.2

-59.8

-53.0

LO

D

(mo

l d

m-3

)

2.5

x 1

0-5

2 x

10

-8

8 x

10

-7

5 x

10

-6

Not

stat

ed

5 x

10

-7

8 x

10

-7

Not

stat

ed

Ele

ctro

de

typ

e PM

E

CW

E

(gra

phit

e)

PM

E

PM

E

PM

E

PM

E

PM

E

PM

E

Mem

bra

ne

ma

teri

al

PV

C

PV

C

PV

C

PV

C

PV

C

PV

C

Sol-

gel

Ion

op

ho

re

Aqu

acy

ano

cob

alt(

III)

-

hep

ta(2

-ph

enyle

thyl)

-

coby

rin

ate1

32

Tet

rap

hen

ylp

orp

hy

rin

ato

cob

alt(

III)

ace

tate

(p-i

sop

rop

yl

der

ivat

ive)

134

Ch

loro

(2-n

itro

-5,1

0,1

5,2

0-

tetr

aph

enylp

orp

hy

rin

ato

)

indiu

m130

Ura

ny

l sa

lop

hen

(4-n

itro

der

ivat

ive)

137

Co

(II)

-sal

en135

Co

(II)

-sal

op

hen

136

Ch

loro

-(5

,10

,15

,20

-

tetr

aph

enylp

orp

hy

rin

ato

)

cob

alt

(III

)107

Table 1.6: Summary of some of the performance characteristics of NO2--selective ionophores

Craig Alexander

69

1.4.6.4. Nitrate

The accurate determination of nitrate is very important in a number of biological,

toxicological and environmental situations139

. As such, a vast number of publications

spanning several decades appear in the literature relating to nitrate sensor development.

This includes numerous attempts at producing selective nitrate ionophores.

Some examples of nitrate ionophores that have been used within ISEs include;

quaternary phosphonium salts140

, quaternary ammonium salts141-143

, a tetra-coordinate

nickel(II) complex144

, bis(2-hydroxyanil)acetylacetone lead(II)145

, a tris(2-

aminoethyl)amine triamide derivative146

, bis(2-hydroxyacetophenone)ethylenediimine

vanadyl (IV)147

, meso-tetrakis[(2-arylphenylurea)-phenyl]porphyrins148

, a tetramethyl

cyclotetra-decanato-nickel(II) complex67

, [3.3.3.3]oxazane149

, Zn(II) complexes150

,

urea-calix[4]arenes151

and a cyclic bis-thiourea152

. The response characteristics of a

selection of nitrate ionophores can be found in Table 1.7.

Craig Alexander

70

log

𝐤𝐍

𝐎𝟑

−,𝐂

𝐥−𝐩

𝐨𝐭

-1.7

(FIM

)

-2.2

(FIM

)

-2.0

(FIM

)

-1.9

(SS

M)

-3.6

(FIM

)

-4.3

(FIM

)

-3.5

(FIM

)

log

𝐤𝐍

𝐎𝟑

−,𝐍

𝐎𝟐−

𝐩𝐨

𝐭

-2.9

(FIM

-2.0

(FIM

)

-1.8

(FIM

)

-1.6

(SS

M)

Not

stat

ed

-4.5

(FIM

)

-1.8

(FIM

)

Res

po

nse

tim

e (s

)

25

10

10

10

25

15

70

Slo

pe

(mV

/dec

)

-51.9

-59.6

-58.8

-54.7

-58.5

-57.8

-47.8

LO

D

(mo

l d

m-3

)

1 x

10

-5

2.5

x 1

0-6

1 x

10

-5

2.3

x 1

0-6

1 x

10

-6

5 x

10

-6

5.6

x 1

0-6

Ele

ctro

de

typ

e ISF

ET

PM

E

PM

E

PM

E

PM

E

CW

E

(pla

tinum

)

PM

E

Mem

bra

ne

ma

teri

al

PV

C

PV

C

PV

C

PV

C

PV

C

PV

C

PV

C

Ion

op

ho

re

Tet

rad

od

ecyla

mm

on

ium

nit

rate

142

5,7

,12

,14

-tet

ram

ethy

l-1

,4,8

,11

-

tetr

aaza

cycl

ote

tra-

dec

a-4

,6,1

1,1

3-

tetr

aen

e n

ick

el(I

I)144

Bis

(2-h

yd

rox

anil

)ace

tyla

ceto

ne

lead

(II)

145

Tri

s[2

-(4

-ter

t-

bu

tylb

enzo

yl)

amin

oet

hyl]

amin

e146

Bis

(2-

hy

roxy

acet

oph

eno

ne)

ethy

len

edii

min

e

van

adyl

(IV

)147

(6,8

,15

,17

-tet

ram

ethy

l-

7H

,16

H,5

,9,1

4,1

8-t

etra

azid

ob

enzo

[b,i

]-

cycl

ote

trad

ecan

ato

-(-2

)K4-

N,N

’,N

’’,N

’’’)

nic

kel

(II)

67

(9,1

1,2

0,2

2-

tetr

ahyd

rote

trab

enzo

[d,f

,k,m

][1

,3,8

,10

]t

etra

azac

ycl

ote

trad

ecin

e-10

-21

-

dit

hio

ne1

52

Table 1.7: Summary of some of the performance characteristics of NO3--selective ionophores

Craig Alexander

71

A review of the literature shows that a multitude of ionophores for the four previously

discussed ions have been synthesised and tested for their possible use within

potentiometric ISEs. Several of these are now available to purchase commercially,

along with relevant polymers, plasticisers, ionic additives and solvents153

. This allows

ionophore-based sensors to be produced without the requirement for specialist

chemical synthesis.

One of the major downfalls of potentiometric sensors is the requirement of a suitable

external reference electrode for them to function. Miniaturised reference electrodes have

been developed77

that can be used to produce a stable potential to measure against

whilst being simple to integrate within an appropriately-sized device for multi-analyte

sensing. However, if the reference electrode was to fail, this would negate the response

from each of the selective indicator electrodes. Reference electrodes are also susceptible

to potential drift over time which could lead to a false response. For the purpose of

home aquarium monitoring, it would be more beneficial to produce a sensor array that

measured the target analyte concentration directly. One method that would allow this is

to combine the recognition element of the sensor with an impedimetric transducer.

Craig Alexander

72

1.5 Impedimetric Interdigitated Electrode Chemical Sensors

The purpose of this section is to discuss the use of interdigitated electrodes (IDEs) as

impedimetric chemical sensors, including the underlying theory of operation and means

of fabrication, with regards to utilising such devices within sensors for aquatic water

quality monitoring.

1.5.1 Electrical Impedance

Electrical impedance (Z) describes the total opposition of an electrical circuit to an

alternating current (AC) when a voltage is applied. Simply, impedance is defined as the

ratio of the AC voltage (VAC) to the AC current (IAC)27

, as shown in Equation 1.28,

where j is the imaginary unit of impedance (√-1). A sinusoidal AC signal is

characterised by its amplitude (Vm or Im), frequency (f) or angular velocity (ω = 2πf) and

phase angle (ϕ). This is represented diagrammatically in Figure 1.12.

𝐙 =

𝐕𝐀𝐂

𝐈𝐀𝐂= |𝐙|𝐞𝐱𝐩(𝒋𝜙) Equation 1.28

Figure 1.12: A typical AC sine wave voltage and current. Adapted from Bănică et al.

27

Craig Alexander

73

The frequency of the sine wave has units of Hertz (Hz) and indicates the number of

cycles per second. The amplitude is often reported either as a peak-to-peak value (2Vm)

or a root mean square value (Vrms = Vm/√2).

Impedance is a complex parameter that is defined by both real and imaginary parts. For

the purpose of this thesis, conductance (G) and capacitance (C) measurements will be

taken to evaluate the sensor response. Conductance is related to impedance according to

Equation 1.29, where R is the resistance, X is the reactance, Y is the admittance

(which is the reciprocal of Z) and B is the susceptance.

𝒁 = 𝐑 + 𝒋𝐗, 𝐘 = 𝐆 + 𝒋𝐁 Equation 1.29

Conductance is measured in the SI unit of siemens (S), the reciprocal of one ohm.

Capacitance is defined as the ability of a body to store electrical charge. The SI unit of

capacitance is the farad (F). The impedance of a capacitor is dependent on the signal

frequency and is related to the reactance as shown in Equation 1.30.

𝐗 = (𝟐𝛑𝒇𝐂)−𝟏 Equation 1.30

Electrochemical impedance spectroscopy (EIS) is a powerful analytical tool that can be

used to provide information on the physical and chemical properties of a material. One

very useful application of EIS is as a transduction method for chemical sensors and

biosensors, by monitoring changes in the electrical properties that occur due to

interactions between a receptor and the target analyte154

. Electrical impedance

measurements are generally carried out by applying an AC signal between two

electrodes. A spectrum is obtained by performing these measurements at a number of

frequencies over a particular range.

Craig Alexander

74

One popular method that is often employed to obtain impedance measurements for

sensor applications is to use a pair of IDEs as the transducer.

1.5.2 Interdigitated Electrodes

IDEs consist of two co-planar electrodes with interlocking digits, forming a ‘comb-like’

structure, on an insulating substrate. A schematic representation of a typical IDE design

is shown in Figure 1.13.

Figure 1.13: Schematic diagram of a typical IDE design, reproduced from Mosaic Industries

155

An IDE is generally characterised by its geometric parameters including the width of

the individual electrode digits (W), the distance of the digit separation (S), the length of

the digits (L) and the number of digits at each electrode (N)154

. When excited with an

AC voltage, an electric field is generated between the adjacent electrode digits of

opposing charge156

. The characteristic penetration depth of the resulting electric field is

dictated by the IDE geometry. It is generally accepted that the majority of the electric

field is enclosed within a distance perpendicular to the surface of the electrodes equal to

that between the centres of adjacent digits, i.e. W + S 154

. This is illustrated in

Figure 1.14.

Craig Alexander

75

Figure 1.14: Cross-section schematic representation of the electric field penetration depth from a

typical IDE when an AC voltage is applied. Adapted from Bratov et al.157

IDEs are utilised in many different ways for a variety of purposes, such as in

telecommunications and biotechnology158

; however, it is their potential use as

transduction elements for chemical sensing applications that are of particular interest

with respect to the experimental work in this thesis.

1.5.3 IDEs for Chemical Sensing Applications

Numerous chemical sensors and biosensors have been described that utilise IDE

transducers159

. The sensing mechanism is usually achieved by placing a sensitive

membrane layer over the digits of the IDEs, or by immobilising receptors in the

interdigital spaces. EIS is used to monitor changes in the electrical properties due to

selective interactions between the receptors and the target analyte.

1.5.3.1. Ion-Selective Conductometric Microsensors

One IDE-based chemical sensor of particular relevance is the ion-selective

conductometric microsensor (ISCOM), first described by Cammann et al.156, 160

. An

ISCOM is produced by coating an IDE transducer with a thin polymeric membrane

containing an ionophore, as used in conventional ISEs. The IDE is used to measure the

bulk conductance (G) of the ion-selective membrane, where the magnitude of G relates

directly to the concentration of the target ion in the test solution156

. The main advantage

Craig Alexander

76

over a conventional ISE is that no external reference electrode is required for the

measurement. Camman et al. have described ISCOMs for the determination of K+, Ca

2+,

NH4+, Li

+ and pH

160. Jaffrezic-Renault et al. produced a NH4

+-selective conductometric

microsensor by depositing a nonactin-based PVC membrane over an IDE transducer161

,

and Port et al. produced ISCOMs for the environmental monitoring of caesium162

.

All ISCOMs that have been reported have appeared to perform comparably in term of

analytical characteristics when compared with their potentiometric counterparts. A

schematic diagram of an ISCOM is shown in Figure 1.15.

Figure 1.15: Schematic representation of an ISCOM

162

As well as not requiring an external reference electrode, IDE-based sensors have the

advantage of being easily miniaturised and mass-produced using microelectronic

fabrication techniques.

1.5.4 IDE Fabrication Techniques

The production of high-quality sensors with micrometer-sized features, such as the

digits of an IDE, requires an appropriate fabrication technique. Two common

microelectronic fabrication techniques that are often utilised for the production of

miniaturised chemical sensors are photolithographic methods and screen-printing.

Craig Alexander

77

1.5.4.1. Photolithography

Sensor fabrication using photolithography involves transferring the desired electrode

pattern into a photoresist layer by irradiating the substrate with ultraviolet (UV) light

through a suitable photomask stencil (although ‘mask-less’ methods are available as

alternatives)154

, to produce thin-film electrodes. This can be carried out either by

chemical etching or lift-off techniques. The processes of chemical etching (a) and lift-

off (b) are illustrated in Figure 1.16.

Chemical etching first involves the deposition of a thin-layer of the required metal, by

techniques such as vacuum evaporation or sputtering, onto a suitable substrate, which is

usually a glass or ceramic material. A photoresist chemical is then coated over the

surface of the metal, usually by way of spin-coating. A photomask is placed over the

substrate and exposed to UV light, which is then developed in a suitable solvent,

leaving behind the desired electrode pattern in non-UV exposed photoresist. The

uncoated metal layer is then chemically etched away and the photoresist removed, to

leave only the electrode design in metal163

.

When the lift-off technique is employed, the photoresist chemical is first coated onto the

surface of the substrate and the electrode pattern is UV-irradiated directly into this layer.

Upon development of the photoresist, the electrode pattern is formed directly on the

substrate. The desired metal is then deposited over the surface of the substrate,

following which the remaining photoresist layer can be removed, leaving behind only

the metal electrode design on the substrate163

.

Craig Alexander

78

Figure 1.16: Photolithographic preparation of thin-film electrodes using (a) chemical etching and

(b) lift-off procedures163

Photolithography provides a route to producing IDEs with excellent resolution, allowing

for very small digit widths and interdigital spacings. Photolithographically-prepared

IDEs with nano-scaled features have been described with digit widths and spacings of

several hundred nanometers164

; although, typical dimensions are in the range of

1–10 µm154

. This method for producing IDEs is not particularly cost-effective; and

requires a significant amount of specialist equipment. It is also largely limited to

inflexible substrates such as glass or ceramic, due to the harsh fabrication conditions

that are required, i.e. the use of various chemicals and high temperatures.

Craig Alexander

79

1.5.4.2. Screen-Printing

Screen-printing is a thick-film technology used for the production of planar chemical

sensors. The process of screen-printing involves forcing a thixotropic ink or paste

through a mesh screen, containing a stencil of the desired electrode design, onto a

contacting substrate with a squeegee68

. The composition of the inks can be altered to

produce conductive prints, by using inks which contain carbon-graphite or precious

metals such as gold or platinum, whilst dielectric pastes can be used to cover conductive

tracks and define sensing areas. Often one design will contain several layers, where

each screen design is printed sequentially to produce the final sensor165

. Once printed,

the inks are dried in an oven to remove the residual solvent. A schematic representation

of the screen-printing process is shown in Figure 1.17.

Figure 1.17: Schematic diagram of screen-printing for the fabrication of thick-film

electrochemical sensors68

Screen-printing provides a cost-effective route for high-throughput production of

multi-layered chemical sensors. The main costs involved are the screen production and

the purchase of the relevant inks and substrates, after which a large number of prints can

be performed at a relatively low running cost. Sensor designs can be printed onto

Craig Alexander

80

flexible, polymer substrates, providing that compatible inks are used68

. Therefore

screen-printing is a particularly attractive fabrication technique for disposable

sensing applications.

The main disadvantage of using screen-printing as the chosen fabrication technique,

particularly for the production of IDEs, is the much lower resolution that is achievable

compared with lithographic methods. Screen-printing is limited to a resolution of

around 200 µm154

, whilst the ink may expand slightly on drying which restricts designs

with features that are close together. As such, any impedimetric IDE device that is

produced via screen-printing technology would require a much thicker sensing layer to

contain the resulting electric field.

1.5.5 Membrane Coating Techniques

With the use of planar sensors which contain a membrane layer, such as ISCOMs, a

suitable coating technique must be selected to afford a homogenous and reproducible

deposition of the polymeric material, such as PVC or sol-gel, which forms the ionic

recognition layer of the sensor.

1.5.5.1. Spin-Coating

Spin-coating involves depositing a solution of a polymeric material onto the chosen

substrate and spinning at a high speed. This results in even spreading and evaporation of

the solvent to produce a uniform film166

. The thickness of the resulting film is

dependent on factors such as the viscosity of the solution and the chosen spin-speed.

Craig Alexander

81

1.5.5.2. Dip-Coating

Dip-coating generally consists of immersing the substrate into the polymer solution

followed by its vertical withdrawal, to produce a uniform film as the solvent evaporates.

The thickness of the resulting film depends on the immersion time, solution viscosity

and rate of withdrawal167

.

1.5.5.3. Drop-Coating

Both spin and dip-coating are wasteful techniques with regards to the polymer solution,

and hence the selective receptors which it contains. In spin-coating, a large quantity of

the polymeric solution is dispersed away from the target substrate; whereas with

dip-coating it is deposited onto both sides of a planar substrate, not just the desired area,

i.e. the sensing electrodes. An alternative method is to simply drop-cast the membrane

solution onto the substrate. Here the liquid simply spreads over the surface of the

substrate and the solvent evaporates to leave a thin-film. It is more difficult to control

the uniformity with the drop-casting technique but wastage of the polymer material is

minimised. Drop-coating is also limited to small areas of less than 1 cm2 27

. For coating

a large number of sensors on a commercial scale, it may also be possible for

drop-coating of the polymeric material to be automated by using small-volume liquid

robotic dispensing systems168

.

1.5.5.4. Screen-Printing

As mentioned in section 1.4.4.2, screen-printing has previously been utilised to deposit

the ion-selective membrane layer onto the conductive components of planar ISEs. This

provides a route for the manufacturing of fully screen-printed devices, and allows for a

controlled, accurate and reproducible deposition of the membrane material to produce a

Craig Alexander

82

homogeneous layer. The simplest method for this appears to be by incorporating the

selective components and plasticisers into a conventional, low-temperature curing,

dielectric screen-printing paste, as described by Koncki et al.75, 76

.

1.6 Aims and Objectives

The overall aim of this multi-disciplinary project was to develop low cost, easy to

fabricate ion-selective sensors for the determination of four aquarium-significant ionic

analytes (H+, NH4

+, NO2

- and NO3

-) with a view to producing a prototype commercial

device for use within artificial freshwater aquaria. The project aimed to create a novel,

multi-analyte sensing device by bringing together aspects of some established

electroanalytical chemistry techniques, such as ISEs, with some more recent

developments in chemical sensors, for example interdigitated impedimetric

microsensors, and micro-fabrication procedures such as screen-printing and

photolithography.

Craig Alexander

83

2 Materials and Methods

2.1 Preparation of Ion-Selective Membranes

2.1.1 Materials

The ionophores used throughout this work were: tetradodecylammonium nitrate

(TDAN), Nitrate Ionophore V (NO3V), Ammonium Ionophore I (NH4I), Hydrogen

Ionophore III (HIII), Hydrogen Ionophore V (HV) and Nitrite Ionophore I (NO2I).

These, along with high molecular weight PVC (viscosity 3.5–5.0 mPa.s), THF (reagent

grade ≥ 99.9%, containing 250 ppm butylated hydroxytoluene (BHT) as inhibitor), the

plasticiser o-nitrophenyloctyl ether (NPOE) and the ionic additives:

tridodecylmethylammonium chloride (TDMACl), sodium tetraphenylborate,

sodium tetrakis (4-fluorophenyl) borate dihydrate and potassium tetrakis

[3,5-bis(trifluoromethyl)phenyl] borate were purchased from Fluka’s Selectophore™

range for sensoric applications (Sigma Aldrich, Gillingham, UK). Bis (2-ethylhexyl)

sebacate (97%), also used as a plasticiser, was purchased from Acros Organics (Fisher

Scientific, Loughborough, UK). The ionic additive potassium tetrakis (4-chlorophenyl)

borate (98%) was purchased from Alfa Aesar (Johnson Matthey, Heysham, UK).

The sol-gel precursors, MTES (99%), DEDMS (97%) and TEOS (98%) were purchased

from Sigma Aldrich, as was PEG 600. Ethanol, used as the solvent for sol-gel

preparation, was analytical reagent grade and purchased from Fisher Scientific. 1.0 M

volumetric standard grade HCl was purchased from Sigma Aldrich, and was diluted to

0.1 M using deionised water (18.2 MΩ.cm), dispensed from an Elga Maxima system

(High Wycombe, UK), prior to use.

Dielectric screen-printing paste (Gwent Electronic Materials, Pontypool, UK) was

provided by Dr. Craig Banks’ research group at Manchester Metropolitan University.

Craig Alexander

84

2.1.2 Polyvinyl Chloride Membranes

PVC ion-selective membrane ‘cocktails’ were prepared according to previously

reported methods110, 117, 121, 132, 142, 152

. A typical PVC membrane composition contained

ionophore (1% w/w), high molecular weight PVC (31–33% w/w), plasticiser

(64–68% w/w) and, if required, a cationic or anionic additive at a molar ratio of

approximately 1:2, with respect to the ionophore (0.2–0.6% w/w). The combined mass

of the membrane components, totalling 500 mg, was dissolved into 5 ml THF to

produce a 10% w/v solution. Compositions of each component within specific

ion-selective membranes for each target ion can be found in Table 2.1. Details of the

chemical structures and IUPAC names of each of the commercial ionophores used can

be found in Appendix 10.2.

Target

Ion Ionophore Plasticiser Additive PVC

NO3-

5 mg tetradodecylammonium

nitrate

(TDAN)

330 mg bis (2-ethylhexyl)

sebacate N/A 165 mg

NO3-

5 mg Nitrate Ionophore V

(NO3V)

328 mg o-nitrophenyloctyl

ether (NPOE)

3 mg

tridodecylmethylammonium

chloride (TDMACl)

164 mg

NH4+

5 mg Ammonium Ionophore I

(NH4I) 338 mg NPOE

2 mg potassium tetrakis (4-

chlorophenyl) borate 155 mg

H+ 5 mg Hydrogen Ionophore III

(HI)

335 mg bis (2-ethylhexyl)

sebacate 2 mg sodium tetraphenylborate 158 mg

H+

5 mg Hydrogen Ionophore V

(HV) 330 mg NPOE

1 mg sodium tetrakis (4-

fluorophenyl) borate dihydrate 164 mg

NO2- 5 mg Nitrite Ionophore I

(NO2I) 328 mg NPOE

2 mg potassium tetrakis [3,5-

bis(trifluoromethyl)phenyl]

borate

165 mg

Table 2.1: Composition of each component required to produce PVC ion-selective membranes

containing 1% w/w ionophore. Each membrane was produced with a total mass of 500 mg

Craig Alexander

85

2.1.3 Sol-Gel Membranes

Two different sol-gel ion-selective membranes were prepared, based on ones previously

reported for use within CWEs97, 101

, using the precursor materials of MTES and a

combination of DEDMS and TEOS in a 3:1 ratio. The sol-gel membranes totalled 5.6

ml, and contained 20 mg ionophore along with ionic additive in the same ratio as for

PVC membranes, and were prepared according to the following general procedures.

2.1.3.1. MTES

3.6 ml MTES, 1.12 ml deionised water, 0.8 ml ethanol and 80 µl 0.1 M HCl were mixed

together for 15 minutes. 20 mg ionophore, along with additive (if required) and 40 µl of

PEG 600, to prevent the production of a brittle membrane, were added to the mixture

and stirred overnight.

2.1.3.2. DEDMS/TEOS

2 ml DEDMS, 0.72 ml TEOS, 2.24 ml ethanol and 0.64 ml 0.1 M HCl were mixed

together for 15 minutes. 20 mg ionophore, along with additive (if required) was added

and mixed for a further 10 minutes. The mixture was allowed to stand at 70 °C on a

hotplate for 1 hour prior to being left overnight under stirring.

All PVC and sol-gel membrane ‘cocktails’ were stored in a refrigerator at 4 °C and used

within 3 months of preparation.

Craig Alexander

86

2.1.4 Dielectric Screen-Printing Paste Membrane

A nitrate-selective membrane suitable for screen-printing was prepared by incorporating

ionophore (1% w/w) and plasticiser (22% w/w) into a commercial dielectric screen-

printing paste (77% w/w). 100 mg of the nitrate ionophore TDAN was weighed out and

2.2 g of NPOE was added. This was mixed for 10 minutes followed by the addition of

7.7 g of dielectric paste. NPOE was used as the plasticiser, as bis (2-ethylhexyl)

sebacate was observed to not mix homogenously with the dielectric paste.

All PVC, sol-gel and dielectric paste membrane ‘cocktails’ were also prepared without

ionophore or ionic additive present, as ‘blank’ membranes to be tested as a control.

Craig Alexander

87

2.2 Construction of Ion-Selective Electrodes

2.2.1 Materials

Internal Ag/AgCl reference electrodes for use within PMEs were prepared using

1.0 mm diameter silver wire (99.9%; Sigma Aldrich), which was cleaned using ethanol

and rinsed with deionised water prior to use. Sodium hypochlorite solution

(Sigma Aldrich) was reagent grade with 10–15% available chlorine. Electrode bodies

were fashioned from 13 mm outer diameter, 10 mm inner diameter acrylic tubing (RS

Components, Northamptonshire, UK). Lids for the ISE bodies were fashioned from lids

for volumetric flasks (size 10/19). BNC (F) to bare end leads (Pomona Electronics, WA,

USA) were purchased from Farnell (Leeds, UK).

2.2.2 Polymer Membrane Electrodes

Ion-selective membranes for use within a PME were prepared by casting the PVC

‘cocktail’ described in section 2.1.2 into a 60 mm glass petri dish. The dish was covered

with filter paper and left to stand overnight to allow the evaporation of THF from the

solution. A 12 mm disc of the resulting membrane was cut-out using a cork borer, and

affixed to one end of the electrode body using Glu & Fix all-purpose, clear, extra strong

adhesive (Bostik, Stafford, UK) and allowed to dry overnight. 10 ml of the appropriate

internal filling solution was added to the tube, a lid was attached to the exposed end,

and glued into place. Details of each specific internal filling solution are shown

in Table 2.2.

Craig Alexander

88

Target ion Internal filling solution

NO3- 0.01 M KCl + 0.01 M KNO3

NH4+

0.01 M NH4Cl

H+

pH 7.0 buffer solution* + 0.01 M KCl

Table 2.2: Details of internal filling solutions used within PMEs for each target ion.

*The composition of the pH buffer solution, used as internal filling solution for H+-ISEs, is

described in section 2.3.2.4.1

An internal Ag/AgCl reference electrode was prepared by soaking a 100 mm length of

silver wire in sodium hypochlorite for 15 minutes. One end was soldered onto a lead

connected to the central pin of a female BNC connector and placed inside the ISE body

through a 2 mm hole which had previously been drilled in the lid. The BNC connector

was attached to the measurement instrumentation using a 1 m co-axial BNC lead.

2.2.3 Coated-Wire Electrodes

A 20 mm Ag/AgCl reference electrode was prepared by soaking a silver wire in sodium

hypochlorite for 15 minutes. One end was attached to a BNC lead using a solder sleeve,

and the joint was covered in heat-shrink to leave 10 mm of the electrode exposed.

The ion-selective membrane was coated onto the Ag/AgCl electrode via dip-coating of

either the PVC or sol-gel solution. Each electrode was dipped into the viscous liquid a

total of ten times by hand, allowing several minutes between coatings. Following

dip-coating, PVC CWEs were dried in air at room temperature overnight and sol-gel

CWEs were dried in an oven at 70 °C overnight. All CWEs were rinsed with deionised

water prior to use.

All PMEs and CWEs were conditioned overnight in a 1000 ppm solution of the target

ion prior to testing, with the exception of the pH ISEs, which were conditioned

overnight in a pH 7.0 buffer solution (as described in section 2.3.2.4.1).

Craig Alexander

89

2.3 Potentiometric Measurements

2.3.1 Measurement Instrumentation

An attempt was made to build a bespoke 16-channel system for obtaining

potentiometric measurements using PHTX-22 pH/ORP preamplifiers (Omega,

Manchester, UK), controlled using an in-house written LabVIEW program (National

Instruments, Austin, TX, USA). This system is fully described in Appendix 10.3;

however, there were several irresolvable complications with the instrumentation and

software, therefore the results obtained are not included in this thesis. This system was

replaced with a commercial ELIT 4-channel ion analyser (NICO2000 Ltd., Middlesex,

UK), which was supplied with the relevant software, connected to a PC and used to

obtain all potentiometric data from the ISEs described in this thesis.

2.3.2 Sensor Testing

2.3.2.1. Materials

The salts used to produce stock solutions of target ions and interfering ions were:

potassium nitrate, potassium nitrite, potassium chloride, ammonium chloride, sodium

chloride, magnesium chloride hexahydrate and calcium chloride dehydrate. All of these

were of at least 99.0% reagent grade purity and purchased from various suppliers. Boric

acid (99.99%), citric acid monohydrate (99.5%) and disodium phosphate dihydrate

(Na2HPO4.2H2O) (>99.0%) were used to produce the pH buffer stock solution and were

purchased from Sigma Aldrich. 1.0 M volumetric standard NaOH was purchased from

Fluka and used as received. Ammonium sulfate (99.5% purity; VWR, Leicestershire,

UK) and copper (II) sulfate (>99.0% purity; Sigma Aldrich) salts were used to produce

ISABs. Double-junction silver/silver chloride reference electrodes were purchased from

Spectronic Camspec Ltd. (Leeds, UK). Lithium acetate dihydrate (reagent grade; Sigma

Craig Alexander

90

Aldrich) was used to produce the reference electrode outer filling solution at a

concentration of 0.1 M. All solutions were prepared using ultrapure deionised water.

Potentiometric experiments were conducted at ambient temperature. Temperature

measurements were recorded using an ELIT 8701 temperature probe, which was placed

into the test sample and connected to the 4-channel ion analyser. The prepared

electrodes were tested alongside commercial half-cells for each target ion, to confirm

the validity of the test sample solutions. Ammonium and nitrate-selective half-cells

were purchased from Scientific Laboratory Supplies (Nottingham, UK). A pH electrode

half-cell was purchased from VWR.

2.3.2.2. Nitrate ISEs

2.3.2.2.1. Stock Solutions

A 10,000 ppm NO3- stock solution was prepared by dissolving 16.31 g KNO3 in 1 litre

of deionised water. For selectivity experiments, a 10,000 ppm NO2- stock solution was

prepared by dissolving 18.50 g KNO2 in 1 litre of deionised water, and a 10,000 ppm

Cl- stock solution was prepared by dissolving 21.03 g KCl in 1 litre of deionised water.

Ammonium sulfate (2 M) was used as ISAB, and was prepared by dissolving 66.07 g in

250 ml deionised water.

2.3.2.2.2. Calibrations

Potentiometric nitrate ISEs were calibrated over the range 0.1–10,000 ppm NO3-.

500 ml working standard solutions of 1000, 100, 10, 1 and 0.1 ppm NO3- were prepared

from serial dilutions of the stock solution described in section 2.3.2.2.1, and 10 ml

ISAB was added to each standard solution prior to testing.

Craig Alexander

91

The electrode under test was placed into the first calibration solution, starting with

0.1 ppm NO3-, along with a double-junction Ag/AgCl reference electrode (0.1 M

lithium acetate outer solution) and the temperature sensor. The solution was stirred at a

constant rate. Once a stable measurement was obtained, the electrode was rinsed and

placed into the next calibration solution. This process was repeated until a reading had

been obtained for each of the six NO3- standard solutions. All potentiometric NO3

- ISEs

were prepared in duplicate and tested in parallel to establish reproducibility.

2.3.2.2.3. Selectivity Determination

Selectivity coefficients for nitrate against the interfering ions, chloride and nitrite, were

determined using the FIM. Selectivity data were obtained for nitrite and chloride as

these are seen as the most likely anion interferences in aquaria. The interfering anion

concentrations were fixed at 1000 ppm, and the test solutions were prepared by mixing

appropriate amounts of nitrate stock solution with interfering ion stock solution and

10 ml ISAB. This mixture was diluted to 500 ml with deionised water. The required

volumes of each stock solution are outlined in Table 2.3.

Required nitrate

concentration (ppm)

Volume of 10,000 ppm

NO3- stock solution (ml)


interfering ion solution (ml)

0.1 0.005 50

1 0.05 50

10 0.5 50

100 5 50

1000 50 50

Table 2.3: Volume of respective stock solutions required to produce 500 ml nitrate calibration

solutions with fixed 1000 ppm interfering ions, for selectivity coefficient determination

The electrode under test was first placed into the solution with the lowest nitrate

concentration, along with a double-junction Ag/AgCl reference electrode and the

temperature sensor. The solution was stirred at a constant rate. Once a stable

Craig Alexander

92

measurement was obtained, the electrode was rinsed and placed into the next solution.

This process was repeated until the ISE had been tested in each of the six NO3- standard

solutions containing the fixed interfering anion.

2.3.2.3. Ammonium ISEs


A 10,000 ppm NH4+ stock solution was prepared by dissolving 29.70 g NH4Cl in 1 litre

of deionised water. For selectivity experiments, 1 litre stock solutions of each

interfering cation (K+, Na

+, Mg

2+ and Ca

2+) were prepared at a concentration of

10,000 ppm by dissolving 19.07 g KCl, 25.42 g NaCl, 83.63 g MgCl2.6H2O and 36.70 g

CaCl2.2H2O in deionised water. Copper (II) sulphate (1 M) was used as ISAB, and was

prepared by dissolving 159.60 g in 1 litre of deionised water.


Potentiometric ammonium ISEs were calibrated over the range 0.1–10,000 ppm NH4+.

Solutions of 1000, 100, 10, 1 and 0.1 ppm NH4+ were prepared from serial dilutions of

the stock solution described in section 2.3.2.3.1. 50 ml ISAB was added to each

standard solution prior to testing.

The electrode under test was placed into the first calibration solution, starting with

0.1 ppm NH4+, along with a double-junction Ag/AgCl reference electrode (0.1 M

lithium acetate outer solution) and the temperature sensor. The solution was stirred at a

constant rate. Once a stable measurement was obtained, the electrode was rinsed and

placed into the next calibration solution. This process was repeated until the ISE had

been tested in each of the six NH4+ standard solutions. All potentiometric NH4

+ ISEs

were prepared in duplicate and tested in parallel to establish reproducibility.

Craig Alexander

93


Selectivity coefficients for NH4+ against the interfering ions potassium, sodium,

magnesium and calcium were determined using the FIM. The interfering cation

concentrations were fixed at 1000 ppm, and the test solutions were prepared by mixing

appropriate amounts of ammonium ion stock solution with interfering ion stock solution

and 50 ml ISAB. This mixture was diluted to 500 ml with deionised water. The required

volumes of each stock solution are outlined in Table 2.4.

Required ammonium

concentration (ppm)


NH4+ stock solution (ml)


interfering ion solution (ml)

0.1 0.005 50

1 0.05 50

10 0.5 50

100 5 50

1000 50 50

Table 2.4: Volume of respective stock solutions required to produce 500 ml ammonium calibration

solutions with fixed 1000 ppm interfering ions, for selectivity coefficient determination

The electrode under test was first placed into the solution with the lowest ammonium

concentration, along with a double-junction Ag/AgCl reference electrode and the

temperature sensor. The solution was stirred at a constant rate. Once a stable

measurement was obtained, the electrode was rinsed and placed into the next solution.

This process was repeated until the ISE had been tested in each of the six NH4+ standard

solutions containing the fixed interfering cation.

Craig Alexander

94

2.3.2.4. pH ISEs


A ‘universal’ pH buffer, as described by Ostling and Virtama169

, was prepared to test

the response of potentiometric pH-ISEs. A stock solution was prepared by weighing out

8.903 g disodium phosphate dihydrate, 7.00 g citric acid monohydrate and 3.54 g boric

acid into a 1 litre flask. 243 ml of 1 M NaOH was added and then filled to the mark with

deionised water. 1.0 M stock solutions of the interfering cations Na+, K

+ and Mg

2+ were

prepared for selectivity coefficient determination by dissolving the appropriate amount

of each respective chloride salt in 1 litre of deionised water.


Calibration standards of pH 2.5, 5.0, 7.0, 8.0, 9.0 and 11.5 were prepared by dispensing

50 ml of the buffer stock solution, described in section 2.3.2.4.1, into a 250 ml flask,

adding the required volume of 0.1 M HCl, as stated in Table 2.5, and filling to the mark

with deionised water.

Desired pH Required volume of 0.1 M HCl (ml)

2.5 158.125

5.0 115.450

7.0 83.775

8.0 71.750

9.0 61.200

11.5 26.875

Table 2.5: Volume of 0.1 M HCl required to add to 50 ml stock solution to produce 250 ml pH

buffer calibration solutions

Craig Alexander

95

The pH values of the resulting solutions were checked using a calibrated bench-top pH

meter (pH211 microprocessor pH meter, HANNA Instruments, Bedfordshire, UK) and

adjusted as necessary using either 0.1 M NaOH or 0.1 M HCl.

The electrode under test was placed into the first calibration buffer solution, starting

with pH 11.5, along with a double-junction Ag/AgCl reference electrode (0.1 M lithium

acetate outer solution) and the temperature sensor. Once a stable measurement was

obtained, the electrode was rinsed and placed into the next calibration solution. This

process was repeated until the ISE had been tested in each of the six buffer solutions.

All potentiometric pH ISEs were prepared in duplicate and tested in parallel to establish

reproducibility.


Selectivity coefficients for H+ against the interfering ions potassium, sodium and

magnesium were determined using the FIM. Buffer solutions containing 0.1 M of the

interfering cation were prepared in the same manner as the 250 ml calibration standards,

as shown above in Table 2.5. However, 25 ml of the respective 1.0 M interfering cation

stock solution was also added prior to filling with deionised water.

2.3.2.5. Nitrite ISEs

Due to the cost of commercial nitrite ionophores, NO2- selective electrodes were not

prepared for potentiometric testing. Previously published research investigating the

response characteristics of the commercially-available ionophore NO2I found that a

PVC membrane containing 1% w/w of the ionophore resulted in log KPot values of -3.6

against interfering NO3- and -3.7 against interfering Cl

-, using the SSM

132.

Craig Alexander

96

2.4 Fabrication of Interdigitated Electrodes

2.4.1 Lift-off Photolithography

2.4.1.1. Materials

Photolithographic patterns were produced using a SF-100 ‘mask-less’ direct projection

exposure system (Intelligent Micro Patterning LLC, St. Petersburg, FL, USA). This

system directly projects a .bmp file onto the chosen substrate without the requirement

for a conventional photomask. Megaposit SPR-220 (Rohm Haas, Philadelphia, PA,

USA) is a commercial positive photoresist that was used in the IDE fabrication process.

Its chemical composition is shown in Table 2.6.

Chemical component CAS number % concentration

Ethyl lactate 97-64-3 30.0–52.0

Anisole 100-66-3 15.0–25.0

Diazo photoactive compound - 1.00–10.0

Cresol novolak resin - 14.0–40.0

Cresol 1319-77-3 0.01–0.99

2-methyl butyl acetate 624-41-9 1.00–5.00

n-amyl acetate 628-63-7 2.00–7.00

Organic siloxane surfactant - 0.01–0.10

Table 2.6: Chemical composition of Megaposit SPR-220 positive photoresist170

Megaposit MF-26A developer solution (Rohm Haas) was used as received without any

further dilution. Its chemical composition is shown in Table 2.7.

Chemical component CAS number % concentration

Water 7732-18-5 >95.0

Tetramethylammonium hydroxide 75-59-2 2.30

Polyglycol - <1.00

Table 2.7: Chemical composition of Megaposit MF-26A developer solution171

Craig Alexander

97

A KW-4A spin-coater (Chemat Technology, CA, USA), connected to a vacuum pump,

was used to evenly coat the substrate material with photoresist. A KW-4AH hot plate

(Chemat Technology) was used to bake the substrates to cure the thin-film photoresist

layer. E-beam deposition of metal layers was carried out using an Edwards Auto500

(Edwards, Crawley, UK). Gold and titanium metal pellets were 99.99% SuperVac®

grade (Testbourne, Hampshire, UK). Sulfuric acid (S.G. 1.83, >95% purity) was

purchased from Fisher Scientific. 30% hydrogen peroxide and acetone were purchased

from Sigma Aldrich.

2.4.1.2. Sensor Design

When designing the IDEs several factors with regards to the geometric and spatial

parameters had to be kept in mind. First of all, due to the in-house equipment available

for producing the electrodes using photolithography, the electrode geometry was limited

to fairly conservative feature sizes of at least 1 pixel (~15 µm) wide. As screen-printing

was also to be used as a fabrication technique for producing IDEs, the sensors were

designed with this in mind. This was to allow direct comparison of the sensing

performance of devices produced using the two different fabrication processes. The

electrodes required a suitable number of digits to produce a large enough sensing area,

but also needed to be of a suitable size so that at least four sensors could be placed into

an appropriately-sized prototype device. Due to the constraints of the SF-100

photolithography system which was used, the electrode design was limited to a total

area of 768 x 1024 pixels (approximately 11.5 x 15.4 mm).

Taking the above factors into account, the IDE sensors were designed with 25 pairs of

digits at each electrode. The digits were 9 mm long and 60 µm wide, with an inter-digit

spacing of 60 µm, giving a sensing area footprint of around 10 mm2. 60 µm was chosen

Craig Alexander

98

as it was hoped this was a large enough feature size to be obtainable using modern

screen-printing techniques, whilst being suitably small enough to produce an electric

field that would be sensitive to an ion-selective membrane deposited on top of the

electrodes. The final design can be seen in Figure 2.1. This sensor will be referred to as

‘IDE Design 1’ throughout this thesis.

Figure 2.1: Schematic representation of IDE Design 1 – Measurements not annotated are the width

of the conductive tracks (0.5 mm) and the distance between the end of each digit and the adjacent

conductive track (0.25 mm)

2.4.1.3. Fabrication Process

IDE Design 1 was fabricated in-house using a lift-off, ‘mask-less’ photolithography

technique followed by e-beam deposition of gold metal according to the following

procedure. 76 x 26 x 1 mm glass microscope slides (VWR) were cleaned by submersing

in a ‘Piranha’ solution, prepared by mixing 3 parts concentrated sulfuric acid with 1 part

hydrogen peroxide, for 15 minutes prior to use. The slides were rinsed with deionised

water and baked on a hotplate at 110 °C to dry. Approximately 2 ml of SPR-220

positive photoresist was deposited onto the clean glass slides, which were spin-coated at

3000 rpm for 30 seconds, to produce a photoresist layer around 7.5 µm thick172

. The

Craig Alexander

99

coated slides were ‘soft baked’ at 110 °C for 90 seconds, to ensure evaporation of any

solvents from the photoresist. Once baked, the slides required a rehydration period of at

least one hour, prior to patterning of the electrode design; however they were generally

allowed to hydrate overnight at this stage.

A .bmp file of the desired electrode design was loaded into a computer connected to the

SF-100 photolithography system. After ensuring the stage was level and the design was

focused, one of the coated microscope slides was placed under the light source.

Preliminary experiments suggested that an exposure time to the SF-100 light source of

60 seconds was sufficient. A total of three designs per microscope slide were patterned

into the photoresist. Each slide was left for at least 1 hour after exposure, following

which a post-exposure bake (PEB) was performed for 90 seconds at 110 °C. After the

PEB, each of the slides were allowed to rehydrate for at least one hour prior to

developing.

Once the patterned designs were suitably hydrated, each slide was placed into the

developer solution for approximately 2 minutes, with agitation, until no more of the

exposed photoresist was observed to be leaving the surface of the microscope slide.

After developing, each slide was rinsed thoroughly with deionised water and dried with

a nitrogen gun.

Following the development stage, the slides were placed face down into the Edwards

Auto500 for electron beam deposition of gold metal. After a suitable vacuum had been

obtained, a 4 nm titanium metal adhesion layer was deposited followed by a 50 nm

layer of gold metal.

Finally, the excess photoresist was removed by sonicating the slides in acetone for

approximately 2 minutes, leaving behind the gold electrode designs. Three devices were

Craig Alexander

100

produced per glass slide; the individual sensors were separated using a diamond scribe.

A microscope image of the electrode digits is shown in Figure 2.2.

Figure 2.2: Microscope image (4x) of the digits of IDE Design 1, fabricated in-house using a

mask-less, lift-off photolithographic technique followed by e-beam deposition of gold metal

2.4.1.4. Reduced Geometry IDEs

Gold IDEs with smaller features than the ones prepared in-house were provided by

MicruX Fluidics (Oviedo, Spain). This design, shown in Figure 2.3, consisted of 15

pairs of digits at each electrode, with a length of 5 mm and a digit width and spacing of

10 µm, on a Pyrex substrate. The sensor contained a reference and auxiliary electrode,

neither of which were connected during the impedimetric experiments. A layer of SU-8

negative photoresist was placed down to define the sensing area and protect the

conductive tracks. This sensor will be referred to as ‘IDE Design 2’ throughout this

thesis.

Craig Alexander

101

Figure 2.3: IDE Design 2. WE1 and WE2 refer to the two individual electrodes which form the

IDEs, RE and AE are the reference and auxillary electrodes, respectively, which were not

connected during the experiments. The red shaded areas highlight the exposed sensing and

connection areas that were not coated with SU-8 epoxy passivation layer173

2.4.2 Screen-Printing

2.4.2.1. Materials

A first attempt at producing screen-printed IDEs was carried out in collaboration with

Dr. Barry Haggett’s research group at the University of Bedfordshire. However, no

sensors that were suitable for testing were produced from this work. The issues

experienced with these sensors will be discussed in Chapter 6.

Screen-printing inks were purchased from Gwent Electronic Materials. The electrodes

were printed using a carbon-graphite ink for fine-line printing (C2110309D5), and the

Craig Alexander

102

sensing areas were defined using a low water ingress polymer dielectric

paste (D2020823D2).

The screens were purchased from DEK (Weymouth, UK). The mesh material of each

screen was polyester, and each screen contained six separate designs with varying

geometric features (Figure 2.4 and Table 2.8), with digit widths and spacings of either

60 or 100 µm. The screen for the IDE designs had a mesh thickness of 70 µm, a mesh

angle of 45° and a mesh count of 230. The screen for the protective dielectric layer had

a mesh thickness of 77 µm, a mesh angle of 45° and a mesh count of 175. Each screen

was mounted onto the frame with a tension of 22.5 N/cm.

96% alumina tiles (AD-96), with dimensions of 101.6 x 101.6 mm, and a thickness of

0.63 mm, were used as the substrate material for the first batches of screen-printed

electrodes, and were purchased from CoorsTek (Fife, UK). The substrate was laser-

scribed to allow the individual sensors to be easily separated.

Further screen-printed electrodes were provided by Dr. Craig Banks’ research group at

Manchester Metropolitan University. These designs printed effectively and will be

referred to throughout this thesis as ‘SP IDE Design 1’. The materials used in this work

were proprietary knowledge (Kanichi Research Ltd., Liverpool, UK).

2.4.2.2. Sensor Design

All sensor designs were produced using AutoCAD 2012 (version 18.2, Autodesk Ltd,

Farnborough, UK). The screen designs of the first attempt at producing screen-printed

IDEs are shown in Figure 2.4, while the geometric parameters of each individual sensor

are summarised in Table 2.8. Each screen contains the stencil of 6 different IDEs so that

Craig Alexander

103

a range of geometries could be tested, with digit widths and inter-digit spacings of either

60 µm or 100 µm, and digit lengths of 1, 5 or 10 mm.

Figure 2.4: Schematic representation of the screens used to produce 6 different IDE designs. The

parameters of each IDE A-F are summarised in Table 2.8. Figure 2.4(a) shows the electrode

geometry layer and Figure 2.4(b) shows the dielectric paste window which defines the sensing area

Design

Number of

digits at each

electrode

Digit width

(µm)

Inter-digit

spacing (µm)

Digit length

(mm)

A 12 60 60 5

B 12 100 100 5

C 12 60 60 1

D 25 60 60 5

E 25 100 100 5

F 25 100 100 10

Table 2.8: Geometric parameters of each IDE design shown in Figure 2.4

Due to the fabrication issues experienced with the first batch of screen-printed IDEs, to

be discussed later in Chapter 6, SP IDE Design 1 was constructed with much more

conservative feature sizes than the initial attempts.

SP IDE Design 1 was produced with 20 digits at each electrode, 10 mm long with a

width of 100 µm and an inter-digit spacing of 150 µm. The screen was produced so that

Craig Alexander

104

24 identical sensors were produced on a single substrate. The final electrode design is

shown in Figure 2.5.

Figure 2.5: Schematic representation of SP IDE Design 1. Measurements not annotated are the

width of the conductive tracks (1 mm) and the distance between the end of each digit and the

adjacent conductive track (0.5 mm)

2.4.2.3. Fabrication Process

2.4.2.3.1. Attempt One

The IDEs were produced as two-layer designs consisting of carbon electrodes and a

dielectric insulating window, using a microDEK 1760RS screen-printer (DEK). Each

layer was printed onto the alumina substrate, and cured at 80 °C in a fan oven for 30

minutes to remove residual solvent from the printed inks.

2.4.2.3.2. SP IDE Design 1

SP IDE Design 1 was produced using a three-layered design and is outlined in

Figure 2.6. Each sensor consists of a carbon electrode layer, an ion-selective membrane

layer and a dielectric passivation layer. Polyester screens were used; however, the exact

information is proprietary and therefore cannot be stated. A microDEK 1760RS

Craig Alexander

105

screen-printer (DEK) was used to produce the sensors, according to the

following procedure.

The IDE geometry was defined using a carbon-graphite ink formulation (carbon

conductive ink, Gwent Electronic Materials) that was screen-printed onto a

100 x 100 mm polyester flexible film substrate (Autostat, 250 µm thickness). This layer

was cured in a fan oven at 60 °C for 30 minutes (see Figure 2.6A). A microscope image

of the electrode digits is shown in Figure 2.7.

A layer of modified dielectric paste ion-selective membrane, described previously in

section 2.1.4, was printed as a rectangular block over the digits of the electrodes to form

the sensing area. This layer was cured in a fan oven at 90 °C for 60 minutes (see Figure

2.6B).

Finally, a layer of unmodified dielectric paste (Gwent Electronic Materials Ltd) was

printed to cover the connections and define the sensing area. After curing at 60 °C for

30 minutes the screen-printed sensor was ready to use (see Figure 2.6C).

Figure 2.6: Outline of the screen-printing fabrication process of SP IDE Design 1. (A) The first

layer represents the IDE geometry, connectors and conductive tracks. (B) The modified dielectric

paste ion-selective membrane layer printed over the electrode digits (dark blue). (C) The insulating

window printed to define the sensing area and cover the conductive tracks (light blue)

Craig Alexander

106

Figure 2.7: Microscope image (4x) of the electrode digits of SP IDE Design 1

2.5 Construction of Ion-Selective Impedimetric Microsensors

2.5.1 Photolithographically-prepared IDE Sensors

2.5.1.1. IDE Design 1

Gold IDEs were first cleaned with acetone, rinsed with deionised water and dried using

a nitrogen gun prior to use.

The ion-selective membrane was deposited onto the surface of the digits of the IDEs via

spin-coating. The desired membrane ‘cocktail’ (PVC or sol-gel; 50 µl) was dispensed

and spin-coated at 2000 rpm for 30 seconds. Sensors with PVC membranes were left

overnight to allow evaporation of any residual THF, whilst sensors with sol-gel

membranes were baked in an oven at 70 °C overnight.

Craig Alexander

107

Electrical connections to the IDEs were made by attaching a 0.5 mm non-insulated

copper bootlace ferrule (MultiComp, Farnell, UK) to the conductive pads using

CircuitWorks two-part, silver-loaded epoxy adhesive (Chemtronics, Kennesaw, GA,

USA), which was allowed to cure overnight. Two wires were soldered onto a female

BNC connector, which were inserted into the two ferrules and crimped tight. The

exposed conductive connectors of the sensor were covered with two-part, fast-drying

epoxy (Devcon, FL, USA). The conductive tracks were covered with electrical

insulating tape, which was also used to define a window around the sensing area.

2.5.1.2. IDE Design 2

IDE Design 2 was cleaned, using acetone, in the same way as IDE Design 1. The sensor

was supplied with cables attached to WE1 and WE2 (see Figure 2.3), RE and AE were

not connected during the experiments.

Ion-selective membranes were deposited onto the exposed sensing area via spin-coating

or drop-coating. For both coating techniques, 1 µl of the desired membrane ‘cocktail’

was dispensed onto the sensing area, with the spin-coated membranes spun at 2000 rpm

for 30 seconds. As for IDE Design 1, sensors with PVC membranes were left overnight

to allow evaporation of any residual THF, whilst sensors with sol-gel membranes were

dried in an oven at 70 °C overnight.

2.5.2 Screen-Printed IDEs

2.5.2.1. SP IDE Design 1

Screen-printed ion-selective microsensors had a membrane layer printed onto the IDE

digits during the fabrication process (see section 2.4.2.3) and therefore required no

additional modification prior to testing. Each individual sensor to be tested was simply

Craig Alexander

108

cut away from the master substrate using scissors. Electrical connections to the IDEs

were made in the same way as for IDE Design 1.

All IDE devices were stored in pure water for at least 24 hours prior to testing, to allow

sufficient soaking of the ion-selective membrane.

2.6 Impedance Measurements

2.6.1 Measurement Instrumentation

Impedimetric measurements were acquired using an Agilent E4980A LCR meter

(Agilent Technologies, Cheshire, UK), which measures two electrical properties

simultaneously across the chosen frequency range. It was controlled using an in-house

written LabVIEW program, the front panel of which is shown in Figure 2.8. The

conductive pads of the sensors were connected to the instrument using an Agilent

16085B terminal adaptor and a 1 m insulated BNC lead.

Figure 2.8: Front panel of the LabVIEW program used to control the Agilent E4980A LCR meter

Craig Alexander

109

The LCR meter used throughout this project is generally intended for use as a

front-panel controlled instrument, which would not be particularly suitable for the

experimental work described in this thesis. The LabVIEW program allowed the user to

select which impedance parameters of the sensor were to be monitored, along with the

frequency range, the number of steps along the range and the average factor of each

measurement. A function was also added to repeat the scan after a given interval, which

was particularly useful for the purpose of this work. At the completion of each scan, the

results were outputted as a Microsoft Excel workbook file.

2.6.2 Sensor Testing

The appropriate background solution (200 ml) was added to a beaker and stirred at a

constant rate. A beaker heater connected to a proportional-integral-derivative (PID)

controller was wrapped around the vessel to hold the test sample at the required

temperature. To prevent evaporation of the test sample solution, Parafilm all-purpose

laboratory film (Bemis Flexible Packaging, WI, USA) was placed over the top of the

beaker, to which a small incision was made to allow sensor access. The sensor to be

tested was submerged into the test vessel using a clamp and retort stand. Electrical

measurements over the full frequency range (20 Hz–2 MHz), at the selected input

amplitude, were taken at 10 minute intervals using the LCR meter until a stable baseline

was obtained, generally 1 hour. Once a steady baseline was reached, aliquots of the

target ion were added at various concentration intervals over the range of interest. Five

readings were taken at each concentration, two minutes apart, prior to the addition of the

next aliquot. Figure 2.9 shows a schematic diagram of the typical experimental set-up.

Specific methodology for each individual ion sensor is discussed in section 2.7.

Craig Alexander

110

Figure 2.9: Schematic diagram of the experimental set-up for testing IDE ion sensors using the

Agilent E4980A LCR meter

2.7 Ion-Selective Impedimetric Microsensors Methodology

2.7.1 Materials

Lithium nitrate (>99%) was purchased from Acros Organics. Magnesium sulfate,

disodium hydrogen phosphate and sodium dihydrogen phosphate monohydrate all had a

purity of at least 99.0% and were purchased from Sigma Aldrich. A low-range TDS

meter (ATP Instrumentation, Leicestershire, UK) was used to estimate the background

ionic strength of the test solutions. An API “Freshwater Master Test Kit” (Mars

Fishcare, PA, USA) was used to estimate the concentrations of target ions already

present within tap and aquarium water test solutions.

2.7.2 Nitrate Sensors

2.7.2.1. Stock Solutions

Unless otherwise stated, aliquots of the KNO3 stock solution described in section

2.3.2.2.1 were added to the test vessel to adjust nitrate concentration throughout. For

experiments where an alternative counter ion was required, a 10,000 ppm NO3- stock

solution was prepared by dissolving 11.12 g LiNO3 in 1 litre of deionised water.

Agilent E4980A LCR meter

Agilent 16085B terminal adaptor

Insulated 1 m BNC lead

Test solution

IDE under test

Retort stand and clamp

Craig Alexander

111

A stock solution of 10,000 ppm magnesium sulfate was produced by dissolving 5.00 g

in 500 ml of deionised water. From this, 10, 20 and 50 ml aliquots were placed into

separate 1 litre flasks and filled with deionised water, to produce 100, 200 and 500 ppm

solutions of MgSO4, respectively.

For selectivity experiments, the stock solutions of nitrite and chloride described

previously (section 2.3.2.2.1) were used.

Unless otherwise stated, all background solutions were stored in a water bath at 25 °C,

prior to testing.

2.7.2.2. Pure Water Calibrations

The nitrate sensor under test was submerged into a beaker containing 200 ml of

deionised water, stirring at a constant rate. Once a stable baseline was obtained, serial

additions of NO3- over the concentration range 1–100 ppm were added to the test vessel.

This concentration range was chosen as it is typical of that expected within a freshwater

aquarium, and covers twice the acceptable limit. NO3-

(1 ppm) was added and five

readings taken at 2 minute intervals until the next concentration (10 ppm) was added.

This process was repeated for 20, 30, 40, 50 and 100 ppm NO3-.

The effect of the counter ion on the sensor response was observed by repeating the

above experiment; however aliquots of the LiNO3 stock solution were added to the test

vessel over the same nitrate concentration range.

The effect of increasing the temperature of the test solution on the sensor response was

also observed by storing the background deionised water and NO3- stock solution at

30 °C prior to testing. The temperature of the PID-controlled beaker heater was also

increased to 30 °C.

Craig Alexander

112

2.7.2.3. Background Effects

Magnesium sulfate of varying concentrations was used as an ionic strength buffer to test

the effect of background ion concentration on the response of the impedimetric nitrate

sensors. MgSO4 solutions of 100, 200 and 500 ppm were tested. These background

concentrations were chosen as 100–200 ppm would be close to the expected TDS value

found in a freshwater aquarium, whilst 500 ppm should provide a background ionic

strength much higher than would be generally observed. As for the calibrations in pure

water, aliquots of nitrate added to the test vessel were over the concentration range of

1–100 ppm.

As many aquarium hobbyists will not have access to purified water, and therefore will

fill aquaria with tap water, it was deemed necessary to test the sensor response by

adding known concentrations of nitrate to tap water. Prior to testing, a handheld TDS

meter was used to estimate the background ionic strength of the sample. The calibration

was performed as described above (section 2.7.2.2). Known concentrations of nitrate

were also added to a sample of water taken from a live aquarium. Prior to testing the

sample, a handheld TDS meter was used to estimate the background ionic strength, and

the initial nitrate concentration in the aquatic water was approximated by with a

standard aquarium test kit and a nitrate ISE.

2.7.2.4. Selectivity

Selectivity of the impedimetric nitrate sensors was deduced using slightly modified

versions of two different mixed solution methods that are traditionally used when

determining selectivity coefficients of potentiometric ISEs (FIM and FPM).

Craig Alexander

113

The key anions found within a freshwater aquarium that could cause a potential

interference to a nitrate-selective sensor were identified as nitrite and chloride. As

discussed earlier in section 1.1.1.2, nitrite levels in an aquarium should not exceed 0.2

ppm. Chloride is generally found in tap water at a concentration of <250 ppm.

A value of 10 ppm nitrite was chosen as the background concentration for carrying out

the FIM. The sensor was added to pure water and, once a stable baseline was observed,

10 ppm NO2- was added to the test vessel. Five measurements were taken at 2 minute

intervals, followed by additions of nitrate in the concentration range 1–100 ppm as

before. For the FPM, the sensor was added to pure water until a stable baseline was

observed, followed by the addition of 50 ppm NO3-. Five measurements were taken at

2 minute intervals, followed by the addition of nitrite at a concentration of 1, 10 and

20 ppm.

A value of 50 ppm chloride was chosen as the background concentration for carrying

out the FIM. The sensor was added to pure water and, once a stable baseline was

observed, 50 ppm Cl- was added to the test vessel. Five measurements were taken at

2 minute intervals, followed by additions of nitrate in the concentration range

1–100 ppm as before. For the FPM, the sensor was added to pure water until a stable

baseline was observed, followed by the addition of 50 ppm NO3-. Five measurements

were taken at 2 minute intervals, followed by the addition of chloride at a concentration

of 10, 50 and 100 ppm.

The response of a sensor coated in a membrane prepared without ionophore or ionic

additive present was established by carrying out a calibration over the concentration

range of 1–100 ppm NO3- in pure water, as a control.

Craig Alexander

114

2.7.2.5. Reproducibility

To determine the device-to-device reproducibility of the impedimetric nitrate sensors,

each sensor was produced in triplicate and a pure water calibration using KNO3 over the

concentration range 1–100 ppm NO3- was performed.

Ammonium Sensors


Unless otherwise stated, aliquots of the NH4Cl stock solution described in section

2.3.2.2.1 were added to the test vessel to adjust ammonium concentration during sensor

calibration experiments throughout.

The interfering ion stock solutions of Na+, K

+, Mg

2+ and Ca

2+ are described in section

2.3.2.3.1. Unless otherwise stated, prior to testing, all background solutions were stored

in a water bath at 25 °C.

2.7.2.7. Measurements in Water

Due to time constraints, it was not possible to obtain a full data set for impedimetric

ammonium sensors. The ammonium sensor under test was submerged into a beaker

containing 200 ml of the deionised water, stirring at a constant rate. Once a stable

baseline was obtained, a single addition of NH4+ at a concentration of 50 ppm was

added to the test vessel.


Selectivity of the impedimetric ammonium sensors was deduced using both the FIM.

The cation interferences that were investigated were Na+, K

+ and Mg

2+.

Craig Alexander

115

The fixed interfering cation concentration was 50 ppm. The sensor was added to 200 ml

of deionised water, and once a stable baseline was observed, 50 ppm interfering ion was

added to the test vessel. Once a stable measurement was reached, 50 ppm of ammonium

was added to the test vessel.


additive present was established by carrying out a measurement of 50 ppm NH4+ in a

deionised water baseline, as a control.

2.7.3 pH Sensors


For pH experiments, phosphate buffers of varying pH values were produced from stock

solutions of 0.1 M anhydrous disodium hydrogen phosphate (Na2HPO4) and 0.1 M

sodium dihydrogen phosphate monohydrate (NaH2PO4.H2O). Na2HPO4 (14.20 g) and

NaH2PO4.H2O (13.80 g) were dissolved in separate 1 litre flasks.

As stated in section 1.1.2, a typical aquarium would generally have a pH around neutral.

Therefore the chosen range for testing the impedimteric pH sensors was between

pH 6.0–8.5. Phosphate buffer solutions (0.01 M) were prepared by mixing the amounts

of 0.1 M stock solutions (described in section 2.7.2.1) shown in Table 2.9 and making

up to 500 ml with deionised water. The pH was confirmed using a bench top pH meter

and adjusted as necessary using drop-wise additions of the respective stock solutions.

Craig Alexander

116

Desired pH 0.1 M Na2HPO4 (ml) 0.1 M NaH2PO4.H2O (ml)

6.0 6.0 44.0

6.5 15.2 34.8

7.0 28.9 21.1

7.5 40.5 9.5

8.0 46.6 3.4

8.5 49.0 1.0

Table 2.9: Composition of each phosphate buffer stock solution required to produce 0.1 M solutions

of each buffer at the required pH

pH 8.5 is outside the published range of this buffer composition, and therefore required

more 0.1 M sodium hydroxide to adjust it to the correct pH value. Hence, the

background ionic strength of this buffer would be slightly higher than the rest.

For impedimetric measurements, a low conductivity solution is required. The final

sample solution was produced by taking a 20 ml aliquot of the buffer at the pH to be

tested and diluting to 200 ml with deionised water. This produced a buffer solution with

a concentration of approximately 1.0 mM. The final pH was recorded using a bench top

pH meter and the background ionic strength estimated using a handheld TDS meter,

prior to addition of nitrate over the concentration range 1–100 ppm. This process was

repeated for each of the buffers stated in Table 2.9.

Unless otherwise stated, prior to testing, all background solutions were stored in a water

bath at 25 °C.

2.7.3.2. Calibrations

Impedimetric pH sensors were calibrated over the range pH 6.0–8.5. The sensor under

test was added to the first calibration solution, starting with pH 8.5, with measurements

taken at 5 minute intervals for a total of 30 minutes. The electrode was rinsed with

Craig Alexander

117

deionised water and added to the next buffer solution, pH 8.0. This process was

repeated for pH 7.5, 7.0, 6.5 and 6.0.

2.7.4 Nitrite Sensors


Unless otherwise stated, aliquots of the KNO2 stock solution described in section

2.3.2.2.1 were added to the test vessel to adjust nitrite concentration throughout.

For selectivity experiments, the stock solutions of nitrate and chloride described

previously in section 2.3.2.2.1 were used. Unless otherwise stated, prior to testing, all

background solutions were stored in a water bath at 25 °C.

2.7.4.2. Measurements in Water

The nitrite sensor under test was submerged into a beaker containing 200 ml of

deionised water, stirring at a constant rate. Once a stable baseline was obtained, an

aliquot of NO2- stock solution was added to the test vessel to give a concentration of

5 ppm.


The selectivity of the impedimetric nitrite sensors was determined against interfering

nitrate and chloride anions at a fixed concentration of 50 ppm. A value of 50 ppm

nitrate was chosen as the background concentration for carrying out the FIM. A value

of 50 ppm chloride was chosen as the background concentration for carrying out the

FIM.

Craig Alexander

118


additive present was established by carrying out a measurement of 5 ppm NO2-

in a

deionised water background, as a control.

Craig Alexander

119

3 Potentiometric Characterisation of Ionophores

This chapter evaluates the analytical performance characteristics of several

commercially-available ionophores experimentally for each of the identified key

aquarium ionic analytes, within conventional potentiometric ISEs. It was of great

importance to understand the behaviour of these ionophores within established chemical

sensing methodologies, to ascertain their potential suitability for use within a freshwater

aquarium monitoring device.

3.1 Nitrate-Selective Electrodes

3.1.1 Nitrate PMEs

Nitrate PMEs were prepared using PVC membranes containing 1% w/w of the

commercial ionophores TDAN and Nitrate Ionophore V (NO3V). The calibration

results over the range 0.1–10,000 ppm NO3- are shown in Figures 3.1 (TDAN) and

Figure 3.2 (NO3V). Selectivity data are shown in Figure 3.3 (TDAN) and

Figure 3.4 (NO3V).

Figure 3.1: Calibration graph of two nitrate-selective PMEs prepared using a PVC membrane

containing 1% w/w TDAN as the ionophore. The calibration results of a commercial

nitrate-selective half-cell tested in the same solutions are also shown

TDAN PME 1 y = -50.65x + 350.85

R² = 0.9972

TDAN PME 2y = -52.24x + 359.06

R² = 0.9981

Commercial nitrate half-cell y = -52.69x + 415.02

R² = 0.9951

0

50

100

150

200

250

300

350

400

450

500

-2 -1 0 1 2 3 4 5

E (m

V)

log NO3- concentration (ppm)

TDAN PME 1

TDAN PME 2

Commercial nitratehalf-cell

Craig Alexander

120

The two prepared TDAN PMEs exhibited a linear range (R2 >0.995) over 1–1000 ppm

NO3- with near-Nernstian slopes of -50.7 and -52.2 mV/dec., respectively. The recorded

temperature for each measurement was between 21.1 and 22.5 °C, therefore the

temperature effects on the results can be considered negligible. The device-to-device

reproducibility of the TDAN PMEs was defined as the relative standard deviation

(%RSD) of the two slopes from the linear range of the calibration graphs, which was

calculated to be 2.19%. Extrapolation of the outliers to the linear section gives the lower

detection limits (LDLs) and upper detection limits (UDLs) for the ISE. These were

calculated for TDAN PME 1 as shown in Equations 3.1 and 3.2.

𝐋𝐃𝐋 (𝐩𝐩𝐦) = 𝟏𝟎(

𝟑𝟓𝟔.𝟓 𝐦𝐕−𝟑𝟓𝟎.𝟖𝟓−𝟓𝟎.𝟔𝟓

)

= 0.77 ppm NO3-

Equation 3.1

𝐔𝐃𝐋 (𝐩𝐩𝐦) = 𝟏𝟎(

𝟏𝟕𝟖.𝟓 𝐦𝐕−𝟑𝟓𝟎.𝟖𝟓−𝟓𝟎.𝟔𝟓

)

= 2540 ppm NO3-

Equation 3.2

The LDLs and UDLs for TDAN PME 2 were calculated as 0.93 and 2130 ppm NO3-,

respectively.

Craig Alexander

121

Figure 3.2: Calibration graph of two nitrate-selective PMEs prepared using a PVC membrane

containing 1% w/w NO3V as the ionophore

The two prepared NO3V PMEs exhibited a linear range (R2 >0.995) over 1–10,000 ppm

NO3- with near-Nernsitan slopes of -54.9 and -55.2 mV/dec., respectively. The recorded

temperature for each measurement was between 20.5 and 22.2 °C. The %RSD of the

two slopes was calculated as 0.36%. The LDLs were calculated as 0.57 ppm for NO3V

PME 1 and 0.56 ppm for NO3V PME 2. The UDLs were calculated as 8010 ppm for

NO3V PME 1 and 7980 ppm for NO3V PME 2.

NO3V PME 1y = -54.92x + 220.9

R² = 0.9965

NO3V PME 2 y = -55.2x + 222.3

R² = 0.9964

0

50

100

150

200

250

-2 -1 0 1 2 3 4 5

E (m

V)


NO3VPME 1

NO3VPME 2

Craig Alexander

122

Figure 3.3: Selectivity determination of TDAN PME 1 using the FIM in 1000 ppm Cl

- and

1000 ppm NO2-. The response of a PME prepared with a ‘blank’ membrane containing no TDAN

is also shown

The potentiometric selectivity coefficients of TDAN PME 1 for interfering NO2- and Cl

-

anions were calculated as illustrated in Figure 1.8. For NO2-, extrapolating the outliers

to the linear section of the calibration curve gives a CA value of 30.3 ppm to enter into

Equation 1.22, and Cl- gives a CA value of 7.3 ppm. The CB value for the interfering

cations was fixed at 1000 ppm. The KPot values were calculated as shown in

Equations 3.3 and 3.4.

𝐊𝐍𝐎𝟑−,𝐍𝐎𝟐

− 𝐏𝐨𝐭 =

𝟑𝟎. 𝟑 𝐩𝐩𝐦

𝟏𝟎𝟎𝟎 𝐩𝐩𝐦= 𝟎. 𝟎𝟑𝟎𝟑 Equation 3.3

𝐥𝐨𝐠𝟏𝟎(𝐊𝐍𝐎𝟑−,𝐍𝐎𝟐

−𝐏𝐨𝐭 ) = −𝟏. 𝟓𝟐 Equation 3.4

Log10 (KNO3−,Cl−

Pot ) was calculated as -2.14. No Nernstian response behaviour was

observed from the ‘blank’ membrane, which was prepared without TDAN present

as the ionophore.

TDAN PME 1 y = -50.65x + 350.85

R² = 0.9972

0

50

100

150

200

250

300

350

400

450

-2 -1 0 1 2 3 4 5

E (m

V)


Pure Nitrate

1000 ppm Cl-

1000 ppm NO2-

Blank membrane

Craig Alexander

123

Figure 3.4: Selectivity determination of NO3V PME 1 using the FIM in 1000 ppm Cl

- and 1000 ppm

NO2-. The response of a PME prepared with a ‘blank’ membrane containing no NO3V as the

ionophore or TDMACl as the ionic additive is also shown

The logarithmic potentiometric selectivity coefficients of NO3V PME 1 for interfering

NO2- and Cl

- anions were calculated as -1.32 and -2.19, respectively. No Nernstian

response behaviour was observed from the ‘blank’ membrane, which was prepared

without NO3V present as the ionophore or TDMACl present as the ionic additive.

NO3V PME 1y = -54.92x + 220.9

R² = 0.9965

0

50

100

150

200

250

-2 -1 0 1 2 3 4 5

E (m

V)


Pure Nitrate

1000 ppm Cl-

1000 ppm NO2-

Blank membrane

Craig Alexander

124

3.1.2 Nitrate CWEs

CWEs, described in section 2.2.3, were prepared as nitrate sensors by dip-coating

Ag/AgCl electrodes by hand into either a PVC or sol-gel solution containing TDAN as

the ionophore. The calibration results for the PVC membrane electrodes are shown in

Figure 3.5, and the sol-gel membrane calibration results are shown in Figure 3.6. FIM

selectivity data are shown in Figure 3.7 (PVC CWE), Figure 3.8 (MTES CWE) and

Figure 3.9 (DEDMS CWE).

Figure 3.5: Calibration graph of two nitrate-selective CWEs prepared by dip-coating the electrode

into a PVC membrane ‘cocktail’ containing 1% w/w TDAN as the ionophore

Both prepared PVC CWEs exhibited reasonable linearity (R2 >0.96) over the

concentration range of 1–1000 ppm NO3-, with a slope that was much greater than the

expected Nernstian value of -59.2 mV/dec. The slope for TDAN PVC CWE 1 was

-77.09 mV/dec. and the slope for TDAN PVC CWE 2 was -71.47 mV/dec. The

recorded temperature for each measurement was between 21.2 and 22.8 °C. The %RSD

of the two slopes was calculated as 5.35%. The LDL for TDAN PVC CWE 1 was

calculated as 1.5 ppm, and for TDAN PVC CWE 2 it was calculated as 0.5 ppm. The

TDAN PVC CWE 2y = -71.47x + 337.48

R² = 0.9804

TDAN PVC CWE 1y = -77.09x + 353.91

R² = 0.9597

0

50

100

150

200

250

300

350

400

450

500

-2 -1 0 1 2 3 4 5

E (m

V)


TDAN PVC CWE 1

TDAN PVC CWE 2

Craig Alexander

125

UDLs were calculated as 3240 ppm for TDAN PVC CWE 1 and 9530 ppm for

TDAN PVC CWE 2.

Figure 3.6: Calibration graph of four nitrate-selective sol-gel CWEs (two MTES and two DEDMS)

containing TDAN as the ionophore

The two sol-gel CWEs that were prepared from the silica precursor MTES containing

TDAN as the nitrate ionophore exhibited a linear range (R2 >0.99) between

10–1000 ppm NO3-. The recorded temperature for each measurement was between 21.3

and 22.1 °C. The %RSD of the two slopes was calculated as 0.58%; however, there

was a large discrepancy between devices of around 20 mV between the obtained

potential values at each measured nitrate concentration. TDAN MTES CWE 1 had a

slope of -48.25 mV/dec. and TDAN MTES CWE 2 had a slope of -48.65 mV/dec. The

LDLs were calculated as 8.4 ppm for TDAN MTES CWE 1 and 8.3 ppm for TDAN

MTES CWE 2. The UDLs were calculated as 2890 ppm for TDAN MTES CWE 1 and

5560 ppm for TDAN MTES CWE 2.

The two sol-gel CWEs that were prepared from the 3:1 ratio of the silica precursors

DEDMS and TEOS containing TDAN as nitrate ionophore exhibited a linear range

TDAN MTES CWE 1y = -48.25x + 286.17

R² = 0.9913TDAN MTES CWE 2y = -48.65x + 269.8

R² = 0.9936

TDAN DEDMS CWE 1y = -39.32x + 364.35

R² = 0.991

TDAN DEDMS CWE 2y = -43.91x + 380.75

R² = 0.9991

0

50

100

150

200

250

300

350

400

450

-2 -1 0 1 2 3 4 5

E (m

V)


TDAN MTES CWE 1

TDAN MTES CWE 2

TDAN DEDMS CWE 1

TDAN DEDMS CWE 2

Craig Alexander

126

(R2 >0.99) between 10–10,000 ppm NO3

-. The DEDMS electrodes were tested

alongside the MTES electrodes and therefore the recorded temperature for each

measurement was the same as for the MTES CWEs above. The %RSD of the two

slopes was calculated as 7.8%. TDAN DEDMS CWE 1 had a slope of -39.32 mV/dec.

and TDAN DEDMS CWE 2 had a slope of -43.91 mV/dec. The LDLs were calculated

as 3.1 ppm for TDAN DEDMS CWE 1 and 4.5 ppm for TDAN DEDMS CWE 2. The

UDLs were calculated as 7760 ppm for TDAN DEDMS CWE 1 and 9750 ppm for

TDAN DEDMS CWE 2.

Figure 3.7: Selectivity determination of TDAN PVC CWE 1 using the FIM in 1000 ppm Cl

- and

1000 ppm NO2-. The response of a CWE prepared with a ‘blank’ membrane containing no TDAN is

also shown

The logarithmic potentiometric selectivity coefficients of TDAN PVC CWE 1 for

interfering NO2- and Cl

- anions were calculated as -0.65 and -1.38, respectively. A

Nernstian response was not observed from the ‘blank’ membrane, which was prepared

without TDAN present as the ionophore; however, there did appear to be some linearity

with increasing nitrate concentration. This suggested that the obtained response was not

exclusively due to increasing nitrate concentration within the test sample and some

TDAN PVC CWE 1y = -71.47x + 337.48

R² = 0.9804

0

50

100

150

200

250

300

350

400

450

-2 -1 0 1 2 3 4 5

E (m

V)


Pure Nitrate

1000 ppm Cl-

1000 ppm NO2-

Blank PVC CWE

Craig Alexander

127

matrix interferences were observed. This therefore explained why a response that was

much greater than Nernstian was obtained for this ISE during the calibration

experiments.

Figure 3.8: Selectivity determination of TDAN MTES CWE 1 using the FIM in 1000 ppm Cl

- and


also shown

The logarithmic potentiometric selectivity coefficients of TDAN MTES CWE 1 for


- anions were calculated as 0.11 and -0.81, respectively. A

reasonably linear response, although not strictly Nernstian, was observed from a ‘blank’

MTES membrane which did not contain TDAN as the ionophore.

TDAN MTES CWE 1 y = -48.25x + 286.17

R² = 0.9913

0

50

100

150

200

250

300

350

400

-2 -1 0 1 2 3 4 5

E (m

V)


Pure Nitrate

1000 ppm Cl-

1000 ppm NO2-

Blank MTES CWE

Craig Alexander

128

Figure 3.9: Selectivity determination of TDAN DEDMS CWE 1 using the FIM in 1000 ppm Cl

- and


also shown

The logarithmic potentiometric selectivity coefficients of TDAN DEDMS CWE 1 for


- anions were calculated as 0.55 and -0.73, respectively. The

‘blank’ DEDMS membrane which did not contain TDAN as the ionophore

demonstrated an almost equivalent response to the target nitrate ion. This suggested that

the selective ionophore within the membrane was not the sole cause of the response, and

that a non-selective response was achieved through matrix interferences.

Due to the poor analytical performance of the TDAN CWEs compared with the PMEs,

which were prepared with the same ionophore, further CWEs were not prepared using

NO3V or with ionophores for the other target analytes. Neither PVC nor the sol-gels

demonstrated good response characteristics when used as the membrane support matrix

for the ionophore TDAN. The CWEs demonstrated considerably inferior selectivity

coefficients against interfering nitrite and chloride anions compared with the PMEs. A

much more prominent response to the target nitrate ion was observed when a CWE with

a ‘blank’ membrane, which did not contain any ionophore, was tested. The CWEs also

TDAN DEDMS CWE 1y = -39.32x + 364.35

R² = 0.991

0

50

100

150

200

250

300

350

400

450

-2 -1 0 1 2 3 4 5

E (m

V)


Pure Nitrate

1000 ppm Cl-

1000 ppm NO2-

Blank DEDMS CWE

Craig Alexander

129

exhibited poor device-to-device reproducibility and less favourable detection limits than

the PMEs.

3.2 Ammonium-Selective Electrodes

Figure 3.10 shows the calibration results for an ammonium-selective PME over the

range 0.1–10,000 ppm NH4+, prepared with the NH4I PVC membrane described in

Table 2.1, containing potassium tetrakis (4-chlorophenyl) borate in a 1:2 mole ratio

(with respect to the ionophore), as the ionic additive. The FIM selectivity data for this

electrode against the interfering cations Na+, K

+, Mg

2+ and Ca

2+ are shown in

Figure 3.11.

Figure 3.10: Calibration graph of two ammonium-selective PMEs prepared using a PVC

membrane containing 1% w/w NH4I as the ionophore

The ammonium PMEs showed reasonable linearity (R2 >0.975) over the concentration

range 10–10,000 ppm NH4+, and exhibited sub-Nernstian slopes of 47.6 mV/dec. for

NH4I PME 1 and 41.5 mV/dec. for NH4I PME 2. The recorded temperature for each

measurement was between 20.9 and 22.7 °C. The %RSD of the slopes was calculated

as 9.61%. The LDLs were calculated as 6.4 ppm and 10.3 ppm NH4+, respectively.

NH4I PME 1y = 47.6x - 40.6

R² = 0.9873

NH4I PME 2y = 41.54x - 18.45

R² = 0.9776

-100

-50

0

50

100

150

200

-2 -1 0 1 2 3 4 5

E (m

V)

log NH4+ concentration (ppm)

NH4I PME 1

NH4I PME 2

Craig Alexander

130

Figure 3.11: Selectivity determination of NH4I PME 1 using the FIM in 1000 ppm of the interfering

cations sodium, potassium, magnesium and calcium

The potentiometric selectivity coefficients were calculated as before (section 3.1.1), for

the interfering cations K+, Na

+, Mg

2+ and Ca

2+. The calculated log KNH4

+,B+Pot values are

summarised in Table 3.1.

Interfering cation (B+) log 𝐊𝐍𝐇𝟒

+,𝐁+𝐏𝐨𝐭

K+ -0.08

Na+ -1.21

Mg2+

-1.90

Ca2+

-2.09

Table 3.1: Potentiometric selectivity coefficients of NH4I PME 1

It has previously been reported that although NH4I (nonactin) is a neutral ionophore,

adding an anion exchanger to the membrane can actually lead to a reduction in its

selectivity and potentiometric performance as an ISE174

. The membrane was therefore

prepared again, with the same ratios of NH4I, plasticiser and PVC; however, potassium

tetrakis (4-chlorophenyl) borate was not added to the polymeric ‘cocktail’ solution. The

calibration results for the PMEs prepared using this alternative membrane composition

are shown in Figure 3.12. The FIM selectivity data for interfering cations Na+, K

+, Mg

2+

NH4I PME 1y = 47.6x - 40.6

R² = 0.9873

-100

-50

0

50

100

150

200

-2 -1 0 1 2 3 4 5

E (m

V)


Pure Ammonium

1000 ppm Na+

1000 ppm K+

1000 ppm Mg2+

1000 ppm Ca2+

Craig Alexander

131

and Ca2+

obtained using the alternative membrane composition are shown in

Figure 3.13.

Figure 3.12: Calibration graph of two ammonium-selective PMEs prepared using a PVC

membrane containing 1% w/w NH4I as the ionophore, with the ionic additive omitted. The

calibration results of a commercial ammonium-selective half-cell tested in the same solutions are

also shown

When the ammonium-selective membrane was prepared with the omission of potassium

tetrakis (4-chlorophenyl) borate, the PMEs exhibited good linearity (R2 >0.995) over the

concentration range 1–10,000 ppm NH4+, and near-Nernstian slopes of 50 mV/dec. for

NH4I PME 1 and 47.7 mV/dec. for NH4I PME 2. The recorded temperature for each

measurement was between 20.4 and 22.5 °C. The %RSD of the slopes was calculated

as 3.36%. The LDLs were calculated as 0.5 ppm and 0.8 ppm NH4+, respectively.

NH4I PME 2y = 47.69x - 45.86

R² = 0.995

NH4I PME 1y = 50.01x - 53.04

R² = 0.9965

Commercial ammonium half-celly = 55.81x + 93.1

R² = 0.9997

-150

-100

-50

0

50

100

150

200

250

300

350

-2 -1 0 1 2 3 4 5

E (m

V)


NH4I PME 1

NH4I PME 2

Commercial ammoniumhalf-cell

Craig Alexander

132

Figure 3.13: Selectivity determination of NH4I PME 1 (ionic additive omitted) using the FIM in

1000 ppm of the interfering cations sodium, potassium, magnesium and calcium. The response of a

PME prepared with a ‘blank’ membrane containing neither NH4I nor ionic additive is also shown

The potentiometric selectivity coefficients for the alternative NH4I PME 1, prepared

with a membrane that did not contain potassium tetrakis (4-chlorophenyl) borate as

anion exchanger, are summarised in Table 3.2. There was no Nernstian response

observed from the ‘blank’ electrode with the membrane that did not contain NH4I as the

ionophore.

Interfering cation (B+) log 𝐊𝐍𝐇𝟒

+,𝐁+𝐏𝐨𝐭

K+ -1.25

Na+ -2.59

Mg2+

-2.60

Ca2+

-3.07

Table 3.2: Potentiometric selectivity coefficients of NH4I PME 1 (ionic additive omitted)

NH4I PME 1y = 50.01x - 53.04

R² = 0.9965

-100

-50

0

50

100

150

200

-2 -1 0 1 2 3 4 5

E (m

V)


Pure Ammonium

1000 ppm Na+

1000 ppm K+

1000 ppm Mg2+

1000 ppm Ca2+

BLANK NH4I

Craig Alexander

133

3.3 Hydrogen Ion-Selective Electrodes

Figure 3.14 shows the calibration results for a hydrogen ion-selective PME prepared

with the commercial ionophore HIII and Figure 3.15 shows the calibration results for a

hydrogen ion-selective PME prepared with the commercial ionophore HV. Both

ionophores were prepared within a PVC membrane as described in Table 2.1, and tested

over the pH range 2.5–11.5. The FIM selectivity data for the HIII PME are shown in

Figure 3.16, and for the HV PME in Figure 3.17, against the interfering cations Na+, K

+

and Mg2+

.

Figure 3.14: Calibration graph of two hydrogen ion-selective PMEs prepared using a PVC

membrane containing 1% w/w HIII as the ionophore. The calibration results of a commercial pH

half-cell tested in the same solutions are also shown

The HIII PMEs demonstrated excellent linearity (R2 >0.99) over the full tested pH

range of 2.5–11.5, with slopes of -46.70 mV/dec. for HIII PME 1 and -51.11 mv/dec.

for HIII PME 2. The recorded temperature for each measurement was between 20.3 and

21.8 °C. The %RSD of the slopes was calculated as 6.38%. The LDLs were calculated

as pH 10.8 for HIII PME 1 and pH 11.4 for HIII PME 2. The UDLs were calculated as

pH 1.8 for HIII PME 1 and pH 2.6 for HIII PME 2.

HIII PME 1y = -46.698x + 371.5

R² = 0.9999

HIII PME 2y = -51.111x + 401.31

R² = 0.998

Commercial pH half-celly = -56.165x + 336.35

R² = 0.9997

-400

-300

-200

-100

0

100

200

300

400

0 2 4 6 8 10 12 14E (m

V)

pH

HIII PME 1

HIII PME 2

CommercialpH half-cell

Craig Alexander

134

Figure 3.15: Calibration graph of two hydrogen ion-selective PMEs prepared using a PVC

membrane containing 1% w/w HV as the ionophore

The HV PMEs demonstrated excellent linearity (R2 >0.99) over the pH range of

5.0–11.5, with sub-Nernstian slopes of -39.80 mV/dec. for HV PME 1 and -39.89

mv/dec. for HV PME 2. The HV electrodes were tested alongside the HIII ones and

therefore the recorded temperature for each measurement was the same as for the HIII

PMEs above. The %RSD of the slopes was calculated as 0.16%. The LDLs were

calculated as pH 11.6 for HV PME 1 and pH 11.7 for HV PME 2. The UDL were

calculated as pH 4.4 for HV PME 1 and pH 4.6 for HV PME 2.

HV PME 2y = -39.887x + 327.92

R² = 0.9977

HV PME 1y = -39.796x + 324.39

R² = 0.9959

-200

-150

-100

-50

0

50

100

150

200

250

0 2 4 6 8 10 12 14

E (m

V)

pH

HV PME 1

HV PME 2

Craig Alexander

135

Figure 3.16: Selectivity determination of HIII PME 1 using the FIM in 0.1 M of the interfering

cations sodium, potassium and magnesium. The response of a PME prepared with a ‘blank’

membrane containing neither HIII nor ionic additive is also shown

The potentiometric selectivity coefficients for HIII PME 1 were calculated as before, for


+ and Mg

2+. The calculated log KH+,B+

Pot values are

summarised in Table 3.3. There was no Nernstian response to the change in hydrogen

ion concentration observed from the ‘blank’ electrode with the membrane that did not

contain HIII as the ionophore.

Interfering cation (B+) log 𝐊𝐇+,𝐁+

𝐏𝐨𝐭

K+ -11.00

Na+ -11.14

Mg2+

-10.78

Table 3.3: Potentiometric selectivity coefficients of HIII PME 1

HIII PME 1y = -46.698x + 371.5

R² = 0.9999

-200

-150

-100

-50

0

50

100

150

200

250

300

0 2 4 6 8 10 12 14

E (m

V)

pH

HIII PME 1

0.1 M K+

0.1 M Na+

0.1 M Mg2+

Blank HIII

Craig Alexander

136

Figure 3.17: Selectivity determination of HV PME 1 using the FIM in 0.1 M of the interfering

cations sodium, potassium and magnesium. The response of a PME prepared with a ‘blank’

membrane containing neither HV nor ionic additive is also shown

The potentiometric selectivity coefficients for HV PME 1 were calculated as before, for


+ and Mg

2+. The calculated log KH+,B+

Pot values are

summarised in Table 3.4. There was no Nernstian response to the change in hydrogen

ion concentration observed from the ‘blank’ electrode with the membrane that did not

contain HV as the ionophore.

Interfering cation (B+) log 𝐊𝐇+,𝐁+

𝐏𝐨𝐭

K+ -11.09

Na+ -11.41

Mg2+

-11.21

Table 3.4: Potentiometric selectivity coefficients of HV PME 1

3.4 Conclusions for Chapter 3

Several commercially-available ionophores were tested within potentiometric ISEs to

assess their analytical performance relating to the determination of three

aquarium-significant ions. Where possible, two different ionophores were investigated

HV PME 1y = -39.887x + 327.92

R² = 0.9977

-200

-150

-100

-50

0

50

100

150

200

250

0 2 4 6 8 10 12 14

E (m

V)

pH

HV PME 1

0.1 M K+

0.1 M Na+

0.1 M Mg2+

Blank HV

Craig Alexander

137

for each target ion. Nitrite was not investigated using potentiometric ISEs due to the

cost of commercially-available nitrite ionophores.

For the nitrate anion, ion-selective membranes containing two different ionophores,

TDAN and NO3V, were tested within potentiometric ISEs. When tested within PMEs

with a PVC membrane layer, both ionophores responded in a near-Nernstian fashion

over a range which would make them suitable for determining the nitrate concentration

within a freshwater aquarium. The NO3V PME exhibited a wider linear range, a slope

that fitted the theoretical value more closely and a lower limit of detection than the

TDAN electrode. The NO3V PMEs also showed better device-to-device reproducibility.

The TDAN PME proved to be marginally more selective to nitrate over nitrite than the

NO3V PME (log10(KNO3−,NO2

−Pot ) = -1.52 versus -1.32), whereas the NO3V PME was

slightly more selective over chloride (log10(KNO3−,Cl−

Pot ) = -2.19 versus -2.14).

The ionophore TDAN was also tested within CWEs, using PVC and two different

sol-gels as the membrane support matrix. The PVC CWEs produced a slope that was

much greater than the theoretically expected value, and all of the response

characteristics were unfavourable when compared with the PMEs. Additionally, sol-gel

CWEs did not provide adequate analytical responses to nitrate. Inadequate response

characteristics from the CWEs may have arisen due to insufficient coating and/or poor

adhesion of the membrane material to the Ag/AgCl wire. As the electrodes were

dip-coated by hand, this may have led to an inhomogeneous coating onto the surface of

the electrode, which may have caused the lack of ion-selectivity and overall poor

analytical performance. To overcome this issue, a commercial dip-coating system, for

example a Chemat DipMaster175

, could be used to ensure that aspects of the dip-coating

process, such as immersion time and withdrawal speed are kept constant.

Craig Alexander

138

It was still of great interest to further investigate the use of sol-gels as an alternative

ion-selective membrane material, however it was considered that they may be more

suitable to coating a planar substrate rather than depositing over a wire electrode. The

use of silica gels as ion-selective membranes within impedimetric IDE devices will be

discussed in Chapter 4.

For ammonium, only one commercial ionophore was available to be purchased, NH4I,

which was tested within PMEs. Initially, PVC membranes were used which consisted of

the plasticiser NPOE and potassium tetrakis (4-chlorophenyl) borate as anion

exchanger. It was shown that the analytical performance of NH4+ PMEs, using NH4I as

the ionophore, was improved substantially when potassium tetrakis (4-chlorophenyl)

borate was omitted from the PVC ion-selective membrane. The PMEs prepared without

the ionic additive in the membrane demonstrated a wider linear range, a lower detection

limit, improved device-to-device reproducibility and improved selectivity coefficients

for each of the tested interfering cations (K+, Na

+, Mg

2+ and Ca

2+). Subsequent

ammonium-selective membranes required for further experimentation within

impedimetric IDE devices, described in Chapter 7, were therefore prepared with

potassium tetrakis (4-chlorophenyl) borate omitted from the membrane ‘cocktail’.

For pH determination, two commercial hydrogen ion-selective ionophores, HIII and

HV, were tested within PMEs with PVC membranes. Both ionophores exhibited

excellent linearity over the expected pH range of a freshwater aquarium, along with

very high selectivity coefficients for the tested interfering cations. HIII PME

demonstrated a wider linear range (2.5–11.5 versus 5.0–11.5), whereas HV did not

show linearity in the acidic region, below approximately pH 4.0.

Craig Alexander

139

As mentioned previously, one of the major disadvantages of potentiometric ISEs is the

requirement of an external reference electrode for them to function. Such a set-up would

not be suited for use within a home aquarium monitoring product, particularly with

sensors and reference electrodes containing liquid inner filling solutions. Therefore, the

main focus of this research was adjusted to producing ion-selective sensors which do

not require an external reference electrode. The proposed methodology was to use IDEs

to monitor changes in the electrical properties of an ion-selective membrane due to

selective responses between the target ions and the previously discussed

commercially-available ionophores.

As the intended final use of the prepared ion-selective membranes was as the

recognition layer within an impedimetric IDE device, it was important to bear in mind

that it has previously been reported that even when a particular membrane composition

has been shown to perform adequately within a potentiometric ISE, this does not

necessarily mean it will produce an equivalent selective response when used within an

ISCOM device156

.

It was decided that nitrate would be the principal target ion to be investigated using IDE

sensors. In the case of commercially-available nitrate ionophores, TDAN is

significantly less expensive than NO3V, costing around £30 per 100 mg as opposed to

around £250 per 100 mg, and therefore would offer a much more cost effective option

for nitrate sensing within a commercial product. TDAN also does not require an

additional ionic additive to be added to the membrane. Consequently, initial proof-of-

principle developmental work for nitrate sensing using IDEs was carried out using the

ionophore TDAN.

Craig Alexander

140

4 Nitrate Sensing using IDE Design 1

4.1 Sensor Calibration

It was first important to establish the behaviour of IDE Design 1 in the absence of an

ion-selective membrane to ensure that a response was obtained from changes in nitrate

concentration in the important range for aquarium purposes (1–100 ppm). Figure 4.1

shows capacitance (A) and conductance (B) spectra obtained from an uncoated sensor,

via addition of aliquots of NO3- stock solution to a deionised water background.

Figure 4.1: Conductance (A) and capacitance (B) spectra for the addition of NO3

- to an uncoated

IDE Design 1 (1 Vrms input amplitude). ‘Pure water’ refers to the initial baseline value and

10-60 mins are the values obtained during the baseline settling period

0.00E+00

1.00E-07

2.00E-07

3.00E-07

4.00E-07

5.00E-07

6.00E-07

7.00E-07

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07

Cap

acit

ance

(F)

Frequency (Hz)

Cp Pure water

Cp 10 mins

Cp 20 mins

Cp 30 mins

Cp 40 mins

Cp 50 mins

Cp 60 mins

Cp 1 ppm NO3-

Cp 10 ppm NO3-

Cp 20 ppm NO3-

Cp 30 ppm NO3-

Cp 40 ppm NO3-

Cp 50 ppm NO3-

Cp 100 ppm NO3-

0.00E+00

5.00E-04

1.00E-03

1.50E-03

2.00E-03

2.50E-03

3.00E-03

3.50E-03

4.00E-03

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07

Co

nd

uct

ance

(S)

Frequency (Hz)

G Pure water

G 10 mins

G 20 mins

G 30 mins

G 40 mins

G 50 mins

G 60 mins

G 1 ppm NO3-

G 10 ppm NO3-

G 20 ppm NO3-

G 30 ppm NO3-

G 40 ppm NO3-

G 50 ppm NO3-

G 100 ppm NO3-

A

B

Craig Alexander

141

Six frequencies across each spectra were evaluated: 100 Hz, 1 kHz, 10 kHz, 100 kHz, 1

MHz and 2 MHz. Figure 4.2 shows the change in conductance against time upon

addition of nitrate at each chosen frequency, whilst Figure 4.3 shows the capacitance.

Figure 4.2: Conductance response of uncoated IDE Design 1 upon addition of 1–100 ppm nitrate at

six frequencies.

A – 100 Hz. B – 1 kHz. C – 10 kHz. D – 100 kHz. E – 1 MHz. F – 2 MHz

For the conductance, most of the six extrapolated frequencies, with the exception of the

lowest frequency at 100 Hz, showed a ‘step-wise’ increase to each of the added aliquots

of potassium nitrate stock solution. The response time of the sensor was almost

immediate (<1 minute), as very little drift was observed in any of the measurements.

A B

C

E

D

F

1 hour baseline

1 ppm NO3-

10 ppm NO3-

20 ppm NO3- 30 ppm NO3

-

40 ppm NO3-

50 ppm NO3-

100 ppm NO3-

Craig Alexander

142

Figure 4.3: Capacitance response of uncoated IDE Design 1 upon addition of 1–100 ppm nitrate at

six frequencies.


For the capacitance, the three lowest extrapolated frequencies (100 Hz, 1 kHz and 10

kHz) showed a ‘step-wise’ increase as the test solution conductivity was increased

through the addition of a potassium nitrate stock solution. At 100 kHz, a slight decrease

was noted at low nitrate concentrations (<20 ppm), followed by ‘step-wise’ increases

from 30–100 ppm. At 1 MHz and 2 MHz there was a decrease in the measured

capacitance as potassium nitrate aliquots were added to the test sample.

The response obtained from the deionised water baseline (G0 or C0) was subtracted from

average of the measured response obtained from the five data points obtained from each

nitrate addition (Gm or Cm). The change in conductance (Gm - G0) or capacitance

(Cm - C0) was plotted against log NO3- concentration. Figure 4.4 shows the conductance

A B

C D

E F

Craig Alexander

143

and Figure 4.5 shows the capacitance. By subtracting the baseline from the measured

response this also negated any electrical differences in the measurements caused by

cables or connectors. Therefore, the change in the electrical impedance observed is only

due to the addition of aliquots of target/interfering ion solutions to the initial

background solution.

Figure 4.4: Deionised water baseline-subtracted conductance (Gm – G0) against log NO3

-

concentration for uncoated IDE Design 1 at six frequencies.


At 1 kHz, the addition of 100 ppm NO3- resulted in an increase in the measured

conductance from the baseline deionised water value of 0.87 mS. The Gm - G0 values for

100 ppm NO3- at the other extrapolated frequencies were 2.75 mS at 10 kHz, 3.36 mS at

100 kHz, 3.39 mS at 1 MHz and 3.41 mS at 2 MHz.

A B

C D

E F

Craig Alexander

144

Figure 4.5: Deionised water baseline-subtracted capacitance (Cm – C0) against log NO3

-



At 100 Hz, an increase in the measured capacitance of 436 nF was observed through the

addition of 100 ppm NO3-. At 1 kHz, this resulted in a Cm - C0 value of 169 nF, and at

10 kHz, Cm - C0 = 14.5 nF. At a frequency of 100 kHz, a slight decrease of

approximately 30 pF from the baseline deionised water value was observed up to

20 ppm NO3-, followed by an increase to 290 pF at 100 ppm NO3

-. Both 1 MHz and

2 MHz showed a decrease of 36 pF from the baseline response.

It has previously been reported for similar devices that plotting the logarithm of the

baseline-subtracted response against the logarithm of the ion concentration produces a

linear relationship176

. This information is shown in Figures 4.6 for G and Figure 4.7

for C.

A B

C D

E F

Craig Alexander

145

Figure 4.6: Logarithm of the change in conductance (Gm – G0) against the logarithm of nitrate



The log (Gm–G0) versus log NO3- graph resulted in a linear increase (R

2 >0.98) for each

of the extrapolated frequencies, with the exception of 100 Hz, where a linear response

was not observed.

A B

C D

E F

Craig Alexander

146

Figure 4.7: Logarithm of the change in capacitance (Cm – C0) against the logarithm of nitrate

concentration for uncoated IDE Design 1 at three frequencies (negative values obtained at 100 kHz,

1 MHz and 2 MHz). A – 100 Hz. B – 1 kHz. C – 10 kHz

The log (Cm–C0) versus log NO3- graph resulted in a linear increase (R

2 >0.98) at

100 Hz, 1 kHz and 10 kHz.

It was established that IDE Design 1 was able to respond to changes in the conductivity

of the bulk test solution, through additions of KNO3 to a deionised water background.

The most analytically-relevant information appeared to arise from the change in

conductance, particularly at higher frequencies, as this is where the most marked

responses were observed. Further experimentation was carried out on this device, using

a membrane containing a commercially-available ionophore to coat the sensing area, in

an attempt to impart nitrate selectivity on the sensor. This would ascertain whether IDE

Design 1 was suitable to be used as a nitrate sensor within a device for monitoring the

water quality of freshwater aquaria.

A B

C

Craig Alexander

147

4.2 PVC Membrane Sensors

4.2.1 Calibrations

Figures 4.8A and 4.8B show the conductance and capacitance spectra, respectively, for

the addition of nitrate in the concentration range 1–100 ppm, to IDE Design 1 spin-

coated at 2000 rpm in a PVC membrane containing 1% w/w TDAN as the ionophore.

This particular device will be referred to as ‘TDAN PVC spin-coated IDE Design 1’.


- to TDAN PVC

spin-coated IDE Design 1 (1 Vrms input amplitude). ‘Pure water’ refers to the initial baseline value

and 10-60 mins are the values obtained during the baseline settling period

0.00E+00

5.00E-08

1.00E-07

1.50E-07

2.00E-07

2.50E-07

3.00E-07

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07

Cap

acit

ance

(F)

Frequency (Hz)

Cp Pure water

Cp 10 mins

Cp 20 mins

Cp 30 mins

Cp 40 mins

Cp 50 mins

Cp 60 mins

Cp 1 ppm NO3-

Cp 10 ppm NO3-

Cp 20 ppm NO3-

Cp 30 ppm NO3-

Cp 40 ppm NO3-

Cp 50 ppm NO3-

Cp 100 ppm NO3-

0.00E+00

5.00E-04

1.00E-03

1.50E-03

2.00E-03

2.50E-03

3.00E-03

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07

Con

du

ctan

ce (S

)

Frequency (Hz)

G Pure water

G 10 mins

G 20 mins

G 30 mins

G 40 mins

G 50 mins

G 60 mins

G 1 ppm NO3-

G 10 ppm NO3-

G 20 ppm NO3-

G 30 ppm NO3-

G 40 ppm NO3-

G 50 ppm NO3-

G 100 ppm NO3-

A

B

Craig Alexander

148

When IDE Design 1 was spin-coated with a PVC membrane containing the ionophore

TDAN, it was more difficult to distinguish between each nitrate addition at many of the

extrapolated frequencies when compared to the results for the uncoated device, and the

‘step-wise’ response that was observed was not as prominent. Figure 4.9 shows the

change in conductance against time upon addition of nitrate at each chosen frequency,

whilst Figure 4.10 shows the capacitance.

Figure 4.9: Conductance response of TDAN PVC spin-coated IDE Design 1 upon addition of

1–100 ppm nitrate at six frequencies.


A B

C D

E F

Craig Alexander

149

Figure 4.10: Capacitance response of TDAN PVC spin-coated IDE Design 1 upon addition of



When TDAN PVC was spin-coated on to IDE Design 1, the most prominent response

upon addition of the target nitrate anion to the test vessel was obtained from the

conductance at a frequency of 2 MHz. The deionised water baseline-subtracted response

for this frequency is shown in Figure 4.11.

A B

C D

E F

Craig Alexander

150


-

concentration for TDAN PVC spin-coated IDE Design 1 at 2 MHz

There was a total increase in the measured conductance at 2 MHz of 1.77 mS upon the

addition of 100 ppm NO3- to a deionised water baseline. The logarithmic

baseline-subtracted conductance against log NO3- concentration at 2 MHz is shown in

Figure 4.12.



Craig Alexander

151

Taking the logarithm of the deionised water baseline-subtracted conductance at 2 MHz

and plotting it against log NO3- concentration confirmed the linearity of the response,

with a R2 value of 0.9931.

Although the logarithm of the baseline response versus the log of the nitrate

concentration was used to determine if the calibration was linear, the

baseline-subtracted values were more beneficial to compare responses from different

sensors and for comparing the performance of the same sensor in different sample

matrices. Figure 4.13 shows the baseline-subtracted conductance at 2 MHz for the

addition of nitrate in the concentration range 1–100 ppm for TDAN PVC spin-coated

IDE Design 1, comparing the aforementioned calibration experiment, using a potassium

nitrate stock solution to adjust nitrate concentration, with the response from an

alternative counter ion (lithium) nitrate salt and the effect of the response when the

temperature of the sample solution was increased to from 25 °C to 30 °C.


-

concentration for TDAN PVC spin-coated IDE Design 1 at 2 MHz. Three calibration experiments

are shown: the addition of 1–100 ppm NO3- with K

+ as the counter ion, the addition of 1–100 ppm

NO3- with Li

+ as the counter ion and the addition of 1–100 ppm NO3

- with K

+ as the counter ion at

30 °C

0

0.0005

0.001

0.0015

0.002

0.0025

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


KNO3 Calibration

LiNO3 Calibration

30 °C Calibration

Craig Alexander

152

When lithium nitrate was used to adjust the nitrate concentration in the test vessel there

was a decrease observed in the recorded measurements of around 20% when compared

with the use of potassium nitrate. At 50 ppm NO3-, which is the upper limit within a

freshwater aquarium, a change in the measured conductance of 1.39 mS was observed

when potassium nitrate was used to adjust the nitrate concentration. When lithium

nitrate was added, the Gm - G0 for 50 ppm NO3- was 1.14 mS. This suggested that the

ionic mobility of the counter ion was affecting the response from the sensor, as the less

ionically-mobile Li+

counter ion produced less of a response than when K+ was used.

Furthermore, when the experiment was conducted using KNO3 to adjust the nitrate

concentration at 30 °C, there was an increase of between 13–19% at each concentration

than when the same experiment was conducted at 25 °C. This suggested that changes

within the solution were being observed, as an increase in temperature would cause an

increase in the conductivity of the solution due to increased ionic mobility.

To establish the effect of changes in the background conductivity of the sample

solution, the sensor was tested through additions of nitrate to different test solutions of

varying ionic strength. Figure 4.14 shows the measured G0 values for each of the

tested environments.

Craig Alexander

153

Figure 4.14: Measured baseline conductance value (G0) of TDAN PVC spin-coated IDE Design 1 in

six separate sample matrices at 2 MHz, prior to the addition of nitrate

From Figure 4.14, it was evident that placing the sensor into an ionic solution increased

the observed baseline conductance value at 2 MHz. A 100 ppm solution of MgSO4

produced a G0 value 1.53 mS greater than the deionised water baseline. 200 ppm

MgSO4 was 1.95 mS greater and 500 ppm MgSO4 was 2.15 mS greater than the

deionised water baseline. A sample of tap water gave a TDS reading of 44 ppm when

taken with a handheld TDS meter, and increased the G0 by 0.89 mS. A sample of water

was taken from a live freshwater aquarium, the recorded TDS value was 75 ppm, there

was no detectable existing NO3- when tested with a commercial test kit, and the G0

increased by 1.49 mS compared to the G0 obtained for deionised water. The measured

conductance responses upon addition of NO3- over the range 1–100 ppm to each of

these background solutions are shown in Figure 4.15.

Deionised water

100 ppm MgSO4

200 ppm MgSO4 500 ppm MgSO4 Tap water (44 ppm TDS)

Aquarium water (75 ppm TDS)

Background sample matrix

G0

bas

eli

ne

val

ue a

t 2

MH

z

Craig Alexander

154

Figure 4.15: Measured conductance response (Gm) of TDAN PVC spin-coated IDE Design 1 against

log NO3- concentration in six separate sample matrices at 2 MHz

The corresponding baseline-subtracted conductance responses are shown in Figure 4.16.

Figure 4.16: Baseline-subtracted conductance (Gm – G0) of TDAN PVC spin-coated IDE Design 1

against log NO3- concentration at 2 MHz, in six separate sample matrices

As the overall background ionic strength of the sample solution was increased, the

sensitivity of the response of the sensor to changes in NO3- concentration was reduced.

This suggested that the sensor was heavily influenced by the background ionic strength

of the test solution.

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

0 0.5 1 1.5 2 2.5

Gm

(S)


Pure water calibration (G0)

100 ppm MgSO4

200 ppm MgSO4

500 ppm MgSO4

Tap water (44 ppm TDS)


0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

0.0014

0.0016

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


Pure water calibration (G0)

100 ppm MgSO4

200 ppm MgSO4

500 ppm MgSO4

Tap water (44 ppm TDS)


Craig Alexander

155

Figure 4.17 shows the deionised water baseline-subtracted conductance at 2 MHz for

the addition of NO3- to three separate TDAN PVC spin-coated IDE Design 1 devices.

Figure 4.17: Deionised water baseline-subtracted conductance (Gm – G0) of three TDAN PVC

spin-coated IDE Design 1 devices against log NO3- concentration at 2 MHz, for reproducibility

determination

The device-to-device reproducibility of the impedimetric devices was defined as the

%RSD of the three separate baseline-subtracted conductance measurements obtained for

50 ppm NO3- (upper limit for NO3

- in an aquarium). For TDAN PVC spin-coated IDE

Design 1 at 2 MHz, this was calculated as 17.15%.

4.2.2 Selectivity

The nitrate selectivity of TDAN PVC spin-coated IDE Design 1 was tested using the

FIM, however, the method deviated slightly from the one described in section 2.7.2.4,

as 1 ppm NO2- and 10 ppm Cl

- were added to the test vessel prior to the addition of the

target ion, not 10 ppm NO2- and 50 ppm Cl

- as is stated in Chapter 2. This was to

establish the effect of additions of low concentrations of the interfering ions on the

sensor response. The selectivity data are shown in Figure 4.18.

0

0.0005

0.001

0.0015

0.002

0.0025

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


TDAN IDE Design 1 - 1



Craig Alexander

156

Figure 4.18: Selectivity determination of TDAN PVC spin-coated IDE Design 1. Comparison of the

deionised water baseline-subtracted conductance response at 2 MHz to 1–100 ppm nitrate: when no

interfering anion is present, when a fixed concentration of 1 ppm nitrite is present and when a fixed

concentration of 10 ppm chloride is present. The response to 1–100 ppm nitrate of a ‘blank’

membrane, which did not contain TDAN as the ionophore, is also shown

At a frequency of 2 MHz, an increase in the obtained response was observed when

interfering nitrite and chloride anions were present in the test sample. At 100 ppm NO3-,

the baseline-subtracted conductance was 0.24 mS greater when 1 ppm nitrite was

present, and it was 0.37 mS greater when 10 ppm chloride was present, compared with

the deionised water baseline when no interfering anion was present. A large response

was observed from the ‘blank’ membrane which was prepared without ionophore; at

lower concentrations (<20 ppm) the response of the ‘blank’ was around 50% of the

‘active’ membrane, which did contain ionophore, however at high concentrations

(>50 ppm) the ‘blank’ response was over 100% of that of the ‘active’ membrane.

The response of IDE Design 1, when spin-coated with a PVC membrane containing

TDAN as a nitrate-selective ionophore, appeared to be too susceptible to changes in the

background ionic strength of the test solution, and therefore did not provide adequate

nitrate selectivity. The effect of the membrane composition on the sensor response was

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


KNO3 Calibration

1 ppm Nitrite FIM

10 ppm Chloride FIM

Blank membrane

Craig Alexander

157

investigated by coating the sensing area of IDE Design 1 in an alternative membrane

material based on ionophore-doped sol-gels.

4.3 Sol-gel Membrane Sensors

Sol-gel membranes were investigated as an alternative membrane material for use

within impedimetric IDE devices for nitrate sensing, by incorporating the commercial

ionophore TDAN into the sol-gel mixture during its preparation, as described in section

2.1.3. A 3:1 ratio of the silica precursors DEDMS:TEOS were used as the sol-gel

membrane material. The sol-gels were spin-coated over the sensing area of IDE Design

1 at a speed of 2000 rpm. This particular device will be referred to as ‘TDAN DEDMS

spin-coated IDE Design 1’.

4.3.1.1. DEDMS/TEOS Membrane


Figures 4.19A and 4.19B show the conductance and capacitance spectra, respectively,

for the addition of nitrate in the concentration range 1–100 ppm, to IDE Design 1

spin-coated at 2000 rpm in a sol-gel membrane prepared from the silica pre-cursors

DEDMS/TEOS containing TDAN as the ionophore. The corresponding response versus

time data at each extrapolated frequency is shown in Figure 4.20 (G) and

Figure 4.21 (C).

Craig Alexander

158


- to TDAN

DEDMS spin-coated IDE Design 1 (1 Vrms input amplitude). ‘Pure water’ refers to the initial

baseline value and 10-60 mins are the values obtained during the baseline settling period

0.00E+00

1.00E-08

2.00E-08

3.00E-08

4.00E-08

5.00E-08

6.00E-08

7.00E-08

8.00E-08

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07

Cap

acit

ance

(F)

Frequency (Hz)

Cp Pure water

Cp 10 mins

Cp 20 mins

Cp 30 mins

Cp 40 mins

Cp 50 mins

Cp 60 mins

Cp 1 ppm NO3-

Cp 10 ppm NO3-

Cp 20 ppm NO3-

Cp 30 ppm NO3-

Cp 40 ppm NO3-

Cp 50 ppm NO3-

Cp 100 ppm NO3-

0.00E+00

1.00E-03

2.00E-03

3.00E-03

4.00E-03

5.00E-03

6.00E-03

7.00E-03

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07

Co

nd

uct

ance

(S)

Frequency (Hz)

G Pure water

G 10 mins

G 20 mins

G 30 mins

G 40 mins

G 50 mins

G 60 mins

G 1 ppm NO3-

G 10 ppm NO3-

G 20 ppm NO3-

G 30 ppm NO3-

G 40 ppm NO3-

G 50 ppm NO3-

G 100 ppm NO3-

A

B

Craig Alexander

159

Figure 4.20: Conductance response of TDAN DEDMS spin-coated IDE Design 1 upon addition of



The corresponding logarithmic baseline subtracted conductance versus log nitrate

concentration produced a linear response at 100 kHz, 1 MHz and 2 MHz. These data are

shown in Figure 4.22. At 100 kHz, there was a total increase in the measured

conductance of 2.00 mS upon the addition of 100 ppm NO3- to a deionised water

background. At a frequency of 1 MHz, the addition of 100 ppm NO3- resulted in an

increase in the measured conductance from the deionised water baseline value of

4.82 mS, and at 2 MHz this value was 5.16 mS.

0.00E+00

1.00E-03

2.00E-03

3.00E-03

4.00E-03

5.00E-03

6.00E-03

7.00E-03

0 50 100 150 200

Co

nd

uct

ance

(S)

Time (min)

G @ 2 MHz

0.00E+00

1.00E-03

2.00E-03

3.00E-03

4.00E-03

5.00E-03

6.00E-03

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

G @ 1 MHz

0.00E+00

5.00E-04

1.00E-03

1.50E-03

2.00E-03

2.50E-03

0 50 100 150 200

Co

nd

uct

ance

(S)

Time (min)

G @ 100 kHz

0.00E+00

5.00E-05

1.00E-04

1.50E-04

2.00E-04

2.50E-04

3.00E-04

3.50E-04

4.00E-04

4.50E-04

5.00E-04

0 50 100 150 200

Co

nd

uct

ance

(S)

Time (min)

G @ 10 kHz

0.00E+00

1.00E-05

2.00E-05

3.00E-05

4.00E-05

5.00E-05

6.00E-05

7.00E-05

8.00E-05

9.00E-05

0 50 100 150 200

Co

nd

uct

ance

(S)

Time (min)

G @ 1 kHz

0.00E+00

2.00E-06

4.00E-06

6.00E-06

8.00E-06

1.00E-05

1.20E-05

1.40E-05

1.60E-05

1.80E-05

2.00E-05

0 50 100 150 200

Co

nd

uct

ance

(S)

Time (min)

G @ 100 Hz

A B

C D

E F

Craig Alexander

160

Figure 4.21: Capacitance response of TDAN DEDMS spin-coated IDE Design 1 upon addition of



The corresponding logarithmic baseline subtracted capacitance versus log nitrate

concentration produced a linear response at 1 kHz, 10 kHz and 100 kHz. These data are

shown in Figure 4.23. At 1 kHz, there was a total increase in the measured capacitance

of 21.05 nF upon the addition of 100 ppm NO3- to a deionised water background. This

frequency produced the least stable measurements as there appeared to be some drift

associated between the values obtained at each nitrate concentration, particularly upon

addition of 30, 40 and 50 ppm NO3-. At a frequency of 10 kHz, the addition of 100 ppm

NO3- resulted in an increase in the measured capacitance from the deionised water

baseline value of 9.78 nF, and at 100 kHz this value was 2.60 nF.

0.00E+00

5.00E-11

1.00E-10

1.50E-10

2.00E-10

2.50E-10

3.00E-10

3.50E-10

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

Cp @ 2 MHz

0.00E+00

5.00E-11

1.00E-10

1.50E-10

2.00E-10

2.50E-10

3.00E-10

3.50E-10

4.00E-10

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

Cp @ 1 MHz

0.00E+00

5.00E-10

1.00E-09

1.50E-09

2.00E-09

2.50E-09

3.00E-09

3.50E-09

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

Cp @ 100 kHz

0.00E+00

2.00E-09

4.00E-09

6.00E-09

8.00E-09

1.00E-08

1.20E-08

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

Cp @ 10 kHz

0.00E+00

5.00E-09

1.00E-08

1.50E-08

2.00E-08

2.50E-08

3.00E-08

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

Cp @ 1 kHz

0.00E+00

5.00E-09

1.00E-08

1.50E-08

2.00E-08

2.50E-08

3.00E-08

3.50E-08

4.00E-08

4.50E-08

5.00E-08

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

Cp @ 100 Hz

A B

C D

E F

Craig Alexander

161


concentration for TDAN DEDMS spin-coated IDE Design 1.

A – 100 kHz. B – 1 MHz. C – 2 MHz

The logarithmic baseline-subtracted conductance produced a linear response each of the

three extrapolated frequencies over the concentration range 1–100 ppm NO3-. At

100 kHz the R2 value was 0.9985, at 1 MHz it was 0.9918 and at 2 MHz it was 0.9902.


concentration for TDAN DEDMS spin-coated IDE Design 1.

A – 1 kHz. B – 10 kHz. C – 100 kHz

y = 0.652x - 3.6643R² = 0.9918

-4

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

0

0 0.5 1 1.5 2 2.5

log

(Gm

-G

0) (S

)


y = 0.4892x - 3.6598R² = 0.9985

-4

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

0

0 0.5 1 1.5 2 2.5

log

(Gm

-G

0) (S

)

log NO3- concentration (ppm)A B

y = 0.6759x - 3.6911R² = 0.9902

-4

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

0

0 0.5 1 1.5 2 2.5

log

(Gm

-G

0) (S

)

log NO3- concentration (ppm)C

y = 0.7927x - 10.171R² = 0.9975

-10.4

-10.2

-10

-9.8

-9.6

-9.4

-9.2

-9

-8.8

-8.6

-8.4

0 0.5 1 1.5 2 2.5

log

(Cm

-C

0) (

F)


y = 0.45x - 8.9104R² = 0.9988

-9

-8.9

-8.8

-8.7

-8.6

-8.5

-8.4

-8.3

-8.2

-8.1

-8

-7.9

0 0.5 1 1.5 2 2.5

log

(Cm

-C

0) (

F)


y = 0.3631x - 8.3737R² = 0.9909

-8.5

-8.4

-8.3

-8.2

-8.1

-8

-7.9

-7.8

-7.7

-7.6

0 0.5 1 1.5 2 2.5

log

(Cm

-C

0) (

F)


A B

C

Craig Alexander

162

The logarithmic baseline subtracted capacitance produced a linear response at each of

the three extrapolated frequencies over the concentration range 1–100 ppm NO3-. At

1 kHz the R2 value was 0.9975, at 10 kHz it was 0.9988 and at 2 MHz it was 0.9909.

Figure 4.24 shows the baseline-subtracted conductance at the three extrapolated

frequencies for the addition of nitrate in the concentration range 1–100 ppm for TDAN

DEDMS spin-coated IDE Design 1. This figure compares the calibration experiment

using potassium nitrate to adjust the nitrate concentration with the response for the use

of lithium nitrate, and the effect on the observed response when the temperature of the

sample solution was increased from 25 °C to 30 °C. The effect of the capacitive

response under these conditions is displayed in Figure 4.25.


-

concentration for TDAN DEDMS spin-coated IDE Design 1. Three calibration experiments are

shown: the addition of 1–100 ppm NO3- with K

+ as the counter ion, the addition of 1–100 ppm NO3

-

with Li+ as the counter ion and the addition of 1–100 ppm NO3

- with K

+ as the counter ion at 30 °C

A – 100 kHz. B – 1 MHz. C – 2 MHz

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)

log NO3- concentration

0

0.001

0.002

0.003

0.004

0.005

0.006

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0

0.0005

0.001

0.0015

0.002

0.0025

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


A B

C

Craig Alexander

163

At 100 kHz, there was a decrease observed in the recorded conductance measurements

of around 60% when lithium nitrate was used to adjust the nitrate concentration,

compared with the corresponding value when potassium nitrate was used. At 50 ppm

NO3- a Gm - G0 value of 1.50 mS was observed when potassium nitrate was used to

adjust the nitrate concentration within the test vessel. This value decreased to 0.55 mS

when lithium nitrate was used. An increase in the temperature of the test solution from

25 °C to 30 °C resulted in an increase in the conductance response by approximately

5%. At 1 MHz, the obtained Gm - G0 value for 50 ppm NO3- was reduced from 2.87 mS,

when KNO3 was added, to 1.22 mS when LiNO3 was added. The Gm - G0 value for

50 ppm NO3- at 30 °C was 3.12 mS. At 2 MHz, the obtained Gm - G0 value for 50 ppm

NO3- was reduced from 2.97 mS, when KNO3

was added, to 1.28 mS when LiNO3 was

added. The Gm - G0 value for 50 ppm NO3- at 30 °C was 3.32 mS.


-

concentration for TDAN DEDMS spin-coated IDE Design 1. Three calibration experiments are

shown: the addition of 1–100 ppm NO3- with K

+ as the counter ion, the addition of 1–100 ppm NO3

-

with Li+ as the counter ion and the addition of 1–100 ppm NO3

- with K

+ as the counter ion at 30 °C

A – 1 kHz. B – 10 kHz. C – 100 kHz

0

1E-08

2E-08

3E-08

4E-08

5E-08

6E-08

7E-08

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


0

2E-09

4E-09

6E-09

8E-09

1E-08

1.2E-08

0 0.5 1 1.5 2 2.5

Cm

-C

o (F

)


0

5E-10

1E-09

1.5E-09

2E-09

2.5E-09

3E-09

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


A B

C

Craig Alexander

164

At 1 kHz, there was a decrease observed in the recorded capacitance measurements of

around 65% when lithium nitrate was used to adjust the nitrate concentration, compared

with the corresponding value when potassium nitrate was used. At 50 ppm NO3-, a

Cm - C0 value of 16.85 nF was observed when potassium nitrate was used to adjust the

nitrate concentration within the test vessel. This value decreased to 5.62 nF when

lithium nitrate was used. An increase in the temperature of the test solution from 25 °C

to 30 °C resulted in a large increase in the capacitive response. At high nitrate

concentrations (>50 ppm) the response was over two times greater than what was

recorded from the sensor at 25 °C. At 10 kHz, the obtained Cm - C0 value for 50 ppm

NO3-

was reduced from 7.23 nF when KNO3 was added to 2.37 nF when LiNO3 was

added. The effect of temperature on the capacitive response was less marked than the

response observed at 1 kHz, and a slight decrease was observed compared with the

capacitive measurement at 25 °C. The Cm - C0 value for 50 ppm NO3- at 30 °C was

5.96 nF. At 100 kHz, the obtained Cm - C0 value for 50 ppm NO3- was reduced from

1.56 nF, when KNO3 was added, to 0.71 nF when LiNO3 was added. The Cm - C0 value

for 50 ppm NO3- at 30 °C was 1.55 nF; therefore, little effect on the capacitive response

at 100 kHz was observed from increasing the temperature of the test solution.

Figure 4.26 shows the measured G0 values at each of the three extrapolated frequencies

in four solutions of varying ionic strength. The C0 values are shown in Figure 4.27.

Craig Alexander

165

Figure 4.26: Measured baseline conductance value (G0) of TDAN DEDMS spin-coated IDE Design

1 in six separate sample matrices prior to the addition of nitrate

A – 100 kHz. B – 1 MHz. C – 2 MHz

Placing the TDAN DEDMS spin-coated IDE Design 1 into an ionic solution increased

the observed baseline conductance value at each of the extrapolated frequencies. At

100 kHz, a 100 ppm solution of MgSO4 produced a G0 value 1.59 mS greater than the

deionised water baseline. A 200 ppm solution of MgSO4 was 2.03 mS greater and

500 ppm MgSO4 was 3.01 mS greater than the deionised water baseline. A sample of

tap water gave a TDS reading of 55 ppm when taken with a handheld TDS meter, and

increased the G0 by 8.55 mS. A sample of water was taken from a live freshwater

aquarium, the recorded TDS value was 192 ppm and the result from the commercial test

kit showed approximately 5 ppm NO3- was present; the G0 value was increased by

2.18 mS when compared with the G0 obtained for deionised water.

At 1 MHz, a 100 ppm solution of MgSO4 produced a G0 value 3.17 mS greater than the


500 ppm MgSO4 was 7.96 mS greater than the deionised water baseline. The sample of

0

0.002

0.004

0.006

0.008

0.01

0.012

G0

bas

elin

e va

lue

at 2

MH

z


0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.01

G0

ba

seli

ne

va

lue

at

1 M

Hz

Background sample matrix0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.01

G0

bas

elin

e va

lue

at 1

00 k

Hz


A B

C

Craig Alexander

166

tap water increased the G0 by 8.98 mS and the sample of water taken from an aquarium

increased the G0 by 5.42 mS.

At 2 MHz, a 100 ppm solution of MgSO4 produced a G0 value 3.25 mS greater than the


500 ppm MgSO4 was 9.18 mS greater than the deionised water baseline. The sample of

tap water increased the G0 by 8.92 mS and the sample of water taken from an aquarium

increased the G0 by 5.88 mS.

Figure 4.27: Measured baseline capacitance value (C0) of TDAN DEDMS spin-coated IDE Design 1

in six separate sample matrices prior to the addition of nitrate

A – 1 kHz. B – 10 kHz. C – 100 kHz

An increase in the C0 values was also observed for TDAN DEDMS spin-coated IDE

Design 1. At 1 kHz, a 100 ppm solution of MgSO4 produced a C0 value 36.38 nF

greater than the deionised water baseline. A 200 ppm solution of MgSO4 was 64.59 nF

greater and 500 ppm MgSO4 was 110.49 nF greater than the deionised water baseline.

The sample of tap water increased the C0 by 2.51 nF and the sample of water taken from

an aquarium increased the C0 by 66.45 nF.

0

5E-10

1E-09

1.5E-09

2E-09

2.5E-09

3E-09

3.5E-09

4E-09

C0

bas

eli

ne

val

ue

at 1

00

kH

z


0

2E-09

4E-09

6E-09

8E-09

1E-08

1.2E-08

1.4E-08

1.6E-08

1.8E-08

2E-08

C0

bas

elin

e v

alu

e a

t 1

0 k

Hz

Background sample matrix0.00E+00

2.00E-08

4.00E-08

6.00E-08

8.00E-08

1.00E-07

1.20E-07

1.40E-07

C0

ba

selin

e v

alu

e a

t 1

kH

z


A B

C

Craig Alexander

167

At 10 kHz, a 100 ppm solution of MgSO4 produced a C0 value 6.94 nF greater than the

deionised water baseline. A 200 ppm solution of MgSO4 was 8.03 nF greater and

500 ppm MgSO4 was 16.48 nF greater than the deionised water baseline. The sample of

tap water increased the C0 by 2.14 nF and the sample of water taken from an aquarium

increased the C0 by 9.12 nF.

At 100 kHz, a 100 ppm solution of MgSO4 produced a C0 value 1.59 nF greater than the

deionised water baseline. A 200 ppm solution of MgSO4 was 2.18 nF greater and

500 ppm MgSO4 was 3.02 nF greater than the deionised water baseline. The sample of

tap water increased the C0 by 0.44 nF and the sample of water taken from an aquarium

increased the C0 by 2.42 nF.

As for TDAN PVC spin-coated IDE Design 1, a decrease in the sensitivity of the

response of the TDAN DEDMS spin-coated IDE Design 1 sensor was observed when

nitrate was added to a solution with increasing background ionic strength

(data not shown). This suggested that, similar to the results obtained for IDE Design 1

when a PVC ion-selective membrane was spin-coated over the sensing area, the

DEDMS/TEOS sol-gel membrane sensor was too susceptible to changes in the

background ionic strength of the solution, thus not providing adequate nitrate

selectivity. Selectivity experiments using solutions of likely interfering anions within a

freshwater aquarium were carried out to confirm the lack of selectivity towards nitrate.

4.3.1.1.2. Selectivity

As for the selectivity experiments conducted using TDAN PVC spin-coated IDE Design

1, the FIM was carried out using 1 ppm NO2- and 10 ppm Cl

- to establish the selectivity

Craig Alexander

168

of TDAN DEDMS spin-coated IDE Design 1. The deionised water baseline-subtracted

responses are shown in Figure 4.28 (G) and Figure 4.29 (C).

Figure 4.28: Selectivity determination of TDAN DEDMS spin-coated IDE Design 1 (conductive

response). A comparison of: The deionised water baseline-subtracted conductance responses to

1–100 ppm nitrate when no interfering anion is present, when a fixed concentration of 1 ppm nitrite

is present and when a fixed concentration of 10 ppm chloride is present. The response to 1–100 ppm

nitrate of a ‘blank’ membrane, which did not contain TDAN as the ionophore, is also shown

A – 100 kHz. B – 1 MHz. C – 2 MHz

At a frequency of 100 kHz, there was a notable increase in the obtained conductive

response when interfering nitrite and chloride anions were present, particularly at low

NO3- concentrations. At 1 ppm NO3

-, the deionised water baseline-subtracted

conductance was 0.08 mS greater when 1 ppm nitrite was present and the Gm - G0 value

was 0.52 mS greater than the pure nitrate measurement when 10 ppm chloride was

present.

At 1 MHz, a slight increase of around 0.05 mS was observed at NO3- concentrations

between 1–50 ppm when 1 ppm nitrite was present. At 100 ppm NO3- there was an

increase of 0.22 mS compared to when no interfering anion was present. When 10 ppm

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0 0.5 1 1.5 2 2.5

Gm

-G

0 (S

)


0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0 0.5 1 1.5 2 2.5

Gm

-G

0 (S

)


0

0.0005

0.001

0.0015

0.002

0.0025

0 0.5 1 1.5 2 2.5

Gm

-G

0 (S

)


A B

C

Craig Alexander

169

chloride was present, the conductance response at each nitrate concentration was

increased by approximately 0.6 mS.

At 2 MHz, a slight decrease of around 0.05 mS was observed at NO3- concentrations

between 1–50 ppm when 1 ppm nitrite was present. At 100 ppm NO3- there was an

increase of 0.28 mS compared to when no interfering anion was present. When 10 ppm

chloride was present, the conductance response at each nitrate concentration was

increased by approximately 0.6 mS.

At all three extrapolated frequencies, a large response was observed from a ‘blank’

membrane which was prepared without the ionophore. At 100 ppm NO3-, the Gm - G0

value was greater for the ‘blank’ membrane than the membrane which contained

ionophore.

Figure 4.29: Selectivity determination of TDAN DEDMS spin-coated IDE Design 1 (capacitive

response). A comparison of: The deionised water baseline-subtracted capacitance responses to

1–100 ppm nitrate when no interfering anion is present, when a fixed concentration of 1 ppm nitrite

is present and when a fixed concentration of 10 ppm chloride is present. The response to 1–100 ppm

nitrate of a ‘blank’ membrane, which did not contain TDAN as the ionophore, is also shown

A – 1 kHz. B – 10 kHz. C – 100 kHz

0

5E-10

1E-09

1.5E-09

2E-09

2.5E-09

3E-09

3.5E-09

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


0

2E-09

4E-09

6E-09

8E-09

1E-08

1.2E-08

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


0

5E-09

1E-08

1.5E-08

2E-08

2.5E-08

3E-08

3.5E-08

0 0.5 1 1.5 2 2.5

Cm

-C

0 (F

)


A B

C

Craig Alexander

170

As for the conductive responses, TDAN DEDMS spin-coated IDE Design 1 showed a

substantial increase in the capacitive response, at all three extrapolated frequencies, to

interfering nitrite and chloride anions. An almost equivalent response to NO3- was

observed from the device membrane when it was coated with a ‘blank’ membrane

which did not contain TDAN as the ionophore. This suggested that the response was

heavily influenced by changes in the bulk solution, and not from selective interactions

occurring within the membrane between the target ion and the ionophore. Device-to-

device reproducibility was not obtained for TDAN DEDMS spin coated IDE Design 1.

IDE Design 1 did not show adequate selectivity with either a spin-coated PVC

membrane or a spin-coated DEDMS/TEOS sol-gel membrane. The lack of selectivity

appeared to arise from the IDE transduction element, rather than due to the membrane

composition which was deposited over the sensing area. As such, a decision was made

not to investigate this particular sensor design further with either an alternative

polymeric membrane material or with an alternative nitrate-selective ionophore. When

using the FIM to determine selectivity of nitrate sensors, it was quite challenging to

deduce, at low interfering anion concentrations, whether the obtained response was

influenced by the presence of the interfering species. As such, the concentration of the

interfering anions was increased in subsequent selectivity experiments, as described in

section 2.7.2.4, so that the effect of the interfering species could be stated definitively.


IDE Design 1 was fabricated in-house, using a lift-off photolithography technique

followed by e-beam deposition of gold metal, and tested for its feasibility as an

impedimetric nitrate-selective sensor for use within a freshwater aquarium through

spin-coating of the sensing area with a selective polymeric membrane containing a

Craig Alexander

171

commercially-available nitrate ionophore. TDAN was tested as the nitrate-selective

ionophore; PVC and silica gels from the precursors DEDMS/TEOS were used as the

membrane materials.

Conductance and capacitance spectra from the IDE devices were obtained using an LCR

meter by applying an AC voltage of 1 Vrms over a frequency range of 20 Hz–2 MHz.

Six frequencies across the spectra were extrapolated for further analysis and to establish

a frequency region of interest where an analytically-relevant response was obtained

upon addition of the target ion.

When a PVC membrane containing the ionophore TDAN was spin-coated over the

digits of IDE Design 1, a prominent response to increasing nitrate concentration was

observed from changes in the conductance at 2 MHz. When a DEDMS/TEOS sol-gel

containing the ionophore TDAN was used as the membrane material, a linear response

was observed from the change in capacitance at 1 kHz, 10 kHz and 100 kHz; and from

the change in conductance at 100 kHz, 1 MHz and 2 MHz.

It has been established that IDE Design 1, when coupled with a spin-coated, thin-film

polymer ion-selective membrane, was not suitable for use as a standalone nitrate sensor.

The geometric parameters of this particular design appeared to produce an electric field

with a penetration depth much greater than the thickness of the deposited membrane

layer. Therefore, upon addition of an ionic solution to the test vessel, the observed

response was largely generated by changes in the conductivity of the bulk sample, not

from selective interactions between the target anion and the immobilised ionophore

within the membrane layer. This conclusion can be drawn as a large response was

observed when aliquots of the target ion were added to a device that had been coated

with a ‘blank’ membrane that did not contain a selective ionophore. A response was

also observed from non-target, interfering anions such as chloride and nitrite, and a

Craig Alexander

172

large increase in the baseline measurement was observed as the ionic strength of the

background test sample solution was increased. To overcome this problem, and produce

a more selective sensor, either the thickness of the membrane layer would need to be

increased so that more of the generated electric field is contained within it, or the

geometric parameters of the IDE digits would need to be reduced so that the penetration

depth of the resulting electric field would be shorter and therefore contained within a

thin-film membrane.

The thickness of a spin-coated membrane layer could be increased by reducing the

spin-speed, although it was anticipated that reducing this to less than the current speed

(2000 rpm) would not increase the thickness sufficiently to produce a selective layer,

and it could also compromise the coating efficiency. An alternative coating process to

spin-coating could be used, such as drop-coating, although IDE Design 1 would not be

particularly well-suited to drop-coating due to the relatively large footprint of its

sensing area.

Due to the fabrication equipment that was available for producing microsensors, it was

not possible to produce IDEs with further reduced geometries in-house. A commercial

alternative was sought and IDE Design 2 was purchased from MicruX Fluidics. Its use

as an impedimetric nitrite sensor is described throughout Chapter 5. This has digit

widths and interdigital spacings of 10 µm, and therefore should produce an electric field

with a significantly shorter penetration depth when excited with an AC voltage than

IDE Design 1.

Another method for producing a thicker membrane layer is to screen-print the selective

components onto the IDE digits in a suitable support matrix, such as a dielectric screen-

printing ink. This was carried out by producing fully screen-printed IDEs, SP IDE

Craig Alexander

173

Design 1, and the results of using this device as an impedimetric nitrate sensor are

described throughout Chapter 6.

The in-house mircofabrication facilities that were available resulted in a laborious and

time-consuming fabrication process of IDE Design 1 that was only suitable for

producing relatively small batches of sensors. Small failures on individual devices, such

as broken digits or short-circuits caused by adjoining digits, would render it unsuitable

for use as an impedance sensor, and consequently the success rate of producing

electrodes that were available for testing was very low. This quite often led to several

days of manufacturing resulting in only a handful of useable devices. As a result of

these issues with fabrication and the results obtained with these sensors, a decision was

made to investigate alternative IDE sensor devices and no further testing was carried out

using IDE Design 1.

Craig Alexander

174

5 Nitrate Sensing using IDE Design 2

5.1 Sensor Calibration

The conductance (A) and capacitance (B) spectra for uncoated IDE Design 2 without a

membrane layer present responding to 1–100 ppm NO3- in a deionised water

background are shown in Figure 5.1.


- to an uncoated



0.00E+00

5.00E-08

1.00E-07

1.50E-07

2.00E-07

2.50E-07

3.00E-07

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07

Cap

acit

ance

(F)

Frequency (Hz)

Cp Pure water

Cp 10 mins

Cp 20 mins

Cp 30 mins

Cp 40 mins

Cp 50 mins

Cp 60 mins

Cp 1 ppm NO3-

Cp 10 ppm NO3-

Cp 20 ppm NO3-

Cp 30 ppm NO3-

Cp 40 ppm NO3-

Cp 50 ppm NO3-

Cp 100 ppm NO3-

0.00E+00

2.00E-04

4.00E-04

6.00E-04

8.00E-04

1.00E-03

1.20E-03

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07

Co

nd

uct

ance

(S)

Frequency (Hz)

G Pure water

G 10 mins

G 20 mins

G 30 mins

G 40 mins

G 50 mins

G 60 mins

G 1 ppm NO3-

G 10 ppm NO3-

G 20 ppm NO3-

G 30 ppm NO3-

G 40 ppm NO3-

G 50 ppm NO3-

G 100 ppm NO3-

A

B

Craig Alexander

175

Prior to further testing with IDE Design 2, an amendment to the LabVIEW program that

controlled the LCR meter was made so that the input amplitude could be altered. This

was seen as particularly beneficial for this project as if a response was observed using a

lower input amplitude it would allow for the production of a prototype device which

would require a much lower power consumption to function. Similar ISCOM-type

devices have also been reported using lower amplitudes than the 1 Vrms that was used

for IDE Design 1161, 176

. The deionised water baseline-subtracted results, at six

extrapolated frequencies, for uncoated IDE Design 2 upon the addition of NO3- over the

concentration range 1–100 ppm at amplitudes of 1 Vrms, 100 mVrms, 10 mVrms and

1 mVrms are shown in Figure 5.2 (G) and Figure 5.3 (C).


-

concentration for uncoated IDE Design 2 at six frequencies and varying input amplitudes


0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

0.0007

0.0008

0.0009

0.001

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

0.0007

0.0008

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


-0.00005

0

0.00005

0.0001

0.00015

0.0002

0.00025

0.0003

0.00035

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


-0.00002

0

0.00002

0.00004

0.00006

0.00008

0.0001

0.00012

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


A B

C D

E F

Craig Alexander

176

An poor response to the increase in solution conductivity was observed when a reduced

input amplitude was used at low frequencies (below 10 kHz), compared with the 1 Vrms

experiment; however, the low frequency response was much less stable than at higher

frequencies, even at the higher input amplitude. Therefore, it would not be of analytical

importance to further investigate the low frequency results.


-

concentration for uncoated IDE Design 2 at six frequencies and varying input amplitudes


As for the conductance results, the capacitance response to the potassium nitrate

additions to deionised water were less pronounced at a low frequency (<1 kHz) when a

lower input amplitude was applied. However, a response was observed at 10 kHz,

100 kHz and 1 MHz when lower input amplitudes were used. At 2 MHz, the

capacitance response was erratic and did not produce an analytically-relevant response

at any of the tested amplitudes.

-1.4E-12

-1.2E-12

-1E-12

-8E-13

-6E-13

-4E-13

-2E-13

0

2E-13

4E-13

6E-13

8E-13

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


-2E-12

0

2E-12

4E-12

6E-12

8E-12

1E-11

1.2E-11

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


0

1E-10

2E-10

3E-10

4E-10

5E-10

6E-10

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


0

1E-09

2E-09

3E-09

4E-09

5E-09

6E-09

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


0

1E-08

2E-08

3E-08

4E-08

5E-08

6E-08

0 0.5 1 1.5 2 2.5C

m-

C0

(F)


-1E-07

-5E-08

0

5E-08

0.0000001

1.5E-07

0.0000002

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


A B

C D

E F

Craig Alexander

177

As a response in the change of conductance and capacitance was observed at lower

input amplitudes, which was seen as desirable so that a prototype device with a lower

power consumption could be produced, it was decided that subsequent experiments

using IDE Design 2 would be excited with an input AC amplitude of 10 mVrms. At

1 mVrms, the measurements were unstable and therefore this amplitude was not chosen

for further investigation.

Following the experiments conducted using IDE Design 1, it was realised that is was of

the upmost importance to ascertain the behaviour of a device that was coated with a

‘blank’ membrane that did not contain a selective ionophore. This was reflected in the

results that were obtained using IDE Design 2 as an impedimetric nitrate sensor.

5.2 PVC Membrane Sensors

5.2.1.1. Spin-coated Membranes

Initial experiments with IDE Design 2 as a potential nitrate sensor involved spin-coating

(2000 rpm) the sensing area of the device with 1 µl of a PVC membrane containing

1% w/w TDAN as the ionophore. This device will be referred to as ‘TDAN PVC

spin-coated IDE Design 2’.



the addition of nitrate in the concentration range 1–100 ppm, to TDAN PVC

spin-coated IDE Design 2.

Craig Alexander

178


- to TDAN PVC

spin-coated IDE Design 2 (10 mVrms input amplitude). ‘Pure water’ refers to the initial baseline

value. Interference was observed at 50 Hz so this data has been omitted

Figure 5.5 shows the corresponding change in conductance against time data upon

addition of nitrate at each chosen frequency, whilst Figure 5.6 shows the

capacitance data.

0.00E+00

1.00E-09

2.00E-09

3.00E-09

4.00E-09

5.00E-09

6.00E-09

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07

Cap

acit

ance

(F)

Frequency (Hz)

Cp Pure water

Cp 1 ppm NO3-

Cp 10 ppm NO3-

Cp 20 ppm NO3-

Cp 30 ppm NO3-

Cp 40 ppm NO3-

Cp 50 ppm NO3-

Cp 100 ppm NO3-

0.00E+00

5.00E-06

1.00E-05

1.50E-05

2.00E-05

2.50E-05

3.00E-05

3.50E-05

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07

Co

nd

uct

ance

(S)

Frequency (Hz)

G Pure water

G 1 ppm NO3-

G 10 ppm NO3-

G 20 ppm NO3-

G 30 ppm NO3-

G 40 ppm NO3-

G 50 ppm NO3-

G 100 ppm NO3-

A

B

Craig Alexander

179

Figure 5.5: Conductance response of TDAN PVC spin-coated IDE Design 2 upon addition of



Adding 1–100 ppm NO3- to a deionised water background did not appear to produce a

linear response at any frequency from measuring the conductance of TDAN PVC

spin-coated IDE Design 2.

0.00E+00

5.00E-06

1.00E-05

1.50E-05

2.00E-05

2.50E-05

3.00E-05

3.50E-05

0 20 40 60 80 100 120 140

Co

nd

uct

ance

(S)

Time (min)

0.00E+00

2.00E-06

4.00E-06

6.00E-06

8.00E-06

1.00E-05

1.20E-05

1.40E-05

1.60E-05

1.80E-05

2.00E-05

0 20 40 60 80 100 120 140

Co

nd

uct

ance

(S)

Time (min)

0.00E+00

1.00E-06

2.00E-06

3.00E-06

4.00E-06

5.00E-06

6.00E-06

7.00E-06

8.00E-06

9.00E-06

1.00E-05

0 20 40 60 80 100 120 140

Co

nd

uct

ance

(S)

Time (min)

0.00E+00

1.00E-06

2.00E-06

3.00E-06

4.00E-06

5.00E-06

6.00E-06

7.00E-06

0 20 40 60 80 100 120 140

Co

nd

uct

ance

(S)

Time (min)

0.00E+00

5.00E-07

1.00E-06

1.50E-06

2.00E-06

2.50E-06

3.00E-06

3.50E-06

4.00E-06

0 20 40 60 80 100 120 140

Co

nd

uct

ance

(S)

Time (min)

0.00E+00

2.00E-07

4.00E-07

6.00E-07

8.00E-07

1.00E-06

1.20E-06

1.40E-06

1.60E-06

1.80E-06

0 20 40 60 80 100 120 140

Co

nd

uct

ance

(S)

Time (min)

A B

C D

E F

Craig Alexander

180

Figure 5.6: Capacitance response of TDAN PVC spin-coated IDE Design 2 upon addition of



The most analytically-relevant response from TDAN PVC spin-coated IDE Design 2

was from the capacitance response at a frequency of 2 MHz. The total addition of

100 ppm NO3- to a deionised water background resulted in a Cm – C0 of 1.15 pF. At

1 MHz, a linear increase to nitrate was observed up to 50 ppm NO3-, followed by a

slight decrease at 100 ppm. The logarithmic baseline-subtracted capacitance against log

NO3- concentration at 2 MHz is shown in Figure 5.7.

1.154E-10

1.156E-10

1.158E-10

1.160E-10

1.162E-10

1.164E-10

1.166E-10

1.168E-10

1.170E-10

0 20 40 60 80 100 120 140

Cap

acit

ance

(F)

Time (min)

1.144E-10

1.146E-10

1.148E-10

1.150E-10

1.152E-10

1.154E-10

1.156E-10

1.158E-10

1.160E-10

0 20 40 60 80 100 120 140

Cap

acit

ance

(F)

Time (min)

1.150E-10

1.155E-10

1.160E-10

1.165E-10

1.170E-10

1.175E-10

1.180E-10

1.185E-10

1.190E-10

0 20 40 60 80 100 120 140

Cap

acit

ance

(F)

Time (min)

1.28E-10

1.30E-10

1.32E-10

1.34E-10

1.36E-10

1.38E-10

1.40E-10

1.42E-10

1.44E-10

1.46E-10

1.48E-10

0 20 40 60 80 100 120 140

Cap

acit

ance

(F)

Time (min)

0.00E+00

5.00E-11

1.00E-10

1.50E-10

2.00E-10

2.50E-10

3.00E-10

3.50E-10

4.00E-10

4.50E-10

5.00E-10

0 20 40 60 80 100 120 140

Cap

acit

ance

(F)

Time (min)

0.00E+00

2.00E-10

4.00E-10

6.00E-10

8.00E-10

1.00E-09

1.20E-09

1.40E-09

1.60E-09

1.80E-09

2.00E-09

0 20 40 60 80 100 120 140

Cap

acit

ance

(F)

Time (min)

A B

C D

E F

Craig Alexander

181



Taking the logarithm of the deionised water baseline-subtracted capacitance at 2 MHz

and plotting it against log NO3- concentration produced a reasonably linear response

with a R2 value of 0.9773.

Figure 5.8 shows the deionised water baseline-subtracted capacitance at 2 MHz for the

addition of NO3- to three separate TDAN PVC spin-coated IDE Design 2 devices.

Figure 5.8: Deionised water baseline-subtracted capacitance (Cm – C0) of three TDAN PVC

spin-coated IDE Design 2 devices against log NO3- concentration, for reproducibility determination

y = 1.1092x - 14.091R² = 0.9773

-14.5

-14

-13.5

-13

-12.5

-12

-11.5

0 0.5 1 1.5 2 2.5

log

(Cm

-C

0)

(F)


-2E-13

0

2E-13

4E-13

6E-13

8E-13

1E-12

1.2E-12

1.4E-12

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)





Craig Alexander

182

Poor device-to-device reproducibility was observed from TDAN PVC spin-coated IDE

Design 2. The third tested device showed a decrease below the baseline response up to

20 ppm NO3- before increasing at higher NO3

- concentrations. At 50 ppm NO3

-, the

%RSD was calculated as 50.90%.


As the previously tested device, IDE Design 1, was found to produce a large response to

the target nitrate anion when coated with a ‘blank’ membrane, it was deemed important

to ascertain the response of TDAN PVC IDE Design 2 when coated with a ‘blank’

membrane prior to investigating the effects of interfering anions on the response. The

comparison of the capacitance response at 2 MHz of a TDAN PVC spin-coated IDE

Design 2 device containing the ionophore with a device coated with a ‘blank’

membrane is shown in Figure 5.9.

Figure 5.9: Selectivity determination of TDAN PVC spin-coated IDE Design 2. Comparison of the

deionised water baseline-subtracted capacitance response at 2 MHz to 1–100 ppm nitrate of device

with an ‘active’ membrane, containing 1% w/w TDAN as the ionophore, and a device with a

‘blank’ membrane, which did not contain any ionophore

0

5E-13

1E-12

1.5E-12

2E-12

2.5E-12

3E-12

3.5E-12

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


KNO3 Calibration

Blank membrane

Craig Alexander

183

It was noted that a large response to nitrate was observed when a ‘blank’ PVC

membrane, which did not contain a nitrate-selective ionophore, was spin-coated onto

IDE Design 2. This suggested that the spin-coated membranes were not of an adequate

thickness to ensure a selective response; however, the observed response was largely

caused by non-selective changes in the conductivity of the test solution, rather than

target ion/ionophore interactions within the membrane. Due to the small overall

footprint of IDE Design 2, it was possible to simply drop-coat the membrane solution

over the sensing area, to produce a PVC membrane layer with increased thickness and

the added benefit of not wasting any of the ‘cocktail’ solution. Further testing of IDE

Design 2 was therefore carried out by depositing the PVC ion-selective membrane

‘cocktail’ by drop-coating.

5.2.1.2. Drop-coated Membranes

IDE Design 2 was prepared as a nitrate-selective sensor by drop-coating 1 µl of a

10% w/v PVC membrane ‘cocktail’ containing 1% w/w TDAN as the ionophore onto

the surface of the sensing area. This was to afford a thicker membrane layer and

therefore impart greater ion-selectivity on the sensor than the previously tested

spin-coated membrane electrodes, due to a larger proportion of the generated electric

field from the IDEs being enclosed within the membrane layer. This device will be

referred to as ‘TDAN PVC drop-coated IDE Design 2’.

Craig Alexander

184


Figures 5.10 and 5.11 show the measured conductance and capacitance, respectively,

for a TDAN PVC drop-coated IDE Design 2 against time for a calibration over

1–100 ppm NO3- in a deionised water background. Five extrapolated frequencies from

the resulting spectra are shown. There was no discernible response obtained at 100 Hz

therefore this data has not been displayed.

Figure 5.10: Conductance response versus time of TDAN PVC drop-coated IDE Design 2

(10% w/v) upon addition of 1–100 ppm nitrate at five frequencies.

A – 1 kHz. B – 10 kHz. C – 100 kHz. D – 1 MHz. E – 2 MHz.

Craig Alexander

185

Figure 5.11: Capacitance response versus time of TDAN PVC drop-coated IDE Design 2

(10% w/v) upon addition of 1–100 ppm nitrate at five frequencies.


It was evident from both the conductance and capacitance results that when a calibration

for IDE Design 2 containing a drop-coated membrane was carried out in the previously

described manner, insufficient time had elapsed for a stable measurement to be taken at

each NO3- concentration. As the membrane thickness has been increased due to

depositing it via drop-coating rather than spin-coating as before, the diffusion distance

for the ions to move to into the membrane layer will have also increased, which in turn,

will have resulted in a longer response time for the sensor. From the results shown in

Figure 5.10 and Figure 5.11, it was not possible to accurately deduce the

baseline-subtracted measurement at any frequency for either the conductive or

capacitive response due to the ‘drift’ associated with this longer response time. It was,

therefore, difficult to distinguish between the end of one NO3- concentration addition

and the beginning of the next. It was also noted that with this particular membrane

composition, a much longer pre-soaking period was required to ensure a stable baseline

Craig Alexander

186

was reached quickly during the measurements. Further experimentation was therefore

carried out to establish both the soaking time required and the response time of this

particular membrane composition when drop-coated onto IDE Design 2.

The baseline settling time of TDAN PVC drop-coated IDE Design 2 was established by

placing a newly-prepared sensor into a background sample of deionised water and

monitoring the response every two hours until a stable measurement was reached. These

data are displayed in Figure 5.12 (G) and Figure 5.13 (C).

Figure 5.12: Conductance deionised water baseline settling response of a dry TDAN PVC

drop-coated IDE Design 2 (10% w/v) at five frequencies


At 1 kHz, the conductance response showed a slight increase over the initial 12 hours;

however, the measured response continued to fluctuate throughout the 96 hours over

which the experiment was conducted. At 10 kHz and 100 kHz the conductance response

0.00E+00

1.00E-04

2.00E-04

3.00E-04

4.00E-04

5.00E-04

6.00E-04

0 20 40 60 80 100

Co

nd

uct

ance

(S)

Time (hours)

0.00E+00

5.00E-05

1.00E-04

1.50E-04

2.00E-04

2.50E-04

3.00E-04

3.50E-04

4.00E-04

4.50E-04

5.00E-04

0 20 40 60 80 100

Con

du

ctan

ce (S

)

Time (hours)

0.00E+00

2.00E-05

4.00E-05

6.00E-05

8.00E-05

1.00E-04

1.20E-04

0 20 40 60 80 100

Co

nd

uct

ance

(S)

Time (hours)

0.00E+00

1.00E-06

2.00E-06

3.00E-06

4.00E-06

5.00E-06

6.00E-06

7.00E-06

8.00E-06

9.00E-06

1.00E-05

0 20 40 60 80 100

Co

nd

uct

ance

(S)

Time (hours)

0.00E+00

5.00E-07

1.00E-06

1.50E-06

2.00E-06

2.50E-06

0 20 40 60 80 100

Co

nd

uct

ance

(S)

Time (hours)

A B

C D

E

Craig Alexander

187

was much more stable, although some ‘drift’ was still evident after 96 hours. The

conductance response at 1 MHz and 2 MHz was completely stable after approximately

36 hours.

Figure 5.13: Capacitance deionised water baseline settling response of a dry TDAN PVC

drop-coated IDE Design 2 (10% w/v) at five frequencies


At 1 kHz, the capacitance response showed a slight increase over the initial 24 hours;

however, the measured response continued to fluctuate throughout the 96 hours over

which the experiment was conducted. At 10 kHz and 100 kHz the capacitance response

was much more stable, although some ‘drift’ was still evident after 96 hours. The

capacitance response at 1 MHz increased initially and showed near-stability after eight

hours. There was a slight decrease in the measured response; however, there was little

1.20E-10

1.22E-10

1.24E-10

1.26E-10

1.28E-10

1.30E-10

1.32E-10

1.34E-10

1.36E-10

1.38E-10

1.40E-10

0 20 40 60 80 100

Cap

acit

ance

(F)

Time (hours)

9.00E-11

1.10E-10

1.30E-10

1.50E-10

1.70E-10

0 20 40 60 80 100

Cap

acit

ance

(F)

Time (hours)

0.00E+00

5.00E-11

1.00E-10

1.50E-10

2.00E-10

2.50E-10

3.00E-10

3.50E-10

4.00E-10

0 20 40 60 80 100

Cap

acit

ance

(F)

Time (hours)

0.00E+00

1.00E-10

2.00E-10

3.00E-10

4.00E-10

5.00E-10

6.00E-10

7.00E-10

0 20 40 60 80 100

Cap

acit

ance

(F)

Time (hours)

0.00E+00

1.00E-10

2.00E-10

3.00E-10

4.00E-10

5.00E-10

6.00E-10

7.00E-10

8.00E-10

9.00E-10

0 20 40 60 80 100

Cap

acit

ance

(F)

Time (hours)

A B

C D

E

Craig Alexander

188

drift observed upon completion of the experiment. A similar response to the 1 MHz

results was also observed at 2 MHz.

The response time of TDAN PVC drop-coated IDE Design 2 was established by adding

an aliquot of 50 ppm NO3- to a deionised water background and monitoring the

response until a stable measurement was reached. These data are displayed in

Figure 5.14 (G) and Figure 5.15 (C).

Figure 5.14: Conductance response of TDAN PVC drop-coated IDE Design 2 (10% w/v) upon the

addition of 50 ppm NO3- to a deionised water background at five frequencies. The aliquot of nitrate

was added to the solution after 60 minutes


No observable response was seen from changes in the conductance at lower frequencies

(1 kHz, 10 kHz and 100 kHz) upon the addition of 50 ppm NO3- to a deionised water

background. At 1 MHz and 2 MHz, there was a noticeable increase of approximately

5.20E-04

5.30E-04

5.40E-04

5.50E-04

5.60E-04

5.70E-04

5.80E-04

5.90E-04

0 200 400 600 800 1000 1200 1400

Co

nd

uct

ance

(S)

Time (min)

4.40E-04

4.50E-04

4.60E-04

4.70E-04

4.80E-04

4.90E-04

5.00E-04

5.10E-04

0 200 400 600 800 1000 1200 1400

Co

nd

uct

ance

(S)

Time (min)

8.40E-06

8.60E-06

8.80E-06

9.00E-06

9.20E-06

9.40E-06

9.60E-06

9.80E-06

1.00E-05

1.02E-05

0 200 400 600 800 1000 1200 1400

Co

nd

uct

ance

(S)

Time (min)

0.00E+00

5.00E-07

1.00E-06

1.50E-06

2.00E-06

2.50E-06

0 200 400 600 800 1000 1200 1400

Co

nd

uct

ance

(S)

Time (min)

1.01E-04

1.02E-04

1.03E-04

1.04E-04

1.05E-04

1.06E-04

1.07E-04

1.08E-04

1.09E-04

0 200 400 600 800 1000 1200 1400

Co

nd

uct

ance

(S)

Time (min)

A B

C D

E

Craig Alexander

189

30 µS; however, the response did not settle and was still ‘drifting’ around 18 hours

following the addition of nitrate to the test solution.

Figure 5.15: Capacitance response of TDAN PVC drop-coated IDE Design 2 (10% w/v) upon the




At 1 kHz, very little response was observed upon the addition of 50 ppm nitrate to a

deionised water background. At 10 kHz and 100 kHz the response was continuing to

drift around 18 hours following the addition of 50 ppm NO3-. At 1 MHz, there was a

distinct increase observed to the addition of nitrate. After two hours, the Cm – C0 was

approximately 3.2 pF, and the response remained stable for the remainder of the

experiment. A similar response was also seen at 2 MHz, after two hours an increase in

the baseline response of approximately 1.2 pF was observed and the response remained

stable for the remainder of the experiment.

1.500E-10

1.505E-10

1.510E-10

1.515E-10

1.520E-10

1.525E-10

1.530E-10

1.535E-10

1.540E-10

0 200 400 600 800 1000 1200 1400

Ca

pa

cita

nce

(F)

Time (min)

1.345E-10

1.350E-10

1.355E-10

1.360E-10

1.365E-10

0 200 400 600 800 1000 1200 1400

Ca

pa

cita

nce

(F)

Time (min)

3.60E-10

3.65E-10

3.70E-10

3.75E-10

3.80E-10

3.85E-10

3.90E-10

0 200 400 600 800 1000 1200 1400

Ca

pa

cita

nce

(F)

Time (min)

5.70E-10

5.75E-10

5.80E-10

5.85E-10

5.90E-10

5.95E-10

6.00E-10

6.05E-10

0 200 400 600 800 1000 1200 1400

Ca

pa

cita

nce

(F)

Time (min)

7.60E-10

7.70E-10

7.80E-10

7.90E-10

8.00E-10

8.10E-10

8.20E-10

8.30E-10

8.40E-10

8.50E-10

0 200 400 600 800 1000 1200 1400

Ca

pa

cita

nce

(F)

Time (min)

A B

C D

E

Craig Alexander

190

In an attempt to reduce the response time of the drop-coated membrane sensors, the

PVC ‘cocktail’ solution viscosity was reduced by decreasing the mass of the membrane

components that were dissolved in 5 ml THF. The previously tested 10% w/v ‘cocktail’

was reduced to 5% w/v by dissolving 250 mg of the membrane components into 5 ml

THF, as opposed to 500 mg. A 2% w/v ‘cocktail’ was prepared by dissolving 100 mg of

the membrane components into 5 ml THF. The membrane composition was prepared in

the same ratios of plasticiser: PVC, as described previously (Table 2.1; Chapter 2);

however, the mass of ionophore dissolved into 5 ml THF was kept constant (5 mg) to

ensure that the 1 µl of ‘cocktail’ that was drop-coated contained the same mass of the

ionophore despite the varying solution viscosity. Therefore, the 5% w/v ‘cocktail’

contained 2% w/w ionophore and the 2% w/v ‘cocktail’ contained 5% w/w ionophore.

A 1% w/v ‘cocktail’, prepared by dissolving 50 mg of the membrane components into

5 ml THF, produced a polymeric coating that was unsuitable for use as an ion-selective

membrane. The resulting solution, when cast onto a planar substrate, did not produce a

homogenous coating. It is likely that an insufficient amount of polymer was present

within the ‘cocktail’ to produce a suitable membrane layer.

Once drop-coated onto the electrodes, each membrane composition was allowed to soak

in deionised water for at least 48 hours prior to testing. The 50 ppm NO3- response time

data for IDE Design 2 containing a 5% w/v (2% w/w TDAN) drop-coated PVC

membrane are shown in Figure 5.16 (G) and Figure 5.17 (C).

Craig Alexander

191





At 1 kHz and 10 kHz, very little response was observed from the change in the

measured conductance upon the addition of 50 ppm nitrate to a deionised water

background. At 100 kHz there was a noticeable increase in the conductance response

from the deionised water baseline; however, this response did not adequately settle and

was continuing to drift around 24 hours following the addition of 50 ppm NO3-. A

similar conductance response was observed at a frequency of 1 MHz and 2 MHz.

8.00E-05

9.00E-05

1.00E-04

1.10E-04

1.20E-04

1.30E-04

1.40E-04

1.50E-04

1.60E-04

0 500 1000 1500 2000

Co

nd

uct

ance

(S)

Time (min)

8.00E-05

9.00E-05

1.00E-04

1.10E-04

1.20E-04

1.30E-04

1.40E-04

0 500 1000 1500 2000

Co

nd

uct

ance

(S)

Time (min)

3.80E-05

3.90E-05

4.00E-05

4.10E-05

4.20E-05

4.30E-05

4.40E-05

4.50E-05

4.60E-05

4.70E-05

0 500 1000 1500 2000

Co

nd

uct

ance

(S)

Time (min)

3.60E-06

3.70E-06

3.80E-06

3.90E-06

4.00E-06

4.10E-06

4.20E-06

0 500 1000 1500 2000

Co

nd

uct

ance

(S)

Time (min)

0.00E+00

2.00E-07

4.00E-07

6.00E-07

8.00E-07

1.00E-06

1.20E-06

1.40E-06

0 500 1000 1500 2000

Co

nd

uct

ance

(S)

Time (min)

A B

C D

E

Craig Alexander

192





A similar pattern was observed for the capacitance response time data for TDAN PVC

drop-coated IDE Design 2 (5% w/v). No observable response was seen from changes in

the conductance at 1 kHz. At 10 kHz, 100 kHz, 1 MHz and 2 MHz, there was a

noticeable increase in the capacitance response from the deionised water baseline;

however, this response did not adequately settle and was continuing to drift around

24 hours following the addition of 50 ppm NO3-.

The IDE Design 2 sensor that comprised of a 5% w/v PVC membrane ‘cocktail’ did not

show a marked improvement in the response time for either the conductance or

capacitance when compared with the previously tested 10% w/v membrane. Therefore,

the response time of a sensor drop-coated with the further reduced 2% w/v PVC

1.234E-10

1.236E-10

1.238E-10

1.240E-10

1.242E-10

1.244E-10

1.246E-10

1.248E-10

1.250E-10

0 500 1000 1500 2000

Ca

pa

cita

nce

(F)

Time (min)

1.245E-10

1.250E-10

1.255E-10

1.260E-10

1.265E-10

1.270E-10

1.275E-10

0 500 1000 1500 2000

Ca

pa

cita

nce

(F)

Time (min)

1.75E-10

1.80E-10

1.85E-10

1.90E-10

1.95E-10

2.00E-10

2.05E-10

0 500 1000 1500 2000

Ca

pa

cita

nce

(F)

Time (min)

2.75E-10

2.80E-10

2.85E-10

2.90E-10

2.95E-10

3.00E-10

3.05E-10

0 500 1000 1500 2000

Ca

pa

cita

nce

(F)

Time (min)

3.20E-10

3.30E-10

3.40E-10

3.50E-10

3.60E-10

3.70E-10

3.80E-10

3.90E-10

4.00E-10

0 500 1000 1500 2000

Ca

pa

cita

nce

(F)

Time (min)

A B

C D

E

Craig Alexander

193

membrane solution was investigated. The 50 ppm NO3- response time data for IDE

Design 2 containing a 2% w/v (5% w/w TDAN) drop-coated PVC membrane are shown

in Figure 5.18 (G) and Figure 5.19 (C).





At 1 kHz, there was an initial increase from the deionised water baseline conductance

response of approximately 1.5 µS when 50 ppm NO3- was added; however, the response

appeared unstable throughout the course of the experiment. At 10 kHz, there was an

initial increase of approximately 1.3 µS upon the addition of 50 ppm NO3- to the test

solution. This response was also erratic and continued to increase over the 24 hours

which the experiment was conducted. At 100 kHz, the conductance response continued

to drift around 24 hours following the addition of 50 ppm NO3-. At 1 MHz, the response

5.00E-05

7.00E-05

9.00E-05

1.10E-04

1.30E-04

1.50E-04

1.70E-04

0 200 400 600 800 1000 1200 1400 1600

Co

nd

uct

ance

(S)

Time (min)

5.00E-05

6.00E-05

7.00E-05

8.00E-05

9.00E-05

1.00E-04

1.10E-04

1.20E-04

1.30E-04

1.40E-04

0 200 400 600 800 1000 1200 1400 1600

Co

nd

uct

ance

(S)

Time (min)

3.30E-05

3.50E-05

3.70E-05

3.90E-05

4.10E-05

4.30E-05

4.50E-05

4.70E-05

4.90E-05

5.10E-05

0 200 400 600 800 1000 1200 1400 1600

Co

nd

uct

ance

(S)

Time (min)

1.05E-05

1.10E-05

1.15E-05

1.20E-05

1.25E-05

1.30E-05

0 200 400 600 800 1000 1200 1400 1600

Co

nd

uct

ance

(S)

Time (min)

3.00E-06

3.50E-06

4.00E-06

4.50E-06

5.00E-06

5.50E-06

6.00E-06

0 200 400 600 800 1000 1200 1400 1600

Co

nd

uct

ance

(S)

Time (min)

A B

C D

E

Craig Alexander

194

appeared to become stable at a value of approximately 50 µS above the G0 value.

Approximately 90% of this value was reached within the first six hours. At 2 MHz, a

near-stable response was obtained at approximately 65 µS above the G0 value, which

was reached within around 6 hours.





For the capacitance response of TDAN PVC drop-coated IDE Design 2 (2% w/v), at

1 kHz, there was an initial increase from the deionised water baseline conductance

response of approximately 120 pF when 50 ppm NO3- was added; however, the

response appeared unstable throughout the course of the experiment. At 10 kHz, there

was an initial increase of approximately 11 pF upon the addition of 50 ppm NO3- to the

test solution, although this response continued to increase at the culmination of the

1.260E-10

1.265E-10

1.270E-10

1.275E-10

1.280E-10

1.285E-10

1.290E-10

0 200 400 600 800 1000 1200 1400 1600

Ca

pa

cita

nce

(F)

Time (min)

1.260E-10

1.270E-10

1.280E-10

1.290E-10

1.300E-10

1.310E-10

1.320E-10

0 200 400 600 800 1000 1200 1400 1600

Ca

pa

cita

nce

(F)

Time (min)

1.55E-10

1.60E-10

1.65E-10

1.70E-10

1.75E-10

1.80E-10

1.85E-10

1.90E-10

0 200 400 600 800 1000 1200 1400 1600

Ca

pa

cita

nce

(F)

Time (min)

2.85E-10

2.90E-10

2.95E-10

3.00E-10

3.05E-10

3.10E-10

3.15E-10

3.20E-10

3.25E-10

3.30E-10

0 200 400 600 800 1000 1200 1400 1600

Ca

pa

cita

nce

(F)

Time (min)

5.00E-10

5.50E-10

6.00E-10

6.50E-10

7.00E-10

7.50E-10

8.00E-10

0 200 400 600 800 1000 1200 1400 1600

Ca

pa

cita

nce

(F)

Time (min)

A B

C D

E

Craig Alexander

195

experiment; by 24 hours the capacitance response had reached approximately 35 pF. At

100 kHz, the capacitance response was reasonably stable at 24 hours following the

addition of 50 ppm NO3-, with a value approximately 27 pF greater than the C0 value.

At 1 MHz, the response appeared to become stable at a value of approximately 4.5 pF

above the C0 value, which was reached within 8 hours of the addition of 50 ppm NO3-.

At 2 MHz, there was an increase from C0 of approximately 2.0 pF; however, the

obtained response less stable than what had been observed at 100 kHz and 1 MHz.

The most distinct and stable responses from the addition of 50 ppm NO3- to a deionised

water background solution were seen when the least viscous PVC membrane ‘cocktail’

solution (2% w/v) was used. There was a notable increase in both the measured

conductance and capacitance of this particular membrane composition upon the addition

of 50 ppm NO3-, which also demonstrated the least amount of ‘drift’ in the response

over time. Although several hours were required for the response to be fully stable, 90%

of this final response was reached within 2 hours, and therefore it was deemed that this

particular composition was suitable for additional testing. Further investigation with

IDE Design 2 was therefore carried out using a drop-coated 2% w/v membrane

‘cocktail’ containing 5% w/w TDAN as the nitrate-selective ionophore.

The observed response time for this drop-coated membrane composition was

substantially longer than what had previously been observed when a spin-coated

membrane was used. Therefore, the methodology for conducting the calibration

experiments was adjusted to measure fewer target ion concentrations over the range

1–100 ppm NO3-, with the sensor exposed to each nitrate concentration for a longer

period of time. The subsequent experiments were conducted by adding 1, 20, 50 and

100 ppm NO3- sequentially to a deionised water background test solution and recording

Craig Alexander

196

the response from the sensor at five minute intervals for 1 hour. The resulting response

versus time data for TDAN PVC drop-coated IDE Design 2 (2% w/v) are shown in

Figure 5.20 (G) and Figure 5.21 (C). The response at 1 kHz was completely unstable for

both the measured conductance and capacitance, so this frequency was not

investigated further.

Figure 5.20: Conductance response versus time of TDAN PVC drop-coated IDE Design 2 (2% w/v)

upon addition of 1–100 ppm nitrate at four frequencies.

A – 10 kHz. B – 100 kHz. C – 1 MHz. D – 2 MHz.

At 10 kHz and 100 kHz, it was noted that the conductance response was not adequately

stable for the average measurement at each separate concentration to be recorded.

However, at 1 MHz and 2 MHz the changes in nitrate concentration were distinct and

appeared settled within the 1 hour period over which they were recorded. At 1 MHz,

upon the total addition of 100 ppm NO3-, the Gm – G0 value settled at approximately

57.7 µS. At 2 MHz, upon the total addition of 100 ppm NO3-, the Gm – G0 value settled

at approximately 70.1 µS.

5.48E-03

5.48E-03

5.49E-03

5.49E-03

5.50E-03

5.50E-03

0 50 100 150 200 250 300 350 400

Co

nd

uct

ance

(S)

Time (min)

1 ppm NO3-

5.59E-03

5.60E-03

5.61E-03

5.62E-03

5.63E-03

5.64E-03

5.65E-03

5.66E-03

5.67E-03

0 50 100 150 200 250 300 350 400

Co

nd

uct

ance

(S)

Time (min)

100 ppm NO3-

5.50E-03

5.51E-03

5.52E-03

5.53E-03

5.54E-03

5.55E-03

5.56E-03

5.57E-03

0 50 100 150 200 250 300 350 400

Co

nd

uct

ance

(S)

Time (min)

50 ppm NO3-

5.48E-03

5.48E-03

5.48E-03

5.48E-03

5.48E-03

5.48E-03

5.48E-03

5.48E-03

5.48E-03

0 50 100 150 200 250 300 350 400

Co

nd

uct

ance

(S)

Time (min)

2 hour baseline

A B

C D

20 ppm NO3-

Craig Alexander

197

Figure 5.21: Capacitance response versus time of TDAN PVC drop-coated IDE Design 2 (2% w/v)



At the higher frequencies of 1 MHz and 2 MHz, there was an initial decrease below the

C0 value of approximately 570 fF when 1 ppm NO3- was added to the deionised water

background solution. The response remained lower than C0 until 50 ppm NO3- had been

added. At 1 MHz, upon the total addition of 100 ppm NO3-, the Cm – C0 value settled at

approximately 2.9 pF. At 2 MHz, upon the total addition of 100 ppm NO3-, the Cm – C0

value settled at approximately 806 fF. There did not appear to be a stable capacitance

response at 10 kHz, the response of each addition of nitrate to the deionised water

background solution was indistinguishable. The most prominent capacitance response

for TDAN PVC drop-coated IDE Design 2 was obtained at a frequency of 100 kHz,

where each nitrate addition could be distinguished. Upon the total addition of 100 ppm

NO3-, the Cm – C0 value settled at approximately 27.4 pF.

The corresponding logarithmic baseline-subtracted response against log nitrate

concentration data are shown in Figure 5.22 (G) and Figure 5.23 (C).

1.034E-10

1.036E-10

1.038E-10

1.040E-10

1.042E-10

1.044E-10

1.046E-10

1.048E-10

1.050E-10

1.052E-10

0 50 100 150 200 250 300 350 400

Cap

acit

ance

(F)

Time (min)

1.025E-10

1.030E-10

1.035E-10

1.040E-10

1.045E-10

1.050E-10

1.055E-10

1.060E-10

1.065E-10

1.070E-10

0 50 100 150 200 250 300 350 400

Cap

acit

ance

(F)

Time (min)

8.00E-11

9.00E-11

1.00E-10

1.10E-10

1.20E-10

1.30E-10

1.40E-10

0 50 100 150 200 250 300 350 400

Cap

acit

ance

(F)

Time (min)

8.00E-11

9.00E-11

1.00E-10

1.10E-10

1.20E-10

1.30E-10

1.40E-10

1.50E-10

1.60E-10

1.70E-10

1.80E-10

0 50 100 150 200 250 300 350 400

Cap

acit

ance

(F)

Time (min)

A B

C D

Craig Alexander

198


concentration for TDAN PVC drop-coated IDE Design 2 (2% w/v) at two frequencies.

A –1 MHz. B – 2 MHz.


the two extrapolated frequencies over the concentration range 1–100 ppm NO3-. At

1 MHz the R2 value was 0.9921 and at 2 MHz it was 0.9887.


concentration for TDAN PVC drop-coated IDE Design 2 (2% w/v) at 100 kHz

Taking the logarithm of the deionised water baseline-subtracted capacitance at 100 kHz

and plotting it against log NO3- concentration confirmed the linearity of the response,

with a R2 value 0.9968.

The effect of changing the ionophore within the selective membrane layer was

investigated, to establish the affect this had upon the sensor response. A 2% w/v

membrane ‘cocktail’ containing 5% w/w of the ionophore NO3V was deposited over

y = 0.6483x - 5.6019R² = 0.9921

-6

-5

-4

-3

-2

-1

0

0 0.5 1 1.5 2 2.5

log

Gm

-G

o(S

)


y = 0.6858x - 5.6029R² = 0.9887

-6

-5

-4

-3

-2

-1

0

0 0.5 1 1.5 2 2.5

log

Gm

-G

o(S

)


y = 1.0663x - 12.658R² = 0.9968

-14

-12

-10

-8

-6

-4

-2

0

0 0.5 1 1.5 2 2.5

log

Cm

-C

o (F

)


Craig Alexander

199

the sensing area of IDE Design 2 via drop-coating, and a calibration over the nitrate

concentration 1–100 ppm was performed as before. This device will be referred to as

‘NO3V PVC drop-coated IDE Design 2’. The resulting response versus time data for

NO3V PVC drop-coated IDE Design 2 (2% w/v) are shown in Figure 5.24 (G) and

Figure 5.25 (C).

Figure 5.24: Conductance response versus time of NO3V PVC drop-coated IDE Design 2 (2% w/v)



At 10 kHz, there was an initial increase in the measured conductance upon the addition

of 1 ppm NO3- to the test vessel, which decreased gradually, settling approximately

12 µS above the G0 value. However, upon the addition of further aliquots of NO3

- stock

solution it was not possible to distinguish between 20, 50 and 100 ppm NO3-. At

100 kHz each addition of NO3-

was distinguishable, and appeared to settle over the

1 hour period that the measurement of each concentration was recorded. Upon the total

addition of 100 ppm NO3-, the Gm – G0 value settled at approximately 77.9 µS. At

1 MHz, upon the total addition of 100 ppm NO3-, the Gm – G0 value settled at

approximately 142.5 µS. At 2 MHz, upon the total addition of 100 ppm NO3-, the

Gm – G0 value settled at approximately 175.6 µS.

0.00E+00

5.00E-05

1.00E-04

1.50E-04

2.00E-04

2.50E-04

0 50 100 150 200 250 300 350 400

Co

nd

uct

ance

(S)

Time (min)

0.00E+00

2.00E-05

4.00E-05

6.00E-05

8.00E-05

1.00E-04

1.20E-04

1.40E-04

1.60E-04

1.80E-04

0 50 100 150 200 250 300 350 400

Co

nd

uct

ance

(S)

Time (min)

0.00E+00

1.00E-05

2.00E-05

3.00E-05

4.00E-05

5.00E-05

6.00E-05

7.00E-05

8.00E-05

9.00E-05

1.00E-04

0 50 100 150 200 250 300 350 400

Co

nd

uct

ance

(S)

Time (min)

0.00E+00

5.00E-06

1.00E-05

1.50E-05

2.00E-05

2.50E-05

3.00E-05

3.50E-05

4.00E-05

0 50 100 150 200 250 300 350 400

Co

nd

uct

ance

(S)

Time (min)

A B

C D

Craig Alexander

200

Figure 5.25: Capacitance response versus time of NO3V PVC drop-coated IDE Design 2 (2% w/v)



At 10 kHz, it was possible to distinguish between the measured capacitance values for

each addition of NO3- to the test vessel. Upon the total addition of 100 ppm NO3

-, the

Cm – C0 value settled at approximately 410 pF. It was also possible to distinguish each

addition of NO3- to the test vessel at 100 kHz.

Upon the total addition of 100 ppm NO3

-,

the Cm – C0 value settled at approximately 65.9 pF. At 1 MHz, there was a slight

decrease in the measured capacitance when 1 ppm NO3- was added to the deionised

water background solution, followed by an increase when the subsequent aliquots were

added. Upon the total addition of 100 ppm NO3-, the Cm – C0 value settled at

approximately 8.4 pF. A similar pattern was observed at 2 MHz; however, the response

was less stable at each concentration. Upon the total addition of 100 ppm NO3-, the

Cm – C0 value settled at approximately 5.3 pF.

The corresponding logarithmic baseline-subtracted response against log nitrate

concentration data are shown in Figure 5.26 (G) and Figure 5.27 (C).

1.29E-10

1.30E-10

1.31E-10

1.32E-10

1.33E-10

1.34E-10

1.35E-10

1.36E-10

0 50 100 150 200 250 300 350 400

Ca

pa

cita

nce

(F)

Time (min)

1.28E-10

1.29E-10

1.30E-10

1.31E-10

1.32E-10

1.33E-10

1.34E-10

1.35E-10

1.36E-10

1.37E-10

1.38E-10

1.39E-10

0 50 100 150 200 250 300 350 400

Ca

pa

cita

nce

(F)

Time (min)

9.00E-11

1.10E-10

1.30E-10

1.50E-10

1.70E-10

1.90E-10

2.10E-10

0 50 100 150 200 250 300 350 400

Ca

pa

cita

nce

(F)

Time (min)

1.00E-10

2.00E-10

3.00E-10

4.00E-10

5.00E-10

6.00E-10

7.00E-10

0 50 100 150 200 250 300 350 400

Ca

pa

cita

nce

(F)

Time (min)

A B

C D

Craig Alexander

201


concentration for NO3V PVC drop-coated IDE Design 2 (2% w/v) at three frequencies.

A – 100 kHz. B – 1 MHz. C – 2 MHz.

The logarithmic baseline subtracted conductance produced a linear response at each of

the three extrapolated frequencies over the concentration range 1–100 ppm NO3-. At

100 kHz the R2 value was 0.9953, at 1 MHz it was 0.9928 and at 2 MHz it was 0.9875.


concentration for NO3V PVC drop-coated IDE Design 2 (2% w/v) at two frequencies (negative

values obtained at 1 MHz and 2 MHz).

A – 10 kHz. B – 100 kHz.


the two extrapolated frequencies over the concentration range 1–100 ppm NO3-. At

10 kHz the R2 value was 0.9940 and at 2 MHz it was 0.9988.

y = 0.3261x - 4.7573R² = 0.9953

-4.8

-4.7

-4.6

-4.5

-4.4

-4.3

-4.2

-4.1

-4

0 0.5 1 1.5 2 2.5

log

Gm

-G

o(S

)


y = 0.5312x - 4.8512R² = 0.9875

-6

-5

-4

-3

-2

-1

0

0 0.5 1 1.5 2 2.5lo

g G

m-

Go

(S)


y = 0.4874x - 4.845R² = 0.9928

-6

-5

-4

-3

-2

-1

0

0 0.5 1 1.5 2 2.5

log

Gm

-G

o(S

)


C

y = 1.0269x - 12.206R² = 0.9988

-14

-12

-10

-8

-6

-4

-2

0

0 0.5 1 1.5 2 2.5

log

Cm

-C

o (F

)


y = 0.3546x - 10.071R² = 0.994

-10.2

-10.1

-10

-9.9

-9.8

-9.7

-9.6

-9.5

-9.4

-9.3

0 0.5 1 1.5 2 2.5

log

Cm

-C

o (F

)


Craig Alexander

202


The FIM was used to establish the response of TDAN PVC drop-coated IDE Design 2

and NO3V PVC drop-coated IDE Design 2, through the addition of NO3- over the

concentration range 1–100 ppm in a deionised water background solution containing the

interfering anion at a fixed concentration. The deionised water baseline-subtracted

selectivity data for the ionophore TDAN are shown in Figure 5.28 (G) and Figure

5.29 (C). The selectivity data for the ionophore NO3V are shown in Figure 5.30 (G) and

Figure 5.31 (C).

Figure 5.28: Selectivity determination of TDAN PVC drop-coated IDE Design 2 (2% w/v) using the

FIM (conductive response). Comparison of: The deionised water baseline-subtracted conductance

responses to 1–100 ppm nitrate when no interfering anion is present, when a fixed concentration of

10 ppm nitrite is present and when a fixed concentration of 50 ppm chloride is present. The

response to 1–100 ppm nitrate of a ‘blank’ membrane, which did not contain TDAN as the

ionophore, is also shown

A – 1 MHz. B – 2 MHz.

For TDAN PVC drop-coated IDE Design 2 (2% w/v), the selectivity of the conductance

response was investigated at 1 MHz and 2 MHz, as these were the frequencies that

appeared to show the most analytically-relevant responses during the calibration

experiments. At 1 MHz, when 10 ppm NO2- was present, the baseline-subtracted

conductance for 1 ppm NO3- was 11.61 µS greater compared with when no interfering

anion was present. When 50 ppm Cl- was present, the baseline-subtracted conductance

0.00E+00

2.00E-05

4.00E-05

6.00E-05

8.00E-05

1.00E-04

1.20E-04

1.40E-04

1.60E-04

1.80E-04

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0.00E+00

2.00E-05

4.00E-05

6.00E-05

8.00E-05

1.00E-04

1.20E-04

1.40E-04

1.60E-04

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


A B

Craig Alexander

203

for 1 ppm NO3- was 34.46 µS greater compared with when no interfering anion was

present. A large response was observed for a ‘blank’ membrane that did not contain the

ionophore. At NO3- concentrations above 50 ppm, the response of the ‘blank’

membrane sensor was over four times the response from the membrane that did contain

the ionophore. A similar pattern was also observed for the response at 2 MHz.

Figure 5.29: Selectivity determination of TDAN PVC drop-coated IDE Design 2 (2% w/v) at

100 kHz using the FIM (capacitive response). Comparison of: The deionised water baseline-

subtracted capacitance responses to 1–100 ppm nitrate when no interfering anion is present, when

a fixed concentration of 10 ppm nitrite is present and when a fixed concentration of 50 ppm

chloride is present. The response to 1–100 ppm nitrate of a ‘blank’ membrane, which did not

contain TDAN as the ionophore, is also shown

The capacitance response of TDAN PVC drop-coated IDE Design 2 (2% w/v) was

investigated at 100 kHz. When 10 ppm NO2- was present, the baseline-subtracted

capacitance for 1 ppm NO3- was 6.13 pF greater compared with when no interfering

anion was present. When 50 ppm Cl- was present, the baseline-subtracted capacitance

for 1 ppm NO3- was 18.57 pF greater compared with when no interfering anion was

present. As for the conductance response, there was a substantial response observed

from a ‘blank’ membrane sensor that did not contain TDAN as the ionophore.

0

1E-11

2E-11

3E-11

4E-11

5E-11

6E-11

7E-11

8E-11

9E-11

1E-10

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


Craig Alexander

204

Figure 5.30: Selectivity determination of NO3V PVC drop-coated IDE Design 2 (2% w/v) using the

FIM (conductive response). Comparison of: The deionised water baseline-subtracted conductance





A – 100 kHz. B – 1 MHz. C – 2 MHz.

For NO3V PVC drop-coated IDE Design 2, the selectivity of the conductance response

was investigated at 100 kHz, 1 MHz and 2 MHz, as these were the frequencies that

appeared to show the most analytically-relevant responses during the calibration

experiments. At 100 kHz, when 10 ppm NO2- was present, the baseline-subtracted


anion was present. A reduction in the observed response was seen for the remaining

nitrate concentrations compared with when nitrite was not present. At 50 and 100 ppm

NO3-, the Gm – G0 was approximately 25 µS lower compared to when the interfering

anion was not present. When 50 ppm Cl- was present, the baseline-subtracted


anion was present. However, there was very little response observed from a ‘blank’

membrane that did not contain NO3V as the ionophore or TDMACl as the cation

0.00E+00

5.00E-05

1.00E-04

1.50E-04

2.00E-04

2.50E-04

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0.00E+00

2.00E-05

4.00E-05

6.00E-05

8.00E-05

1.00E-04

1.20E-04

1.40E-04

1.60E-04

1.80E-04

2.00E-04

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0.00E+00

1.00E-05

2.00E-05

3.00E-05

4.00E-05

5.00E-05

6.00E-05

7.00E-05

8.00E-05

9.00E-05

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


A B

C

Craig Alexander

205

exchanger. At 20, 50 and 100 ppm NO3-, the response from the ‘blank’ membrane

sensor was <1% of that from the ‘active’ membrane sensor that did contain the

ionophore and cation exchanger. A similar response was observed at 1 MHz and

2 MHz; however, the ‘blank’ response was greater. At 50 ppm NO3-, the ‘blank’

response was approximately 31% that of the ‘active’ response at 1 MHz, and

approximately 46% at 2 MHz.

Figure 5.31: Selectivity determination of NO3V PVC drop-coated IDE Design 2 (2% w/v) using the

FIM (capacitive response). Comparison of: The deionised water baseline-subtracted capacitance





A – 10 kHz. B – 100 kHz.

The capacitance response of NO3V PVC drop-coated IDE Design 2 (2% w/v) was

investigated at 10 kHz and 100 kHz. At 10 kHz, when 10 ppm NO2- was present, the

baseline-subtracted capacitance for 1 ppm NO3- was 60.08 pF greater compared with

when no interfering anion was present. When 50 ppm Cl- was present, the baseline-

subtracted capacitance for 1 ppm NO3- was 279.23 pF greater compared with when no

interfering anion was present. When a ‘blank’ membrane was tested, there was a

decrease below the C0 value of approximately 150 pF and there was very little

observable change to this value when additional aliquots of nitrate were added to the

test solution. At 100 kHz, when 10 ppm NO2- was present, the baseline-subtracted

0.00E+00

1.00E-11

2.00E-11

3.00E-11

4.00E-11

5.00E-11

6.00E-11

7.00E-11

8.00E-11

9.00E-11

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


-2.00E-10

-1.00E-10

0.00E+00

1.00E-10

2.00E-10

3.00E-10

4.00E-10

5.00E-10

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


A B

Craig Alexander

206

capacitance for 1 ppm NO3- was 8.88 pF greater compared with when no interfering

anion was present. When 50 ppm Cl- was present, the baseline-subtracted capacitance

for 1 ppm NO3- was 55.81 pF greater compared with when no interfering anion was

present. When a ‘blank’ membrane was tested, there was an initial increase of

approximately 6 pF when 1 ppm NO3- was added, this increased to approximately 10 pF

for 20 ppm NO3-; however, no further increase was observed with the subsequent nitrate

additions.

The response from a ‘blank’ NO3V membrane was much less prominent than that

observed from a ‘blank’ TDAN membrane prepared at the same solution viscosity. The

only difference between the two membranes was NPOE was used as the plasticiser with

NO3V whereas bis (2-ethylhexyl) sebacate was used with TDAN. This suggested that

the plasticiser NPOE may be better suited for use within this type of device, possibly

due to providing increased hydrophobicity to the membrane. It is necessary to further

investigate changing the membrane composition to optimise the response from the

sensors, such as investigating the use of different plasticisers.

Due to time constraints, it was not possible to further investigate IDE Design 2 as an

impedimetric nitrate sensor. Further experimentation is required to establish the effects

of changing the sample matrix composition and to establish the device-to-device

reproducibility. Several experiments were conducted to establish the response of this

device when coated with a PVC membrane containing ionophores for other target ions.

This was to establish whether this particular membrane composition was suitable with

IDE Design 2 when alternative ionophores were used for any of the other

aquarium-significant target ions. These results are discussed in Chapter 7.

Craig Alexander

207


IDE Design 2, a commercially-available microfabricated IDE device, was purchased

and tested for its feasibility as a nitrate-selective sensor within a freshwater aquarium,

via spin-coating or drop-coating of the sensing area with a selective polymeric

membrane containing commercial nitrate ionophores. TDAN and NO3V were tested as

the nitrate-selective ionophores; PVC was used as the membrane material.

Prior to conducting any experiments, IDE Design 2 appeared to provide a more

favourable approach to producing impedimetric ion-selective sensors than the

previously discussed IDE Design 1, as it had reduced geometric parameters which in

turn would lead to a reduced penetration depth of the generated electric field.

Several experiments were conducted to excite the IDE device with a reduced input

amplitude than had been used with the previous device (1 Vrms). This was seen as

beneficial for producing a prototype device with bespoke circuitry to power the sensors.

It was concluded that an acceptable response was observed by applying an AC voltage

of 10 mVrms. Therefore, subsequent experiments were conducted using this amplitude.

When a PVC membrane containing 1% w/w of the nitrate ionophore TDAN was

deposited over the surface of the sensing area via spin-coating, a linear response was

obtained through nitrate additions to a deionised water background from the changes in

capacitance at 2 MHz. When a ‘blank’ membrane that did not contain the ionophore

was spin-coated over the IDE digits, a large response was observed which suggested

that the sensor was actually responding to non-selective changes in the conductivity of

the test solution, and not from selective interactions within the membrane. The response

of three spin-coated devices prepared in the same way demonstrated poor

Craig Alexander

208

device-to-device reproducibility when nitrate was added to each sensor. As such, further

testing was not carried out using TDAN PVC spin-coated IDE Design 2, and drop-

coating of the polymeric membrane material was investigated as an alternative

coating method.

Drop-coating was investigated as an alternative coating method in an attempt to produce

a thicker polymeric membrane layer over the sensing area and therefore impart greater

selectivity on the device. When the initial membrane ‘cocktail’ composition of 10% w/v

was drop-coated onto IDE Design 2, an unacceptably long response time to the target

nitrate anion was observed. This suggested that the resulting membrane, once cast, was

of a thickness that was too great in relation to the electrode geometry; the increased

diffusion distance for ions to move into the membrane layer produced a long sensor

response time. It is not likely that a particularly fast response time would be required for

a sensor for aquarium purposes, as the target analytes would generally accumulate over

time as opposed to reaching dangerous levels immediately. However, a response that

was prone to drift would be problematic, as it would be difficult to establish whether a

particular response was due to the presence of the target species or if the measurement

was unsettled.

In an attempt to improve the response time from the drop-coated membrane sensors, the

viscosity of the polymeric ‘cocktail’ that contained the selective ionophore was reduced

by decreasing the mass of the membrane components that were dissolved into 5 ml

THF. Reducing the membrane solution to a concentration of 2% w/v produced a sensor

with more acceptable response characteristics; therefore, this membrane composition

was investigated further.

Craig Alexander

209

When a 2% w/v PVC membrane containing 5% w/w of the ionophore TDAN was

drop-coated over the digits of IDE Design 2, a prominent response to increasing nitrate

concentration was observed from changes in the conductance at 1 MHz and 2 MHz, and

from changes in the capacitance at 100 kHz. Substantial interferences were observed

using this membrane composition from the interfering anions nitrite and chloride, and a

large response was observed when a ‘blank’ membrane was deposited over the sensing

area. NO3V was investigated as an alternative nitrate-selective ionophore within a

2% w/v drop-coated PVC membrane. This membrane composition produced an

analytically-relevant response at 100 kHz, 1 MHz and 2 MHz for the changes in the

measured conductance, and at 10 kHz and 100 kHz for changes in the measured

capacitance. A substantial reduction in the observed response from a ‘blank’ membrane

was observed; however, interference from chloride and nitrite anions was still seen. The

lack of response from a ‘blank’ NO3V membrane suggested that the hydrophobicity of

the deposited membrane is also an important factor to consider to ensure selectivity

from such devices.

For a microfabricated device such as IDE Design 2 to be used as an impedimetric nitrate

sensor, it is apparent that one of the major considerations has to be the thickness of the

polymeric membrane that coats the sensing area in relation to the electrode digit

geometry. By simply increasing the membrane thickness, this results in an unacceptably

long response time with a measurement that is prone to drift. By depositing a membrane

that is too thin over the surface of the electrodes a lack of ion-selectivity is observed due

to the penetration depth of the resulting electric field reaching into the bulk solution.

For the purpose of this work, it would have been beneficial to be able to characterise the

thickness of the deposited membrane to approximate whether it was sufficient to

Craig Alexander

210

enclose the generated electric field. During this work, an attempt was made to

characterise the membrane thickness using a ‘top-down’ scanning electron microscopy

(SEM) approach, however, the results that were obtained did not provide a definitive

answer. Suggestions for alternative routes for characterising membrane thicknesses are

provided in Chapter 8.

The best route for providing a compromise between an acceptable response time

without a loss of selectivity would be to investigate IDE sensors with even further

reduced digit geometry; however, it was not possible to obtain any devices with lower

electrode geometry than IDE Design 2 throughout this project.

IDE Design 2 was a commercial device that was purchased ‘off-the-shelf’ and was not

specifically designed for the purpose for which it was tested. This was due to time and

financial constraints, in an attempt to provide proof-of-principle data from a device with

reduced electrode geometry from the previously tested IDE Design 1. It was much less

expensive and much quicker to obtain this particular electrode device than it would have

been to design and obtain a bespoke sensor. Following on from this proof-of-principle

work, it would have been beneficial to produce a sensor with a custom design.

Unfortunately, time and financial constraints dictated that this was not possible for the

purpose of this project.

IDE Design 2 featured a reference electrode and auxiliary electrode, which, although

not connected during the experimental work, may have provided interference and

affected the obtained response. These additional electrodes would not be included in a

custom design. This particular device also consisted of fifteen digits at each electrode;

the sensitivity of the response could be improved by increasing the number of digit

pairs. Suggestions for further experimental work are discussed in Chapter 8.

Craig Alexander

211

6 Nitrate Sensing using SP IDE Design 1

6.1 Issues Experienced with Initial Sensor Designs

As mentioned previously in section 2.4.2, there were issues experienced during the

fabrication stages of the first screen-printed IDEs which resulted in no suitable devices

available for testing as ion-selective sensors. Figure 6.1A shows a microscope image of

one of the designs with 60 µm digit widths and inter-digit spacing, and Figure 6.1B

shows a design with 100 µm widths and inter-digit spacing.

Figure 6.1: Microscope image (4x) of the carbon screen-printed IDE sensors produced at the

University of Bedfordshire. (A) shows one of the IDEs printed with 60 µm feature sizes. (B) shows

one of the IDEs printed with 100 µm feature sizes

A

B

Craig Alexander

212

It is evident from Figure 6.1 that the electrodes comprising of either 60 µm or 100 µm

line width geometries were not printed successfully; therefore, no data were obtained

from any of these sensors. The feature sizes of both sensors were too small and the ink

had expanded upon drying which resulted in many short-circuits between adjacent

digits, rendering the device unsuitable for use as an impedimetric ion-selective sensor.

Testing was carried out using the larger SP IDE Design 1, which had been designed

with a narrower digit width (100 µm) and a larger space between adjacent digits

(150 µm), and this consequently prevented short-circuits occurring during expansion of

the ink upon drying. A microscope image of the digits of SP IDE Design 1 is shown in

Figure 2.7 (Chapter 2).

6.2 SP IDE Design 1 Calibration

As for the photolithographically prepared IDE Design 1 and IDE Design 2 devices, it

was important to ascertain the response to the target ion of a SP IDE Design 1 sensor

which did not contain a selective membrane layer over the IDE digits. An uncoated SP

IDE Design 1 sensor, which had not had the modified dielectric paste membrane layer

printed over the sensing area, was placed into a deionised water background until a

stable baseline was observed, followed by the addition of NO3- over the concentration

range 1–100 ppm. The resulting conductance (A) and capacitance (B) spectra are shown

in Figure 6.2.

Craig Alexander

213

Figure 6.2: Conductance (A) and capacitance (B) spectra for the addition of NO3- to an uncoated SP



As for IDE Design 2, the response of an uncoated device was tested at a series of

reduced input amplitudes. The deionised water baseline-subtracted results upon the

addition of NO3- to an uncoated SP IDE Design 1 device at amplitudes of 1 Vrms,

100 mVrms, 10 mVrms and 1 mVrms are shown in Figure 6.3 (G) and Figure 6.4 (C).

0.00E+00

2.00E-08

4.00E-08

6.00E-08

8.00E-08

1.00E-07

1.20E-07

1.40E-07

1.60E-07

1.80E-07

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07

Cap

acit

ance

(F)

Frequency (Hz)

Cp Pure water

Cp 10 mins

Cp 30 mins

Cp 60 mins

Cp 90 mins

Cp 100 mins

Cp 110 mins

Cp 120 mins

Cp 1 ppm NO3-

Cp 10 ppm NO3-

Cp 20 ppm NO3-

Cp 30 ppm NO3-

Cp 40 ppm NO3-

Cp 50 ppm NO3-

Cp 100 ppm NO3-

0.00E+00

2.00E-05

4.00E-05

6.00E-05

8.00E-05

1.00E-04

1.20E-04

1.40E-04

1.60E-04

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07

Con

du

ctan

ce (S

)

Frequency (Hz)

G Pure water

G 10 mins

G 30 mins

G 60 mins

G 90 mins

G 100 mins

G 110 mins

G 120 mins

G 1 ppm NO3-

G 10 ppm NO3-

G 20 ppm NO3-

G 30 ppm NO3-

G 40 ppm NO3-

G 50 ppm NO3-

G 100 ppm NO3-

A

B

Craig Alexander

214


-

concentration for uncoated SP IDE Design 1 at six frequencies and varying input amplitudes.


An increase in the measured conductance was observed for 100 Hz, 1 kHz, 10 kHz and

100 kHz as the solution conductivity was increased, at each of the tested input

amplitudes. There was a decrease below the G0 value for the measured conductance at

1 MHz and 2 MHz.

-0.00003

-0.000025

-0.00002

-0.000015

-0.00001

-0.000005

0

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)log NO3

- concentration (ppm)

-0.00003

-0.000025

-0.00002

-0.000015

-0.00001

-0.000005

0

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0

0.00002

0.00004

0.00006

0.00008

0.0001

0.00012

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0

0.00002

0.00004

0.00006

0.00008

0.0001

0.00012

0.00014

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0

0.00002

0.00004

0.00006

0.00008

0.0001

0.00012

0.00014

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0

0.00002

0.00004

0.00006

0.00008

0.0001

0.00012

0.00014

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


A B

C D

E F

Craig Alexander

215


-

concentration for uncoated SP IDE Design 1 at six frequencies and varying input amplitudes


There was no observable analytically-relevant response from the capacitance of

uncoated SP IDE Design 1. A decrease in the measured capacitance was observed as the

solution conductivity was increased through the addition of potassium nitrate to the test

vessel at 1 MHz and 2 MHz.

As a response in the change of conductance was observed at lower input amplitudes,

which was seen as desirable so that a prototype device with a lower power consumption

could be produced, it was decided that subsequent experiments using SP IDE Design 1

would be excited with an input AC amplitude of 10 mVrms, as this amplitude produced a

response that was analytically-valid. At an input amplitude of 1 mVrms an unstable

measurement was obtained.

-1E-10

-9E-11

-8E-11

-7E-11

-6E-11

-5E-11

-4E-11

-3E-11

-2E-11

-1E-11

0

0 0.5 1 1.5 2 2.5

Cm

-C0

(F)


-1E-12

-8E-13

-6E-13

-4E-13

-2E-13

0

2E-13

4E-13

0 0.5 1 1.5 2 2.5

Cm

-C0

(F)


-7E-12

-6E-12

-5E-12

-4E-12

-3E-12

-2E-12

-1E-12

0

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


-1.2E-10

-1E-10

-8E-11

-6E-11

-4E-11

-2E-11

0

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


-3E-10

-2E-10

-1E-10

0

1E-10

2E-10

3E-10

4E-10

5E-10

6E-10

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


-2.5E-08

-2E-08

-1.5E-08

-1E-08

-5E-09

0

5E-09

1E-08

1.5E-08

2E-08

0 0.5 1 1.5 2 2.5

Cm

-C

0(F

)


A B

C D

E F

Craig Alexander

216


the addition of nitrate in the concentration range 1–100 ppm, to SP IDE Design 1 which

had been screen-printed with a modified dielectric paste ion-selective membrane layer

containing 22% w/w NPOE as the plasticiser and 1% w/w TDAN as the ionophore.


- to SP IDE Design

1 with a layer of plasticised dielectric paste containing 1% w/w TDAN as the ionophore (10 mVrms

input amplitude). ‘Pure water’ refers to the initial baseline value and 10-60 mins are the values

obtained during the baseline settling period. Interference at 50 Hz was observed; however this

frequency was not investigated

When this membrane layer was present, the most prominent analytical response from

the sensor was from the conductance at 1 kHz, 10 kHz and 100 kHz. As discussed

0.00E+00

2.00E-09

4.00E-09

6.00E-09

8.00E-09

1.00E-08

1.20E-08

1.40E-08

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07

Cap

acit

ance

(F)

Frequency (Hz)

Cp Pure water

Cp 10 mins

Cp 20 mins

Cp 30 mins

Cp 40 mins

Cp 50 mins

Cp 60 mins

Cp 1 ppm NO3-

Cp 10 ppm NO3-

Cp 20 ppm NO3-

Cp 30 ppm NO3-

Cp 40 ppm NO3-

Cp 50 ppm NO3-

Cp 100 ppm NO3-

0.00E+00

1.00E-05

2.00E-05

3.00E-05

4.00E-05

5.00E-05

6.00E-05

7.00E-05

8.00E-05

9.00E-05

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07

Co

nd

uct

ance

(S)

Frequency (Hz)

G Pure water

G 10 mins

G 20 mins

G 30 mins

G 40 mins

G 50 mins

G 60 mins

G 1 ppm NO3-

G 10 ppm NO3-

G 20 ppm NO3-

G 30 ppm NO3-

G 40 ppm NO3-

G 50 ppm NO3-

G 100 ppm NO3-

A

B

Craig Alexander

217

throughout Chapters 4 and 5, it was important to understand the behaviour of a sensor

comprising of a membrane layer coating the sensing area that did not contain any

selective ionophore. The deionised water baseline-subtracted conductance at 1 kHz,

10 kHz and 100 kHz of an IDE Design 1 device, consisting of a modified dielectric

paste membrane layer that contained TDAN as the ionophore, is compared with a

device consisting of a ‘blank’ dielectric paste membrane, without ionophore present, in

Figure 6.6.

Figure 6.6: Comparison of the conductance response of SP IDE Design 1 when a membrane

containing 1% w/w TDAN was printed over the sensing area with a ‘blank’ membrane which did

not contain any ionophore

A – 1 kHz. B – 10 kHz. C – 100 kHz

Above 1 ppm NO3-, the observed response for the ‘blank’ membrane SP IDE Design 1

device upon addition of the target anion was between 67–89% of that from the

ionophore-containing membrane at 1 kHz; this was around 60% at 10 kHz and between

65–90% at 100 kHz. This suggested that non-selective matrix interferences were

responsible for a large proportion of the observed response upon addition of NO3- to the

test solution.

0

0.000005

0.00001

0.000015

0.00002

0.000025

0.00003

0.000035

0.00004

0.000045

0.00005

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0

0.000005

0.00001

0.000015

0.00002

0.000025

0.00003

0.000035

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0

0.000002

0.000004

0.000006

0.000008

0.00001

0.000012

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


A B

C

Craig Alexander

218

In an attempt to reduce the response that was observed from a device which was coated

with a ‘blank’ modified dielectric paste ion-selective membrane, and therefore improve

the selectivity of the device by reducing matrix interferences, the thickness of the

membrane layer over the sensing area was increased by first printing and curing a

‘build-up’ layer of non-modified dielectric paste. A comparison of the response of three

‘blank’ membranes; one with no ‘build-up’ layer, one with a single ‘build-up’ layer and

one with two ‘build up’ layers, is shown in Figure 6.7.

Figure 6.7: Comparison of the conductance response of SP IDE Design 1 coated with a ‘blank’

membrane containing no ionophore when zero, one or two ‘build-up’ layers of non-modified

dielectric paste were printed prior to the modified layer containing NPOE as the plasticiser.

A – 1 kHz. B – 10 kHz. C – 100 kHz

It was observed at all three frequencies of interest that the conductive response was

substantially reduced when two ‘build-up’ layers of unmodified dielectric paste were

printed and cured over the digits of SP IDE Design 2 prior to the printing of the

modified dielectric paste containing plasticiser. Subsequent experiments were therefore

conducted with devices that had two ‘build-up’ layers printed with a third layer

comprising of modified dielectric paste containing 1% w/w of TDAN as the

0

0.000005

0.00001

0.000015

0.00002

0.000025

0.00003

0.000035

0.00004

0.000045

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0

0.000005

0.00001

0.000015

0.00002

0.000025

0.00003

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


-0.000001

0

0.000001

0.000002

0.000003

0.000004

0.000005

0.000006

0.000007

0.000008

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


A B

C

Craig Alexander

219

nitrate-selective ionophore. This particular device will be referred to as ‘TDAN SP IDE

Design 1’. The conductance spectrum for this device is shown in Figure 6.8.

Figure 6.8: Conductance spectrum for the addition of NO3

- to TDAN SP IDE Design 1 (10 mVrms

input amplitude). ‘Pure water’ refers to the initial baseline value. Interference was observed at

50 Hz so this data has been omitted

The conductance response versus time data at each extrapolated frequency (1 kHz,

10 kHz and 100 kHz), upon the addition of nitrate over the concentration range

1–100 ppm, is shown in Figure 6.9.

Figure 6.9: Conductance response versus time of TDAN SP IDE Design 1 upon addition of

1–100 ppm nitrate at four frequencies. A – 1 kHz. B – 10 kHz. C – 100 kHz.

-2.00E-05

0.00E+00

2.00E-05

4.00E-05

6.00E-05

8.00E-05

1.00E-04

1.20E-04

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07

Co

nd

uct

ance

(S)

Frequency (Hz)

G Pure water

G 1 ppm NO3-

G 10 ppm NO3-

G 20 ppm NO3-

G 30 ppm NO3-

G 40 ppm NO3-

G 50 ppm NO3-

G 100 ppm NO3-

0.00E+00

1.00E-05

2.00E-05

3.00E-05

4.00E-05

5.00E-05

6.00E-05

7.00E-05

0 20 40 60 80 100 120 140

Co

nd

uct

ance

(S)

Time (min)

G @ 100 kHz

0.00E+00

2.00E-06

4.00E-06

6.00E-06

8.00E-06

1.00E-05

1.20E-05

1.40E-05

1.60E-05

1.80E-05

0 20 40 60 80 100 120 140

Co

nd

uct

ance

(S)

Time (min)

G @ 10 kHz

0.00E+00

1.00E-06

2.00E-06

3.00E-06

4.00E-06

5.00E-06

6.00E-06

7.00E-06

0 20 40 60 80 100 120 140

Co

nd

uct

ance

(S)

Time (min)

G @ 1 kHz

A B

C

Craig Alexander

220

The corresponding logarithmic baseline-subtracted conductance versus log nitrate

concentration is shown in Figure 6.10.


concentration for TDAN SP IDE Design 1

A – 1 kHz. B – 10 kHz. C – 100 kHz.

Taking the logarithm of the deionised water baseline-subtracted conductance and

plotting it against log NO3- concentration confirmed the linearity of the response (1 kHz

R2 = 0.9988, 10 kHz R

2 = 0.9929 and 100 kHz R

2 = 0.9862).

Figure 6.11 shows the comparison between adjusting the nitrate concentration within

the test vessel using a KNO3 stock solution and a LiNO3 stock solution. The effect of

temperature on the sensor response was not determined, as it was deemed more

important to ascertain the ion-selectivity of the device before looking at peripheral

characteristics of the response.

y = 0.3635x - 5.1709R² = 0.9862

-5.3

-5.2

-5.1

-5

-4.9

-4.8

-4.7

-4.6

-4.5

-4.4

-4.3

0 0.5 1 1.5 2 2.5

log

(Gm

-G

0)

(S)


y = 0.3881x - 5.5975R² = 0.9929

-5.7

-5.6

-5.5

-5.4

-5.3

-5.2

-5.1

-5

-4.9

-4.8

-4.7

0 0.5 1 1.5 2 2.5

log

(Gm

-G

0)

(S)


y = 0.2591x - 5.8665R² = 0.9988

-5.9

-5.8

-5.7

-5.6

-5.5

-5.4

-5.3

0 0.5 1 1.5 2 2.5

log

(Gm

-G

0)

(S)


C

Craig Alexander

221


-

concentration for TDAN SP IDE Design 1. Two calibration experiments are shown: the addition of

1–100 ppm NO3- with K

+ as the counter ion and the addition of 1–100 ppm NO3

- with Li

+ as the

counter ion

A – 1 kHz. B – 10 kHz. C – 100 kHz.

When lithium nitrate was used to adjust the nitrate concentration in the test vessel, the

marked decrease compared with the potassium nitrate calibration that had been present

for previous devices was not observed. This suggested that the counter ion that was used

with the target ion was producing less of an effect on the observed sensor response. At

1 kHz, the lithium nitrate calibration was slightly higher than the potassium nitrate

calibration, which would not be expected if solution conductivity was heavily

influencing the sensor response. At 50 ppm NO3-, the %RSD between the two

calibration experiments was 7.87%. There was less of a marked difference between the

calibration experiments at 10 kHz and 100 kHz; %RSD between the two measurements

at 50 ppm NO3- was 2.22% at 10 kHz and 4.72% at 100 kHz.

The effect of the changes in background conductivity of the sample solution was

investigated by placing the sensor into solutions of varying ionic strength. Figure 6.12

0

0.000002

0.000004

0.000006

0.000008

0.00001

0.000012

0.000014

0.000016

0.000018

0 0.5 1 1.5 2 2.5

Gm

-G

0 (S

)


0

0.000001

0.000002

0.000003

0.000004

0.000005

0.000006

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


A B

0

0.000005

0.00001

0.000015

0.00002

0.000025

0.00003

0.000035

0.00004

0.000045

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


C

Craig Alexander

222

shows the measured G0 values for each of the tested environments at each of the three

frequencies of interest.

Figure 6.12: Measured baseline conductance value (G0) of TDAN SP IDE Design 1 in four separate

sample matrices prior to the addition of nitrate

A – 1 kHz. B – 10 kHz. C – 100 kHz.

An increase of G0 was seen at all three extrapolated frequencies as the ionic strength of

the background solution was increased. A 100 ppm solution of MgSO4 produced a G0

value that was 2.43 µS greater than the deionised water value at 1 kHz. A 200 ppm

solution of MgSO4 was 2.67 µS greater and 500 ppm MgSO4 was 3.09 µS greater than

the deionised water value. At 10 kHz the G0 values were 11.41 µS greater than

deionised water for 100 ppm MgSO4, 13.15 µS greater for 200 ppm MgSO4 and

14.87 µS greater for 500 ppm MgSO4. At 100 kHz the G0 values were 36.69 µS greater

than deionised water for 100 ppm MgSO4, 45.43 µS greater for 200 ppm MgSO4 and

57.71 µS greater for 500 ppm MgSO4.

0

0.00001

0.00002

0.00003

0.00004

0.00005

0.00006

0.00007

0.00008

0.00009

G0

bas

eli

ne

at

10

0 k

Hz


0

0.000005

0.00001

0.000015

0.00002

0.000025

G0

bas

eli

ne

at

10

kH

z

Background sample matrix0

0.0000005

0.000001

0.0000015

0.000002

0.0000025

0.000003

0.0000035

0.000004

0.0000045

G0

bas

eli

ne

val

ue

at

1 k

Hz


A B

C

Craig Alexander

223

The baseline-subtracted conductance responses at each frequency upon addition of NO3-

over the range 1–100 ppm to each of these background solutions are shown in

Figure 6.13.

Figure 6.13: Baseline-subtracted conductance (Gm – G0) of TDAN SP IDE Design 1 against log NO3

-

concentration in four separate sample matrices

A – 1 kHz. B – 10 kHz. C – 100 kHz.

At each of the extrapolated frequencies, the sensitivity of the sensor to each addition of

NO3- was reduced as the ionic strength of the background solution was increased.

Figure 6.14 shows the deionised water baseline-subtracted conductance at 1 kHz,

10 kHz and 100 kHz, for the addition of NO3- to three separate TDAN SP IDE

Design 1 devices.

0

0.000005

0.00001

0.000015

0.00002

0.000025

0.00003

0.000035

0.00004

0.000045

0.00005

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0

0.000002

0.000004

0.000006

0.000008

0.00001

0.000012

0.000014

0.000016

0 0.5 1 1.5 2 2.5

Gm

-G

0 (S

)


0

0.0000005

0.000001

0.0000015

0.000002

0.0000025

0.000003

0.0000035

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


A B

C

Craig Alexander

224

Figure 6.14: Deionised water baseline-subtracted conductance (Gm – G0) of three TDAN SP IDE

Design 1 devices against log NO3- concentration, for reproducibility determination

A – 1 kHz. B – 10 kHz. C – 100 kHz.

The device-to-device reproducibility, defined as the %RSD of the three obtained

baseline-subtracted conductance measurements at 50 ppm NO3-, was calculated as

27.20% at 1 kHz, 8.26% at 10 kHz and 10.93% at 100 kHz.

6.3 SP IDE Design 1 Selectivity

The FIM was used to establish the response of the TDAN SP IDE Design 1 sensor

through the addition of NO3- over the concentration range 1–100 ppm in a background

solution containing fixed interfering anions. The FPM was also used to deduce the

effect of adding an interfering ion to a deionised water background containing nitrate at

a fixed concentration (50 ppm). The selectivity data of TDAN SP IDE Design 1 are


0

0.000005

0.00001

0.000015

0.00002

0.000025

0.00003

0.000035

0.00004

0.000045

0.00005

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0

0.000002

0.000004

0.000006

0.000008

0.00001

0.000012

0.000014

0.000016

0.000018

0.00002

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0

0.000001

0.000002

0.000003

0.000004

0.000005

0.000006

0.000007

0.000008

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


A B

C

Craig Alexander

225

Figure 6.15: Selectivity determination of TDAN SP IDE Design 1 using the FIM. Comparison of:

The deionised water baseline-subtracted conductance responses to 1–100 ppm nitrate when no

interfering anion is present, when a fixed concentration of 10 ppm nitrite is present and when a

fixed concentration of 50 ppm chloride is present. The response to 1–100 ppm nitrate of a ‘blank’

membrane, which did not contain TDAN as the ionophore, is also shown

A – 1 kHz. B – 10 kHz. C – 100 kHz.

When the FIM was used to establish the selectivity of TDAN SP IDE Design 1, at each

extrapolated frequency, an increase in the conductance response was observed when the

nitrate calibration was carried out as before, however 50 ppm Cl- or 10 ppm NO2

- were

present in the test sample.

At 1 kHz, there was an increase of 5.18 µS from the deionised water G0 value when 50

ppm Cl- was added to the test sample. When the FPM was used, and 50 ppm NO3

- was

added to the test sample, this resulted in an increase in the measured conductance of

4.35 µS. The addition of 10 ppm NO2- to the test vessel resulted in an increase in the

measured conductance of 2.72 µS, compared with 2.47 µS when 10 ppm NO3- was

added to a deionised water background solution.

0

0.00001

0.00002

0.00003

0.00004

0.00005

0.00006

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


-0.000001

0

0.000001

0.000002

0.000003

0.000004

0.000005

0.000006

0.000007

0.000008

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


0

0.000005

0.00001

0.000015

0.00002

0.000025

0 0.5 1 1.5 2 2.5

Gm

-G

0(S

)


A B

C

Craig Alexander

226

At 10 kHz, adding 50 ppm Cl- to the deionised water baseline resulted in a Gm - G0

value of 15.54 µS compared with 13.07 µS which was the Gm – G0 obtained from

50 ppm NO3-. The Gm – G0 value for 10 ppm NO2

- was 6.62 µS compared to 6.14 µS

which was obtained from 50 ppm NO3-.

At 100 kHz, adding 50 ppm Cl- to the deionised water baseline resulted in a Gm - G0

value of 37.37 µS compared with 29.82 µS which was the Gm – G0 obtained from

50 ppm NO3-. The Gm – G0 value for 10 ppm NO2

- was 18.03 µS compared with

14.10 µS which was obtained from 50 ppm NO3-.

As such a large response was obtained from the interfering anions at each extrapolated

frequency. This suggested that the TDAN SP IDE Design 1 was unable to adequately

distinguish between the different anions, and therefore did not provide adequate nitrate

selectivity.

Very little response was observed from the ‘blank’ membrane, which consisted of two

‘build-up’ layers of standard dielectric paste followed by a layer of the modified paste

that contained NPOE as the plasticiser but did not contain TDAN as the ionophore, at

any of the extrapolated frequencies. At 50 ppm NO3-, the ‘blank’ response at 1 kHz was

0.43% that of the conductance measurement when the ionophore was present; at 10 kHz

it was 8.26% and at 100 kHz it was 33.57%.

There was little response obtained when a ‘blank’ membrane, which did not contain any

ionophore, was deposited over the sensing area of the SP IDEs. This suggested that the

origin of the sensor response was from interactions within the membrane layer

containing an immobilised ionophore. As such a large response was obtained from

non-target species and an increase to the baseline G0 value was observed when solutions

with increasing ionic strength were used, this suggested that this particular ionophore,

Craig Alexander

227

TDAN, was not selective to nitrate within this membrane configuration. Unfortunately

it was not possible to obtain any further screen-printed devices with alternative

membrane parameters. Further work is required to produce sensors based on SP IDE

Design 1 that contain alternative plasticisers and/or ionophores within the membrane

layer. Suggestions for further experimental work are discussed in Chapter 8.


An ion-selective impedimetric nitrate sensor based on carbon IDEs was fabricated using

a fully screen-printed process, which included a screen-printed ion-selective membrane

layer coating the sensing area, and tested for its feasibility as a nitrate-selective sensor

for use within a freshwater aquarium. A suitable membrane support matrix for

screen-printing was produced by incorporating a nitrate-selective ionophore (TDAN),

along with the plasticiser NPOE, into a commercial dielectric screen-printing ink. Initial

fabrication issues dictated that a larger geometry was required to produce IDEs via

screen-printing, and eventually it was found that a design consisting of electrode digits

with a width of 100 µm, separated by an interdigital space between adjacent digits of

150 µm, printed effectively and reproducibly.

When a single layer of the modified dielectric screen-printing ink was coated over the

IDEs as the ion-selective membrane layer, a large response to nitrate was observed with

a ‘blank’ membrane that did not contain the ionophore. This suggested that the

penetration depth of the resulting electric field was much greater than the thickness of

the ion-selective membrane. Therefore, non-selective changes in the conductivity of the

bulk solution were observed, rather than changes in the electrical properties of the

membrane due to selective interactions between the target anion and the ionophore

immobilised within it. In an attempt to overcome this issue, a ‘build-up’ layer of

Craig Alexander

228

standard dielectric paste was first printed and cured over the sensing area, followed by

the modified ink, to afford a thicker membrane layer. At certain key mid-range

frequencies (1 kHz–100 kHz) where a linear relationship between the impedimetric

response and nitrate concentration was observed, there was a reasonable reduction in the

magnitude of response from the ‘blank’ membrane. This suggested that the thicker

membrane was containing more of the electric field and therefore changes in sample

matrix conductivity were less significant to the response. By adding a second ‘build-up’

layer prior to the membrane, the ‘blank’ response was even further reduced. At a

frequency of 1 kHz the observed ‘blank’ response was less than 10% of the response

observed at each nitrate concentration compared with the sensor that contained TDAN

as the ionophore.

This layer-by-layer screen-printing approach to depositing the ion-selective membrane

layer of the devices appeared to provide a route for producing disposable, fully

screen-printed, all-solid-state nitrate sensors that do not require a reference electrode.

However, when the selectivity of the three-layer devices was tested, a strong

interference from both nitrite and chloride anions was observed. This suggested that the

TDAN ionophore within this particular membrane composition did not show a strong

selectivity for nitrate when implemented within this type of device, and therefore would

not be suitable for use within an aquarium nitrate sensor. Adding ‘build-up’ layers to the

device did not appear to significantly increase the response time of the electrodes, as the

selective components within the membrane were only held within the thinnest top layer,

so the overall diffusion distance for ions to move into the membrane layer was

not increased.

Craig Alexander

229

Due to the manufacturing costs involved in producing the screen-printed sensors at a

commercial rate, it was not possible to obtain any more IDE Design 1 sensors for

further testing. Additional investigation is required to produce sensors with alternative

ionophores and plasticisers contained within the membrane layer in order to produce a

greater selective response to the target ions. Screen-printing has been shown to be a

feasible route for producing ISCOM-type devices for impedimetric ion-selective

sensing without the requirement of an external reference electrode. It is also suitable for

producing disposable sensors on inexpensive substrates which is particularly attractive

for the purpose of a commercial aquarium sensor. As the sensors are fully

screen-printed, they are ready to use as received without any further modification. The

main drawback of utilising this technique for producing such devices is that a

reasonably large quantity of ionophore is required to produce a sufficient amount of

modified paste for screen-printing (100 mg ionophore per 10 g of membrane material),

some of which would be wasted as it is left behind on the screens following the printing

process. This is particularly unattractive for mass producing such devices as some of the

commercially-available ionophores for aquarium-significant ions are vastly expensive,

for example 100 mg of NO2I would cost in excess of £2300.

For commercial purposes, a comparison between fabrication processes is needed to

establish which method is the most cost-effective for mass-production. The options are

to produce a less expensive carbon IDE device which requires a larger amount of

ionophore using screen-printing, or to purchase commercial microfabricated IDEs, such

as IDE Design 2, which then require coating with a small amount of polymer

ion-selective membrane, and therefore require less ionophore. The final cost-per-device

will depend on how much ionophore is required and the initial cost of the individual

IDE transduction element.

Craig Alexander

230

7 pH, Ammonium and Nitrite Sensing using IDE Design 2

Due to time constraints, it was not possible to carry out all of the necessary experiments

to obtain full data-sets from IDE sensors for pH, ammonium or nitrite sensing. Several

experiments for these target ions were carried out using IDE Design 2 with a

drop-coated 2% w/v PVC membrane ‘cocktail’ containing 5% w/w of the

complimentary ionophore. The primary aims of these investigations were to ascertain

the response of a ‘blank’ membrane that did not contain the ionophore in comparison

with an ‘active’ membrane that did, and to establish the selectivity of the sensors against

interfering ions that are most likely to be present within an aquarium. Much further

research is required to fully validate the individual sensor performance for use within an

aquarium monitoring device. Suggestions for further experimental work are discussed

in Chapter 8.

7.1 Ammonium Sensors

An IDE Design 2 device was drop-coated with a 2% w/v PVC membrane ‘cocktail’

containing 5% w/w of the ionophore NH4I. An aliquot of 50 ppm NH4+ was added to a

deionised water background solution and the response was obtained at 10 minute

intervals over two hours. As with the nitrate experiments conducted using IDE Design 2

(Chapter 5), an input amplitude of 10 mVrms was used. This device will be referred to as

‘NH4I PVC drop-coated IDE Design 2’. The conductance versus time data at five

frequencies of interest are shown in Figure 7.1 and the capacitance versus time data are


Craig Alexander

231

Figure 7.1: Conductance response versus time of NH4I PVC drop-coated IDE Design 2 (2% w/v)

upon the addition of 50 ppm NH4+ to a deionised water background at five frequencies. The aliquot

of ammonium stock solution was added after 120 minutes.


Each of the frequencies of interest showed a distinct increase from the G0 value upon

the addition of 50 ppm NH4+ to a deionised water background. At 1 kHz, the response

appeared to settle after around 90 minutes with a Gm – G0 value of approximately

3.0 µS. The response at 10 kHz also appeared to settle after around 90 minutes, with a

Gm – G0 value of approximately 3.8 µS. At 100 kHz, the response appeared to settle

after around 60 minutes with a Gm – G0 value of approximately 5.9 µS. The

conductance response at 1 MHz showed an immediate increase of approximately 38 µS

before gradually decreasing and settling after around 40 minutes with a Gm – G0 value

of approximately 26 µS. The conductance response at 2 MHz showed an immediate

0.00E+00

2.00E-05

4.00E-05

6.00E-05

8.00E-05

1.00E-04

1.20E-04

0 50 100 150 200 250

Co

nd

uct

ance

(S)

Time (min)

0.00E+00

1.00E-05

2.00E-05

3.00E-05

4.00E-05

5.00E-05

6.00E-05

0 50 100 150 200 250

Co

nd

uct

ance

(S)

Time (min)

0.00E+00

5.00E-07

1.00E-06

1.50E-06

2.00E-06

2.50E-06

3.00E-06

3.50E-06

4.00E-06

4.50E-06

0 50 100 150 200 250

Co

nd

uct

ance

(S)

Time (min)

2 hour deionised water baseline

50 ppm NH4+

0.00E+00

1.00E-06

2.00E-06

3.00E-06

4.00E-06

5.00E-06

6.00E-06

7.00E-06

8.00E-06

9.00E-06

1.00E-05

0 50 100 150 200 250

Co

nd

uct

ance

(S)

Time (min)

0.00E+00

1.00E-06

2.00E-06

3.00E-06

4.00E-06

5.00E-06

6.00E-06

0 50 100 150 200 250

Co

nd

uct

ance

(S)

Time (min)

A B

C D

E

Craig Alexander

232

increase of approximately 50 µS before settling after around 30 minutes with a Gm – G0

value of approximately 70 µS.

Figure 7.2: Capacitance response versus time of NH4I PVC drop-coated IDE Design 2 (2% w/v)

upon the addition of 50 ppm NH4+ to a deionised water background at five frequencies. The aliquot

of ammonium stock solution was added after 120 minutes.


At 1 kHz, the capacitance response increased from the C0 value by approximately

155 pF, however the response appeared unstable and prone to drift. The response at

10 kHz increased by approximately 24 pF; however, this also appeared unstable. At

100 kHz, the response appeared to settle after around 40 minutes with a Cm – C0 value

of approximately 16 pF. The capacitance response at 1 MHz showed an increase of

approximately 12 pF, settling after around 40 minutes. The capacitance response at

2 MHz showed an increase of approximately 11 pF, settling after around 40 minutes.

1.28E-10

1.30E-10

1.32E-10

1.34E-10

1.36E-10

1.38E-10

1.40E-10

1.42E-10

0 50 100 150 200 250

Ca

pa

cita

nce

(F)

Time (min)

1.28E-10

1.30E-10

1.32E-10

1.34E-10

1.36E-10

1.38E-10

1.40E-10

1.42E-10

1.44E-10

1.46E-10

1.48E-10

0 50 100 150 200 250

Ca

pa

cita

nce

(F)

Time (min)

1.35E-10

1.40E-10

1.45E-10

1.50E-10

1.55E-10

1.60E-10

1.65E-10

0 50 100 150 200 250

Ca

pa

cita

nce

(F)

Time (min)

0.00E+00

5.00E-11

1.00E-10

1.50E-10

2.00E-10

2.50E-10

3.00E-10

3.50E-10

4.00E-10

0 50 100 150 200 250

Ca

pa

cita

nce

(F)

Time (min)

A B

C D

E

1.28E-10

1.30E-10

1.32E-10

1.34E-10

1.36E-10

1.38E-10

1.40E-10

1.42E-10

1.44E-10

0 50 100 150 200 250

Ca

pa

cita

nce

(F)

Time (min)

Craig Alexander

233

Selectivity data for NH4I PVC drop-coated IDE Design 2 were obtained using the FIM,

through the addition of 50 ppm of the interfering cation to a deionised water

background. An aliquot of ammonium at a concentration of 50 ppm NH4+ was also

added to a device that had been prepared without the ionophore present. The

baseline-subtracted data at each extrapolated frequency are displayed in Table 7.1 (G)

and Table 7.2 (C).

Gm – G0 (µS)

Ion (50 ppm) Frequency

1 kHz 10 kHz 100 kHz 1 MHz 2 MHz

Ammonium 2.95 3.79 5.91 26.07 70.50

Potassium 1.05 1.29 1.83 41.33 98.86

Sodium 0.39 0.43 0.73 45.71 99.05

Magnesium 0.11 0.03 1.50 46.93 91.22

Ammonium

(blank membrane) 2.36 2.82 3.86 21.72 46.96

Table 7.1: Selectivity determination of NH4I PVC drop-coated IDE Design 2 (2% w/v) (conductive

response). Deionised water baseline-subtracted conductance upon the addition of the 50 ppm of:

The target ammonium ion and interfering cations potassium, sodium and magnesium. The response

from the addition of 50 ppm NH4+ to a device prepared without ionophore present is also shown

At the frequencies 1 kHz, 10 kHz and 100 kHz, the conductance response

characteristics of the sensor appeared to be favourable to ammonium when compared

with the response from each of the interfering cations. However, at the higher

frequencies of 1 MHz and 2 MHz there was an increased response to each of the three

tested interfering cations when compared with the response obtained from the target

ammonium ion. At each frequency of interest, a large response was observed from a

‘blank’ membrane that did not contain the ionophore.

Craig Alexander

234

Cm – C0 (pF)

Ion (50 ppm) Frequency


Ammonium 155.44 23.94 16.10 12.20 10.63

Potassium 51.17 15.72 14.88 11.72 7.28

Sodium 29.10 14.45 15.19 11.04 6.03

Magnesium 23.23 13.85 16.45 6.30 2.41

Ammonium

(blank membrane) 63.74 26.16 12.96 5.15 3.10

Table 7.2: Selectivity determination of NH4I PVC drop-coated IDE Design 2 (2% w/v) (capacitive

response). Deionised water baseline-subtracted capacitance upon the addition of the 50 ppm of: The

target ammonium ion and interfering cations potassium, sodium and magnesium. The response

from the addition of 50 ppm NH4+ to a device prepared without ionophore present is also shown

At 1 kHz, the capacitance response from NH4I drop-coated IDE Design 2 appeared to

be favourable to ammonium when compared with the response from each of the

interfering cations. The response from the ‘blank’ membrane that did not contain the

ionophore was around 40% that of the ‘active’ membrane at 1 kHz. At 10 kHz, the

capacitance response was much higher for ammonium compared with the interfering

cations; however, there was a substantial response from the ‘blank’ membrane. At

100 kHz there was a large response from each of the interfering cations and from the

‘blank’ membrane sensor. At 1 MHz and 2 MHz, substantial interference was observed

from potassium and sodium ions; however, the response from the ‘blank’ membrane

was less pronounced.

7.2 Nitrite Sensors

An IDE Design 2 device was drop-coated with a 2% w/v PVC membrane ‘cocktail’

containing 5% w/w of the ionophore NO2I. An aliquot of 5 ppm NO2- was added to a

deionised water background solution and the response was obtained at 10 minute

intervals over two hours. An input amplitude of 10 mVrms was used. This device will be

referred to as ‘NO2I PVC drop-coated IDE Design 2’. The conductance versus time

Craig Alexander

235

data at five frequencies of interest are shown in Figure 7.3 and the capacitance versus

time data at five frequencies of interest are shown in Figure 7.4.

Figure 7.3: Conductance response versus time of NO2I PVC drop-coated IDE Design 2 (2% w/v)

upon the addition of 5 ppm NO2- to a deionised water background at five frequencies. The aliquot

of nitrite stock solution was added after 60 minutes.


Each of the frequencies of interest showed a distinct increase from the G0 value upon

the addition of 5 ppm NO2- to a deionised water background. At 1 kHz, there was a

response that settled after 10 minutes with a Gm – G0 value of approximately 2.0 µS.

The conductance response at 10 kHz, 100 kHz, 1 MHz and 2 MHz appeared to settle

after around 30 minutes. The Gm – G0 value at 10 kHz was approximately 17 µS. The

Gm – G0 value at 100 kHz was approximately 21 µS. The Gm – G0 value at 1 MHz was

approximately 21 µS. The Gm – G0 value at 2 MHz was approximately 20 µS.

0.00E+00

1.00E-05

2.00E-05

3.00E-05

4.00E-05

5.00E-05

6.00E-05

7.00E-05

0 50 100 150 200 250

Co

nd

uct

ance

(S)

Time (min)

2.00E-06

2.50E-06

3.00E-06

3.50E-06

4.00E-06

4.50E-06

5.00E-06

5.50E-06

6.00E-06

6.50E-06

7.00E-06

0 50 100 150 200 250

Co

nd

uct

ance

(S)

Time (min)

1 hour deionised water baseline

5 ppm NO2-

1.00E-05

1.50E-05

2.00E-05

2.50E-05

3.00E-05

3.50E-05

4.00E-05

4.50E-05

5.00E-05

5.50E-05

0 50 100 150 200 250

Co

nd

uct

ance

(S)

Time (min)

0.00E+00

5.00E-06

1.00E-05

1.50E-05

2.00E-05

2.50E-05

3.00E-05

3.50E-05

0 50 100 150 200 250

Co

nd

uct

ance

(S)

Time (min)

0.00E+00

5.00E-06

1.00E-05

1.50E-05

2.00E-05

2.50E-05

3.00E-05

0 50 100 150 200 250

Co

nd

uct

ance

(S)

Time (min)

A B

C D

E

Craig Alexander

236

Figure 7.4: Capacitance response versus time of NO2I PVC drop-coated IDE Design 2 (2% w/v)

upon the addition of 5 ppm NO2- to a deionised water background at five frequencies. The aliquot

of nitrite stock solution was added after 60 minutes.


At 1 kHz, upon the addition of 5 ppm NO2-, the capacitance response increased from the

C0 value by approximately 1.12 nF, and the measurement appeared stable after 30

minutes. The response at 10 kHz increased by approximately 87.54 pF; and appeared

stable after 30 minutes. At 100 kHz, there was an increase to the C0 value of

approximately 2.2 pF; however, this response appeared unstable. The capacitance

response at 1 MHz and 2 MHz decreased below the C0 value when nitrite was added.

As chloride and nitrate are likely to be present at a higher concentration than the target

nitrite anion within an aquarium, it was important to establish the behaviour of NO2I

PVC drop-coated IDE Design 2 at high concentrations of the interfering anions.

Therefore, selectivity data was obtained through the addition of 50 ppm of the

1.276E-10

1.278E-10

1.280E-10

1.282E-10

1.284E-10

1.286E-10

1.288E-10

1.290E-10

0 50 100 150 200 250

Ca

pa

cita

nce

(F)

Time (min)

1.285E-10

1.286E-10

1.287E-10

1.288E-10

1.289E-10

1.290E-10

1.291E-10

1.292E-10

1.293E-10

1.294E-10

0 50 100 150 200 250

Ca

pa

cita

nce

(F)

Time (min)

0.00E+00

5.00E-11

1.00E-10

1.50E-10

2.00E-10

2.50E-10

3.00E-10

0 50 100 150 200 250

Ca

pa

cita

nce

(F)

Time (min)

0.00E+00

2.00E-10

4.00E-10

6.00E-10

8.00E-10

1.00E-09

1.20E-09

1.40E-09

1.60E-09

1.80E-09

0 50 100 150 200 250

Ca

pa

cita

nce

(F)

Time (min)

A B

C D

E

Craig Alexander

237

interfering anion to a deionised water background. Therefore, it was important to

establish the behaviour of the nitrite sensor when the interfering anions were present in

a greater concentration than that of the target ion. Nitrite at a concentration of 5 ppm

NO2- was also tested using a device that had been prepared without the ionophore

present within the membrane. The baseline-subtracted data at each extrapolated

frequency are displayed in Table 7.3 (G) and Table 7.4 (C).

Gm – G0 (µS)

Ion Frequency


Nitrite (5 ppm) 2.15 17.10 21.39 21.22 19.66

Chloride (50 ppm) 1.34 19.55 25.29 48.00 72.60

Nitrate (50 ppm) 1.2 19.30 25.29 47.64 63.22

Nitrite (5 ppm)

(blank membrane) 0.04 -0.75 9.26 42.84 43.79

Table 7.3: Selectivity determination of NO2I PVC drop-coated IDE Design 2 (2% w/v) (conductive

response). Deionised water baseline-subtracted conductance upon the addition of: 5 ppm of the

target nitrite ion and 50 ppm of the interfering anions chloride and nitrate. The response from the

addition of 5 ppm NO2- to a device prepared without ionophore present is also shown

At 1 kHz the conductance response characteristics of the sensor appeared to be

favourable to nitrite when compared with the response from each of the interfering

anions. There was also very little response from a ‘blank’ membrane that did not

contain the ionophore. At 10 kHz there was an increased response from chloride and

nitrate; however, there was little response from the ‘blank’ membrane. An increased

response was observed for chloride and nitrate at 100 kHz, and the ‘blank’ membrane

device showed an increased response compared to the lower frequency results. At the

higher frequencies of 1 MHz and 2 MHz there was an increased response of both tested

interfering anions when compared to the response obtained from the target nitrite ion.

There was also a substantial response, greater than that of the ‘active’ membrane, from

the ‘blank’ membrane that did not contain the ionophore.

Craig Alexander

238

Cm – C0 (pF)

Ion Frequency


Nitrite (5 ppm) 1117.56 87.54 2.19 - -

Chloride (50 ppm) 1101.79 126.32 10.09 - -

Nitrate (50 ppm) 1040.39 115.16 8.48 - -

Nitrite (5 ppm)

(blank membrane) 15.45 22.67 28.76 - -

Table 7.4: Selectivity determination of NO2I PVC drop-coated IDE Design 2 (2% w/v) (capacitive

response). Deionised water baseline-subtracted capacitance upon the addition of: 5 ppm of the

target nitrite ion and 50 ppm of the interfering anions chloride and nitrate. The response from the

addition of 5 ppm NO2- to a device prepared without ionophore present is also shown

The capacitance response of NO2I PVC drop-coated IDE Design 2 was not investigated

at 1 MHz or 2 MHz, as there was an observed decrease in the measurements when

5 ppm NO2- was added to a deionised water background. At 1 kHz, 50 ppm of each of

the target anions produced a response that was almost equivalent to 5 ppm of the target

nitrite ion. The ‘blank’ membrane produced a response that was <1% of that of the

‘active’ membrane. At 10 kHz, the interfering anions produced a greater response on the

sensor, and an increased response was observed from the ‘blank’ membrane device,

when compared with the 1 kHz response. At 100 kHz, the response from the interfering

anions was even more pronounced and the ‘blank’ membrane response was

substantially greater than the response observed from the ‘active’ membrane that

contained the selective ionophore.

7.3 pH Sensors

Impedimetric pH sensors were prepared by drop-coating a 2% w/v PVC ‘cocktail’

containing 5% w/w of the ionophore onto the sensing area of IDE Design 2. Two

different commercial ionophores were tested, HIII and HV. An input amplitude of

10 mVrms was used. These devices will be referred to as ‘HIII PVC drop-coated IDE

Design 2’ and ‘HV PVC drop-coated IDE Design 2’, respectively.

Craig Alexander

239

The sensors were tested by placing the sensor into a low conductivity buffer solution,

described in Chapter 2 (section 2.7.3.1; Table 2.9), and recording the response at five

minute intervals over 30 minutes. The measurements were carried out in six separate pH

buffer solutions over the likely range found within a freshwater aquarium

(pH 6.5–8.5). As mentioned in section 2.7.3.1, the buffer solutions were prepared by

diluting a 10 mM stock solution at the desired pH value by a factor of 10 in deionised

water. As such, the final pH of the test solution deviated from that of the stock solution

due to a reduced buffering capacity. The final pH of each tested sample solution and the

resulting TDS concentration are shown in Table 7.5.

pH of 10 mM

stock solution

Final pH of diluted

test sample

TDS of diluted

test sample (ppm)

6.00 6.24 68

6.50 6.76 60

7.00 7.24 79

7.50 7.89 86

8.00 8.56 87

8.50 9.04 77

Table 7.5: Final pH and TDS concentration of the diluted buffer solutions used to test the response

of the impedimetric pH sensors

The response versus time data for HIII PVC drop-coated IDE Design 2 are shown in

Figure 7.5 (G) and Figure 7.6 (C). The response versus time data for HV PVC

drop-coated IDE Design 2 are shown in Figure 7.7 (G) and Figure 7.8 (C).

Craig Alexander

240

Figure 7.5: Conductance response versus time of HIII PVC drop-coated IDE Design 2 (2% w/v)

between pH 9.04–6.24 at five frequencies.


The conductance response of HIII PVC drop-coated IDE Design 2 does not appear to

show an analytically-relevant change over the pH range 9.04–6.24 at any of the

extrapolated frequencies. None of the measurements in any of the tested solutions

appeared to produce a stable response over the 30 minutes that they were submerged

within each test solution. Therefore, further analysis of the conductance results obtained

using this device was not possible.

5.96E-03

5.97E-03

5.98E-03

5.99E-03

6.00E-03

6.01E-03

6.02E-03

6.03E-03

6.04E-03

0 50 100 150 200

Co

nd

uct

ance

(S)

Time (min)

5.85E-03

5.86E-03

5.87E-03

5.88E-03

5.89E-03

5.90E-03

5.91E-03

5.92E-03

0 50 100 150 200

Co

nd

uct

ance

(S)

Time (min)

5.79E-03

5.80E-03

5.80E-03

5.81E-03

5.81E-03

5.82E-03

5.82E-03

5.83E-03

5.83E-03

5.84E-03

5.84E-03

0 50 100 150 200

Co

nd

uct

ance

(S)

Time (min)

5.760E-03

5.765E-03

5.770E-03

5.775E-03

5.780E-03

5.785E-03

5.790E-03

5.795E-03

5.800E-03

5.805E-03

5.810E-03

0 50 100 150 200

Co

nd

uct

ance

(S)

Time (min)

5.74E-03

5.75E-03

5.76E-03

5.77E-03

5.78E-03

5.79E-03

5.80E-03

0 50 100 150 200

Co

nd

uct

ance

(S)

Time (min)

pH 9.04

pH 8.56

pH 7.89

pH 7.24

pH 6.76pH 6.24

A B

C D

E

Craig Alexander

241

Figure 7.6: Capacitance response versus time of HIII PVC drop-coated IDE Design 2 (2% w/v)



The capacitance response of HIII PVC drop-coated IDE Design 2 produced a more

stable measurement at each tested pH than the conductance response at several

frequencies. At 1 kHz, there was no discernible response from the capacitance as the

sensor was placed into solutions with varying pH. At 10 kHz, 100 kHz, 1 MHz and

2 MHz, the capacitance response decreased as the pH decreased. This was not as

expected as an increase in the concentration of hydrogen ions within the test solution

would result in an increase in the measured conductance as H+ moved into the selective

membrane layer.

The effect of changing the ionophore on the response was investigated by drop-coating

a 2% w/v PVC membrane containing the ionophore HV onto the sensing area of

IDE Design 2.

1.080E-10

1.085E-10

1.090E-10

1.095E-10

1.100E-10

1.105E-10

1.110E-10

1.115E-10

1.120E-10

1.125E-10

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

1.090E-10

1.100E-10

1.110E-10

1.120E-10

1.130E-10

1.140E-10

1.150E-10

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

1.00E-10

1.05E-10

1.10E-10

1.15E-10

1.20E-10

1.25E-10

1.30E-10

1.35E-10

1.40E-10

1.45E-10

1.50E-10

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

1.50E-10

2.00E-10

2.50E-10

3.00E-10

3.50E-10

4.00E-10

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

6.00E-10

6.50E-10

7.00E-10

7.50E-10

8.00E-10

8.50E-10

9.00E-10

9.50E-10

1.00E-09

1.05E-09

1.10E-09

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

A B

C D

E

Craig Alexander

242

Figure 7.7: Conductance response versus time of HV PVC drop-coated IDE Design 2 (2% w/v)



The conductance response of HV PVC drop-coated IDE Design 2 demonstrated a

reduction in the measured response as the solution pH was decreased at 10 kHz,

100 kHz, 1 MHz and 2 MHz. The observed responses at each frequency were more

stable than the conductance response when HIII was used as the ionophore. As for the

results obtained when HIII was the ionophore, the observed change decreased as the

hydrogen ion concentration in the test solution was increased.

1.00E-04

1.20E-04

1.40E-04

1.60E-04

1.80E-04

2.00E-04

2.20E-04

2.40E-04

2.60E-04

0 50 100 150 200

Con

du

ctan

ce (S

)

Time (min)

1.00E-05

2.00E-05

3.00E-05

4.00E-05

5.00E-05

6.00E-05

7.00E-05

8.00E-05

0 50 100 150 200

Con

du

ctan

ce (S

)

Time (min)

5.00E-06

7.00E-06

9.00E-06

1.10E-05

1.30E-05

1.50E-05

1.70E-05

1.90E-05

2.10E-05

2.30E-05

2.50E-05

0 50 100 150 200

Con

du

ctan

ce (S

)

Time (min)

3.00E-06

3.50E-06

4.00E-06

4.50E-06

5.00E-06

5.50E-06

6.00E-06

6.50E-06

7.00E-06

0 50 100 150 200

Co

nd

uct

ance

(S)

Time (min)

A B

C D

E

6.00E-05

8.00E-05

1.00E-04

1.20E-04

1.40E-04

1.60E-04

1.80E-04

0 50 100 150 200

Con

du

ctan

ce (S

)

Time (min)

Craig Alexander

243

Figure 7.8: Capacitance response versus time of HV PVC drop-coated IDE Design 2 (2% w/v)



The capacitance response of HV PVC drop-coated IDE Design 2 showed a reduction in

the measurement as the pH of the test solution was decreased.

There was a limitation in the method that was used for measuring the response from the

pH sensors, as it required the complete removal of the device from one test sample

before being rinsed and placed into the next. This could have caused impedance effects

from the movement of the coaxial cable connecting the sensor under test to the LCR

meter. As such, the measurements obtained were unreliable and it cannot be definitively

stated that the observed response was a result of changes in the H+ concentration within

the test sample. One possible alternative method for testing impedimetric pH sensors,

without the requirement of withdrawing and submerging the sensor into different

sample solutions, would be to use an exponential dilution system. The sensor under test

1.330E-10

1.335E-10

1.340E-10

1.345E-10

1.350E-10

1.355E-10

1.360E-10

1.365E-10

1.370E-10

1.375E-10

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

E

1.36E-10

1.37E-10

1.38E-10

1.39E-10

1.40E-10

1.41E-10

1.42E-10

1.43E-10

1.44E-10

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

1.40E-10

1.50E-10

1.60E-10

1.70E-10

1.80E-10

1.90E-10

2.00E-10

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

1.00E-10

1.50E-10

2.00E-10

2.50E-10

3.00E-10

3.50E-10

4.00E-10

4.50E-10

5.00E-10

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

6.00E-10

7.00E-10

8.00E-10

9.00E-10

1.00E-09

1.10E-09

1.20E-09

1.30E-09

1.40E-09

1.50E-09

0 50 100 150 200

Cap

acit

ance

(F)

Time (min)

A B

C D

Craig Alexander

244

would be placed into a sample solution of known pH at the highest or lowest required

hydrogen ion concentration. Using a peristaltic pump at a known flow rate, a constant

addition of a pH buffer could then be added to either steadily increase or decrease the

hydrogen ion concentration within the test sample over the desired pH range over time.

Due to time constraints it was not possible to further investigate IDE Design 2 for its

use as a possible pH sensor.


Several experiments were conducted to investigate the use of IDE Design 2 as an

impedimetric sensor for ammonium, nitrite and pH.

When 50 ppm NH4+ was added to a deionised water background, an increase in both the

conductance and capacitance was observed from NH4I PVC drop-coated IDE Design 2

at each of the extrapolated frequencies. The selectivity of this device was investigated

through the addition of 50 ppm of the interfering cations potassium, sodium and

magnesium to a deionised water background, without the target ion present. At lower

frequencies of 1 kHz and 10 kHz there was a decrease in both the conductance and

capacitance response of each of the investigated interfering cations compared with the

response obtained for a solution containing only 50 ppm ammonium. However, there

appeared to be a substantial conductance and capacitance response when 50 ppm NH4+

was added to a solution and tested with a device that contained a ‘blank’ membrane. At

increased frequencies, both the interfering cation and ‘blank’ membrane responses

became more prominent. This suggested that the membrane may have been too thin for

this electrode geometry and therefore changes in the conductivity of the bulk solution

were observed.

Craig Alexander

245

Nitrite was added to a deionised water background and the resulting change in

conductance and capacitance was monitored using NO2I drop-coated IDE Design 2. An

increase in the response of the sensor was observed from the capacitance at all of the

extrapolated frequencies and from the conductance at frequencies <100 kHz. The

selectivity of this device was investigated through the addition of 50 ppm of the

interfering anions chloride and nitrate to a deionised water background, without the

target ion present. At a frequency of 1 kHz, there was a decrease in both the

conductance and capacitance response of each of the investigated interfering cations

compared with the response obtained for a solution containing only 5 ppm nitrite. At

this particular frequency very little response was obtained from the ‘blank’

PVC membrane.

Both the ammonium and nitrite sensors constructed from IDE Design 2 appeared to

demonstrate favourable response characteristics with regards to ion-selectivity at lower

frequencies. Therefore, further investigation is required into the frequency-dependence

of the response of IDE-based sensors, as the interference from the bulk solution was

most marked at the highest investigated frequencies.

Experiments conducted using IDE Design 2, which was coated with a 2% w/v PVC

membrane containing two different ionophores for H+ showed a decrease in the

response as the concentration of hydrogen ions within the test sample was increased.

This was not as expected and may have arisen due to a limitation with the method that

was used, which required the removal of the sensor from the test solution. Further

experimentation is required into the use of IDE devices coated with a polymeric

membrane as impedimetric pH sensors.

Craig Alexander

246

8 Conclusions and Suggestions for Further Work

8.1 Conclusions

The main focus of this thesis was to investigate the use of IDEs coated in a selective

membrane as impedimetric chemical sensors. Three separate devices based on IDEs

were fabricated using either photolithography or screen-printing and tested as

impedimetric ion-selective sensors for the determination of key ionic analytes found

within a freshwater aquarium.

The initial experimental work focused on the characterisation of commercially-available

ionophores for each of the identified aquarium-significant ions within conventional

potentiometric ISEs, to establish their feasibility for use within a freshwater sensing

device. Calibration and selectivity data were obtained from PMEs containing the

ionophores of interest and the results obtained deemed that each membrane composition

would be suitable for further testing within an IDE-based sensor. CWEs for nitrate were

also produced by coating the polymeric membrane over the surface of a Ag/AgCl

electrode, but these did not demonstrate acceptable response characteristics when

compared with PMEs. Sol-gels were investigated as an alternative membrane material

to the conventional PVC membranes; however, they exhibited poor response

characteristics that may have arisen from inadequate adhesion to the electrode.

IDE Design 1 was fabricated in-house using a ‘mask-less’ photolithography technique

followed by e-beam deposition of a 50 nm layer of gold metal. The sensing area of this

device was coated in a polymeric membrane containing a commercially-available nitrate

ionophore via spin-coating. The results from this particular device suggested that

non-selective changes in the bulk conductivity of the test solution were observed. It was

concluded that the electrode geometry of this device, with regards to the digits, was too

Craig Alexander

247

large and therefore the resulting penetration depth of the generated electric field was not

contained within the membrane layer and reached into the bulk test solution. Due to the

lack of selectivity and arduous fabrication process involved with this device, further

investigation was not carried out. Alternative devices were sought which featured either

reduced electrode geometry and/or an increased thickness of the selective

membrane layer.

To produce an impedimetric sensor with reduced electrode geometry, IDE Design 2 was

constructed from a commercial microfabricated IDE transducer. Initially the sensing

area of the device was coated with a PVC membrane via spin-coating; however, a large

response was observed when a device with a ‘blank’ membrane was tested. In an

attempt to overcome this and produce a thicker membrane layer, the polymeric material

was deposited via drop-coating. This also had the advantage of reducing the volume of

membrane solution that is wasted compared with spin-coating. Drop-coating of the PVC

membrane resulted in an increased response time of the sensing device due to an

increased diffusion distance. To improve the sensor response time, the PVC ‘cocktail’

viscosity was reduced by decreasing the mass of polymer and plasticiser that was

dissolved into the THF membrane solvent. A PVC membrane cocktail consisting of

2% w/v of the membrane components appeared to produce a sensor with a more

acceptable response time, so this composition was investigated further. The

nitrate-selective ionophore TDAN was investigated initially; however, when this

ionophore was tested it did not demonstrate adequate ion-selectivity and a response was

observed when a ‘blank’ membrane was deposited. An alternative ionophore, NO3V,

was investigated. The results from this membrane composition also demonstrated

interference from non-target anions; however, the response from the ‘blank’ membrane

was substantially lower than for the TDAN membrane. The difference between the two

Craig Alexander

248

‘blanks’ was the choice of plasticiser within the membrane. NPOE, which was used

with the ionophore NO3V, may have imparted greater hydrophobicity on the PVC

membrane and therefore a lower response was observed when no ionophore was

present.

Several experiments were conducted using IDE Design 2 as a sensor for the other

identified aquarium significant ions: ammonium, nitrite and pH. The sensing area of the

device was drop-coated with a 2% w/v PVC membrane containing 5% w/w of the

respective ionophore. For ammonium, the response of the sensor was ascertained by

measuring the response of a single concentration of the target ion (50 ppm). The

selectivity of the sensor was investigated by measuring the obtained response from

50 ppm of the interfering cations potassium, sodium and magnesium. For nitrite, the

response of the sensor was ascertained by measuring the response of a single

concentration of the target ion (5 ppm). The selectivity of the sensor was investigated by

measuring the obtained response from 50 ppm of the interfering anions chloride and

nitrate. It was observed that the ion-selectivity of both the ammonium and nitrite

sensors was greatest at the lowest investigated frequencies. The responses from the

devices that were coated in ‘blank’ membrane were also lowest in the low frequency

region. The results obtained from IDE Design 2 devices coated in an H+-selective

membrane were inconclusive, although this appeared to be as a result of the method that

was used, not from the devices themselves.

SP IDE Design 1 was produced as a fully screen-printed device which consisted of

carbon IDEs coated with a selective membrane layer. The ion-selective membrane layer

was modified to be suitable for screen-printing by mixing a commercial dielectric

screen-printing paste with NPOE as the plasticiser and TDAN as a

Craig Alexander

249

nitrate-selective ionophore. An initial attempt at producing screen-printed carbon IDE

transducers resulted in devices with many short-circuits as the digits were printed too

close together. An IDE geometry of 100 µm digit width with a 150 µm space between

adjacent digits was found to print effectively. When a single layer of the modified

dielectric paste ion-selective membrane layer was printed over the sensing area, strong

non-selective matrix interferences were observed. These were reduced by printing

‘build-up’ layers of non-modified paste prior to the final sensing layer. This

significantly reduced the observed response from a ‘blank’ membrane, which did not

contain any ionophore. When TDAN was used as the nitrate-selective ionophore, a

strong interference was observed from both chloride and nitrite anions, which would

render this particular membrane composition unsuitable for use within a freshwater

aquarium. Availability issues dictated that it was not possible for further screen-printed

IDE devices to be produced.

Due to the commercial aspect of this project, it is very important to consider the final

cost of producing individual ion-selective sensors based on IDEs, using either

photolithographic or screen-printing fabrication techniques. Screen-printing is generally

seen as the more cost-effective route for the high-throughput, mass-production of

sensing devices; however, due to the cost of commercially-available ionophores, this

may not be the case for this specific application. Both fabrication techniques require

further investigation into producing the best possible ion-selectivity. If both fabrication

techniques prove to produce devices which are viable for use within a freshwater

aquarium monitoring device, then the final cost-per-device needs to be established. This

would be calculated by ascertaining the cost of the transduction element together with

the cost of the recognition layer.

Craig Alexander

250

The use of IDEs as impedimetric sensors for aquarium water quality monitoring

provides several advantages over currently available methods. The subjectivity of wet

chemical testing is removed and the fabrication techniques involved allow for a more

cost-effective alternative to commercial ISEs. IDE devices are also well-suited to

disposable sensing without the requirement of an external reference electrode.

This thesis has described the first use of fully screen-printed ISCOM-type devices

which included both a screen-printed transduction element and a screen-printed

ion-selective recognition layer. This brought together research conducted by

Jaffrezic-Renault et al.161

, Camman et al.156

and Port et al.162

into the development of

ISCOM devices, with the research into screen-printable ion-selective membranes for

potentiometric applications, as described previously by Koncki et al.76

To the author’s

knowledge, this research also describes the first use of ISCOM-type devices with

ionophore-doped silica gels prepared via the sol-gel method as an alternative

ion-selective membrane material.

A substantial amount of further work is required to fully optimise and validate the

individual sensors before moving to the next stage of producing a prototype device. The

purpose of the following section is to discuss some of the potential experimental work

required to achieve this, along with considerations for the design of a prototype

multi-analyte aquarium sensing device.

8.2 Future Work

8.2.1 Individual Sensor Testing

Further investigation into individual ion sensors is required, such as for ammonium. As

mentioned previously in section 1.1.1.1, the ratio between the concentrations of

unionized and ionized ammonia is heavily dependent on both the pH and the

Craig Alexander

251

temperature of the environment. It is therefore important that calibration experiments

are carried out in a background buffer solution at several pH values in the expected pH

range of an aquarium. It is important to establish a suitable concentration range which

provides a wide range of unionized, toxic ammonia at each pH value, around the

acceptable upper limit for aquaria of 0.02 ppm NH3. Table 8.1 shows the unionized

ammonia concentration in the test vessel following the additions of a NH4+ stock

solution over the TAN concentration range 0.25–50 ppm, at pH values between pH 6.0

and 8.5. This would allow the observation of the response of the sensor using the same

TAN concentrations, but at varying ratios of non-toxic NH4+ and toxic NH3.

Ammonium concentration added (ppm)

pH 6.0 pH 6.5 pH 7.0 pH 7.0 (30 °C)

pH 7.5 pH 8.0 pH 8.5

0.25 0.00014 0.00045 0.00141 0.00199 0.0044 0.01342 0.03801

0.5 0.00028 0.00089 0.00282 0.00398 0.00881 0.02683 0.07603

1 0.00057 0.00179 0.00564 0.00796 0.01762 0.05366 0.15205

2 0.00113 0.00358 0.01128 0.01593 0.03523 0.10732 0.3041

5 0.00283 0.00895 0.02819 0.03982 0.08808 0.26831 0.76026

10 0.00567 0.0179 0.05639 0.07965 0.17616 0.53662 1.52052

50 0.02834 0.0895 0.28193 0.39823 0.88079 2.6831 7.60258

Table 8.1: The resulting concentration, in ppm, of unionized ammonia present following additions

of an ammonium stock solution to a test sample at various pH values. The values highlighted in

green are below the acceptable aquarium limits of 0.02 ppm NH3, whereas the values highlighted

in red are above this limit. Unless otherwise stated these concentrations relate to the equilibrium

of the two forms of ammonia at 25 °C

As the measured ammonia concentration is pH-dependent, it is important to ensure that

the ammonium measurement is linked to the pH measurement so that it can be corrected

accordingly. Likewise, pH is temperature dependent and therefore would require the pH

measurement to be linked to a temperature sensor. A suitable algorithm, taking into

account both the pH and temperature of the aquarium, would need to be incorporated

into any software that was written for a commercial device, to convert the measured

NH4+ concentration into its respective NH3 concentration, as this is the value that is

Craig Alexander

252

important for aquarium users to understand. It is also of upmost importance that the

effects of changing the pH within the aquatic environment on the response of sensors

for the other ions, such as nitrate and nitrite, are investigated.

8.2.2 Membrane Characterisation

It has been shown that one of the most important considerations for utilising IDE-based

sensors, which consist of a membrane layer coating the sensing area, is the thickness of

the deposited membrane in relation to the IDE geometry. Unfortunately, throughout this

project it was not possible to characterise any of the deposited membrane layers to

ascertain an exact thickness measurement. It would be very useful to be able to

accurately determine the physical characteristics of the deposited polymeric membrane

so that this information could be used to predict the effects that matrix interferences are

likely to have on the response of the sensor. By using specialist thin-film thickness

measurement equipment, such as the optical instruments offered by Filmetrics177

, or

microscopy techniques, such as atomic force microscopy (AFM), it may be possible to

accurately deduce the thickness of the ion-selective membrane layer.It has also been

shown that it is possible to use alternative coating techniques to increase the thickness

of the ion-selective membrane over the sensing area of the IDEs to contain more of the

generated electric field; however, this results in an increased response time of the sensor

due to an increased diffusion distance.

8.2.3 Membrane Materials

The use of sol-gels as an alternative ion-selective membrane material requires further

investigation. Sol-gels based on silica precursor materials were investigated throughout

this project; however, the results obtained were inconclusive as to their potential

feasibility within an impedimetric aquatic water sensing device. The inconclusive

Craig Alexander

253

results were largely caused by an unsuitable transduction element as opposed to the

selective membrane recognition layer. Further investigation into optimising the

composition of the silica gels is required. The ability to accurately characterise the

thickness of a deposited sol-gel layer, as described earlier, would aid this as it would

allow the user to predict the suitability of the sol-gel as an ion-selective membrane

based on its thickness relative to the membrane geometry. It would be possible to adjust

the viscosity of the sol-gel solution, and therefore the thickness of the final cast

membrane, by adjusting parameters such as the mass of precursor used and the volume

of solvent used during its preparation.

8.2.4 IDE Geometry

It would be most beneficial to investigate the response of an IDE sensor which had the

smallest possible geometry with respect to the digit widths and interdigital spacing. This

would allow the transduction element of the sensor to be coupled with an ion-selective

membrane layer that was very thin without compromising either the ion-selectivity or

response time. For example, if a microfabricated IDE transducer was designed with

digit widths and spacings of 1 µm, then a thin-film membrane of 2 µm would be

required to enclose the resulting electric field when excited with an AC voltage. Such a

thickness would be very achievable using conventional polymeric coating techniques.

Unfortunately, throughout this project, it was not possible to obtain any sensors with an

electrode geometry lower than IDE Design 2, which was purchased from MicruX

Fluidics, and consisted of digit widths and interdigital spacings of 10 µm. IDE Design 2

also appeared to demonstrate poor sensitivity which may have arisen due to the low

number of digits at each electrode (15). It would be advantageous to investigate how the

number of digits at each electrode affects the response of the sensors. This would also

Craig Alexander

254

be benefitted by having the smallest possible digit geometry, as a larger number of

digits could fit into a small sensing area.

8.2.5 Screen-Printed Sensors

Screen-printing has also been demonstrated as an appropriate fabrication technique for

producing ion-selective sensors based on IDEs. A layer-by-layer approach was used to

produce fully screen-printed devices, which included a screen-printed ion-selective

membrane layer by incorporating ionophores and plasticisers into a commercial

dielectric screen-printing paste. Further research is required into adjusting the

components within the recognition layer to maximise the selectivity of the sensor

response, by investigating several combinations of dielectric pastes, plasticisers and

ionophores.

8.2.6 Considerations for a Prototype Device

As the overall aim of this project was to develop sensors which could be implemented

within a prototype commercial device, it was important to consider a suitable design.

The individual sensors for each target ion need to be optimised with regards to the

electrode geometry, membrane composition, optimum frequency and input amplitude.

Once this information has been determined, it would be beneficial to produce bespoke

circuitry to power multiple sensors simultaneously along with a suitable housing for the

prototype. The IDE sensors could be produced as a replaceable transduction element

that is placed into the housing containing the necessary circuitry to operate them.

Screen-printing would likely provide the more cost-effective route for the transduction

element; however, the relatively large sensing area would require more of the often

expensive ionophore to be present within the membrane layer. A commercial IDE

Craig Alexander

255

device which has been fabricated using photolithography would likely be more

expensive than a screen-printed IDE device; however, the smaller overall footprint of its

sensing area would only require a very small amount (1 µl or less) of membrane

material to be fully coated.

A screen-printed device would have the advantage that it could be produced

‘ready-to-use’ with no further modification required, and it is possible to use flexible

substrates which would be better suited to disposable sensing applications. However,

the possibility of automating the deposition of a polymeric membrane on to an IDE

device using robotics could be investigated. The small surface area of a microfabricated

device could ensure that the sensing areas are produced on to an appropriate substrate,

such as glass, and then mounted into a suitable holder which would act as the

replaceable transduction element of the prototype.

8.2.7 Sensor Longevity

As the LCR meter that was used during this project was a single-input device, it did not

lend itself particularly well for use in determining the longevity of the impedimetric

sensors. It would be favourable to do this with a bespoke prototype device that is

capable of monitoring several sensors alongside one another. The sensors could be

placed into a test sample solution containing the target ions at a known, fixed

concentration and the response would be monitored over time. Degradation in the

observed signal would determine the operational lifetime of the sensors.

The operational lifetime of the sensors would be dictated by several factors, such as

leaching of the selective components from the membrane material and biofouling.

Either of these could be overcome by producing a disposable sensing element which

Craig Alexander

256

would require replacement at regular intervals. The device-to-device reproducibility of

the individual sensors could also be more accurately determined using bespoke

circuitry. Once reproducibility had been established, it could be possible to batch

calibrate devices, which would mean individual sensors would not require the final user

to calibrate them.

8.2.8 Data Display

One final consideration is how the results are to be displayed to the user. One option is

to use a screen which is connected directly to the device which could be positioned on

the outside of the user’s aquarium. Another option would be to use an appropriate

transmitter to wirelessly send the measurements to a suitable receiver unit, such as a PC

or smartphone with appropriate software installed.

Craig Alexander

257

9 References

1. http://www.ornamentalfish.org/aquanautmarket/ukmarket.php (accessed: 22/11/2013)

2. Pet Food Manufacturers Association, Pet Population 2013. Available at: http://www.pfma.org.uk/pet-

population/ (accessed: 20/11/2013)

3. European Pet Food Industry, Facts and Figures 2012. Available at: http://www.fediaf.org/facts-figures/

(accessed 20/11/2013)

4. American Pet Products Association, Pet Industry Size and Ownership Statistics 2013/14. Available at

http://www.americanpetproducts.org/press_industrytrends.asp (accessed: 20/11/2013)

5. K. J. Ruff, Freshwater Aquarium Chemistry - A Comprehensive Guide for Tropical Fish Hobbyists,

AquaChem Publishing, 2009.

6. R. Grommen, I. Van Hauteghem, M. Van Wambeke and W. Verstaete, Aquaculture, 2002, 211, 115-

124.

7. K. Emerson, R. C. Russo, R. E. Lund and R. V. Thurston, J. Fish. Res. Board Can., 1975, 32, 2379-

2383.

8. J. J. Keaffaber, R. Palma and K. R. Williams, J. Chem. Educ., 2008, 85, 225-230.

9. H. Roberts and B. Palmeiro, Vet. Clin. N. Am.: Exotic, 2008, 11, 359-374.

10. R. Baird, J. Bottomley and H. Taitz, Water Res., 1979, 13, 181-184.

11. F. B. Eddy, J. Fish Biol., 2005, 67, 1495-1513.

12. Ornamental Aquatic Trade Association (OATA) - Water Quality Criteria, Version 2.0, March 2008.

Available at http://oata.wedidthis.co.uk/wp-content/uploads/2012/08/Water-Quality-Criteria.pdf

(accessed 22/10/2013)

13. H. Kroupova, J. Machova and Z. Svobodova, Vet. Med., 2005, 50, 461-471.

14. J. A. Camargo, A. Alonso and A. Salamanca, Chemosphere, 2005, 58, 1255-1267.

15. R. R. Hensen, OFI Educational Publication 1 - Water Quality in the Ornamental Aquatic Industry,

Ornamental Fish International, 2006.

16. Dissolved Oxygen Fact Sheet - Available at

http://www.waterboards.ca.gov/water_issues/programs/swamp/docs/cwt/guidance/3110en.pdf (accessed:

06/12/2013)

17. Oxygen in the Aquarium - Available at http://www.algone.com/articles/technical-aquarium-

information/oxygen-in-the-aquarium (accessed: 6/12/13)

18. J. H. Tullock, Water Chemistry for the Marine Aquarium, Barron's Educational Series, Inc., 2002.

19. J. Saurina, E. Lopez-Aviles, A. Le Moal and S. Hernandez-Cassou, Anal. Chim. Acta, 2002, 464, 89-

98.

20. Phenol red acid/base equilibria. Available at http://commons.wikimedia.org/wiki/File:Phenol_red_-

_acid_base_equilibria_-_2.gif (accessed: 11/12/2013)

21. UV-Vis Spectrometry - Available at http://ishigirl.tripod.com/pchem/pka.html (accessed: 11/12/2013)

Craig Alexander

258

22. C. W. M.I. Nichols, J. Am. Chem. Soc., 1934, 56, 769-774.

23. H. Verdouw, C. J. A. Van Echteld and E. M. J. Dekkers, Water Res., 1978, 12, 399-402.

24. K. J. Ruff, Freshwater Test Kit Review, AquaChem Publishing, 2010.

25. Biotek® Application Note - Nitric Oxide Determination using the PowerWave 200 Scanning

Microplate Spectrophotometer and KC4 Data Reduction Software - Available at

http://www.biotek.de/resources/docs/PowerWave_200_Nitric_Oxide_Determination.pdf (accessed:

11/12/2013)

26. R. W. Catterall, Chemical Sensors, Oxford University Press, 1997.

27. F.-G. Bănică, Chemical Sensors and Biosensors: Fundamentals and Applications, John Wiley & Sons

Ltd., 2012.

28. U. E. Spichiger-Keller, Chemical Sensors and Biosensors for Medical and Biological Applications,

Wiley-VCH, 1998.

29. B. R. Eggins, Chemical Sensors and Biosensors, John Wiley & Sons Ltd., 2002.

30. C. M. A. Brett, Electroanalysis, Oxford University Press, 1998.

31. F. J. H. D.A. Skoog, S.R. Crouch, Principles of Instrumental Analysis, Thomson Brooks/Cole, 2007.

32. K. N. Mikhelson, Ion-Selective Electrodes - Lecture Notes in Chemistry, Springer-Verlag, Berlin,

2013.

33. R. W. Cattrall, Chemical Sensors, Oxford University Press, 1997.

34. P. Buhlmann, Ion-Selective Electrodes with Ionophore-Doped Sensing Membranes, John Wiley and

Sons Ltd., 2012.

35. A. K. Singh, G. Bhattacharjee, M. Singh and S. Chandra, Electroanal., 1997, 9, 1005-1008.

36. E. Malinowska, V. Oklejas, R. W. Hower, R. B. Brown and M. E. Meyerhoff, Sensor. Actuat., B-

Chem, 1996, 33, 161-167.

37. S. Y. Yun, Y. K. Hong, B. K. Oh, G. S. Cha, H. Nam, S. B. Lae and J. I. Jin, Anal. Chem., 1997, 69,

868-873.

38. R. D. Johnson and L. G. Bachas, Anal. Bioanal. Chem., 2003, 376, 328-341.

39. A. Craggs, G. J. Moody and J. D. R. Thomas, J. Chem. Educ., 1974, 51, 541-544.

40. U. Schaller, E. Bakker, U. E. Spichiger and E. Pretsch, Anal. Chem., 1994, 66, 391-398.

41. F. Faridbod, M. R. Ganjali, R. Dinarvand and P. Norouzi, Afr. J. Biotechnol., 2007, 6, 2960-2987.

42. R. Eugster, T. Rosatzin, B. Rusterholz, B. Aebersold, U. Pedrazza, D. Ruegg, A. Schmid, U. E.

Spichiger and W. Simon, Anal. Chim. Acta, 1994, 289, 1-13.

43. Cristina Mihali and Nora Vaum (2012). Use of Plasticizers for Electrochemical Sensors, Recent

Advances in Plasticizers, Dr. Mohammad Luqman (Ed.), InTech. Available from:

http://www.intechopen.com/books/recent-advances-in-plasticizers/use-of-plasticizers-for-

electrochemical-sensors (accessed: 19/12/2013)

Craig Alexander

259

44. Mohsen M. Zareh (2012). Plasticizers and Their Role in Membrane Selective Electrodes, Recent

Advances in Plasticizers, Dr. Mohammad Luqman (Ed.), InTech. Available from:

http://www.intechopen.com/books/recent-advances-in-plasticizers/plasticizers-and-their-role-in-

membrane-selective-electrodes (accessed: 19/12/2013)

45. E. Bakker, R. Meruva, E. Pretsch and M. Meyerhoff, Anal. Chem., 1994, 66, 3021-3030.

46. E. Lindner and B. D. Pendley, Anal. Chim. Acta, 2013, 762, 1-13.

47. G. G. Guilbault, R. A. Durst, M. S. Frant, H. Freiser, E. H. Hansen, T. S. Light, E. Pungor, G.

Rechnitz, N. M. Rice, T. J. Rohm, W. Simon and J. D. R. Thomas, Pure Appl. Chem., 1976, 48, 127-132.

48. Y. Umezawa, K. Umezawa and H. Sato, Pure Appl. Chem., 1995, 67, 507-518.

49. K. Umezawa, P. Buhlmann, Y. Umezawa, K. Tohda and S. Amemiya, Pure Appl. Chem., 2000, 72,

1851-2082.

50. P. Kane and D. Diamond, Talanta, 1997, 44, 1847-1858.

51. E. Bakker, R. K. Maruva, E. Pretsch and M. E. Mayerhoff, Anal. Chem., 1994, 66, 3021-3030.

52. V. P. Y. Gadzekpo and G. D. Christian, Anal. Chim. Acta, 1984, 164, 279-282.

53. F. Deyhimi, Talanta, 1999, 50, 1129-1134.

54. H. X. Wang and M. Pu, Chin. Chem. Lett., 2002, 13, 355-358.

55. A. Radu, T. Radu, C. McGraw, P. Dillingham, S. Anastasova-Ivanova and D. Diamond, J. Serb.

Chem. Soc., 2013, 78, 1729-1761.

56. G. J. Moody, R. B. Oke and J. D. R. Thomas, Analyst, 1970, 95, 910-918.

57. E. Bakker, P. Bühlmann and E. Pretsch, Chem. Rev., 1997, 97, 3083-3132.

58. P. W. Alexander, T. Dimitrakopoulos and D. B. Hibbert, Electroanal., 1998, 10, 707-712.

59. R. W. Cattrall and H. Freiser, Anal. Chem., 1971, 43, 1905-1906.

60. H. James, G. Carmack and H. Freiser, Anal. Chem., 1972, 44, 856-857.

61. H. Freiser, J. Chem. Soc., Faraday Trans., 1986, 82, 1217-1221.

62. A. Abbaspour, A. Izadyar and H. Sharghi, Anal. Chim. Acta, 2004, 525, 91-96.

63. A. Shokrollahi, A. Abbaspour, M. Ghaedi, A. N. Haghighi, A. H. Kianfar and M. Ranjbar, Talanta,

2011, 84, 34-41.

64. H. R. Zare, M. Salavati-Niassary, F. Memarzadeh, M. Mazloum and N. Nasirizadeh, Anal. Sci., 2004,

20, 815-819.

65. M. M. Ardakany, A. A. Ensafi, H. Naeimi, A. Dastanpour and A. Shamlli, Sensor. Actuat., B-Chem,

2003, 96, 441-445.

66. M. Aghamohammadi and N. Alizadeh, Anal. Chim. Acta, 2003, 480, 299-306.

67. M. M. Ardakani, A. Dastanpour and M. Salavati-Niasari, J. Electroanal. Chem., 2004, 568, 1-6.

68. J. P. Metters, R. O. Kadara and C. E. Banks, Analyst, 2011, 136, 1067-1076.

Craig Alexander

260

69. R. W. Hower, J. H. Shin, G. S. Cha, R. K. Meruva, M. E. Meyerhoff and R. B. Brown, Sensor.

Actuat., B-Chem, 1996, 33, 168-172.

70. G. S. Cha, D. Liu, M. E. Meyerhoff, H. C. Cantor, A. R. Midgley, H. D. Goldberg and R. B. Brown,

Anal. Chem., 1991, 63, 1666-1672.

71. H. D. Goldberg, R. B. Brown, D. P. Liu and M. E. Meyerhoff, Sensor. Actuat., B-Chem., 1994, 21,

171-183.

72. S. Walsh, D. Diamond, J. McLaughlin, E. McAdams, D. Woolfson, D. Jones and M. Bonner,

Electroanal., 1997, 9, 1318-1324.

73. R. Zielińska, E. Mulik, A. Michalska, S. Achmatowicz and M. Maj-Zurawska, Anal. Chim. Acta,

2002, 451, 243-249.

74. A. E. Musa, F. J. Del Campo, N. Abramova, M. A. Alonso-Lomillo, O. Domínguez-Renedo, M. J.

Arcos-Martínez, M. Brivio, D. Snakenborg, O. Geschke and J. P. Kutter, Electroanal., 2011, 23, 115-

121.

75. R. Koncki, S. Głab, J. Dziwulska, I. Palchetti and M. Mascini, Anal. Chim. Acta, 1999, 385, 451-459.

76. L. Tymecki, S. Glab and R. Koncki, Sensors, 2006, 6, 390-396.

77. Ł. Tymecki, E. Zwierkowska and R. Koncki, Anal. Chim. Acta, 2004, 526, 3-11.

78. U. Lange, N. V. Roznyatovskaya and V. M. Mirsky, Anal. Chim. Acta, 2008, 614, 1-26.

79. J. Bobacka, Electroanal., 2006, 18, 7-18.

80. C. Jimenez-Jorquera, J. Orozco and A. Baldi, Sensors, 2010, 10, 61-83.

81. U. Oesch and W. Simon, Anal. Chem., 1980, 52, 692-700.

82. P. Bühlmann, Y. Umezawa, S. Rondinini, A. Vertova, A. Pigliucci and L. Bertesago, Anal. Chem.,

2000, 72, 1843-1852.

83. C. A. Staples, R. Guinn, K. Kramarz and M. Lampi, Hum. Ecol. Risk Assess., 2011, 17, 1057-1076.

84. M. Collinson, Crit. Rev. Anal. Chem., 1999, 29, 289-311.

85. A. M. Buckley and M. Greenblatt, J. Chem. Educ., 1994, 71, 599-602.

86. M. M. Collinson, TrAC, Trends Anal. Chem., 2002, 21, 30-38.

87. K. Kimura, T. Sunagawa and M. Yokoyama, Chem. Lett., 1995, 967.

88. K. Kimura, T. Sunagawa and M. Yokoyama, Chem. Commun., 1996, 745.

89. K. Kimura, T. Sunagawa and M. Yokoyama, Anal. Chem., 1997, 69, 2379-2383.

90. K. Kimura, T. Sunagawa, S. Yajima, S. Miyake and M. Yokoyama, Anal. Chem., 1998, 70, 4309-

4313.

91. K. Kimura, S. Yajima, K. Okamoto and M. Yokoyama, J. Mater. Chem., 2000, 10, 1819-1824.

92. K. Kimura, H. Takase, S. Yajima and M. Yokoyama, Analyst, 1999, 124, 517-520.

93. S. Yajima, S. Uchida, Y. Hori and K. Kimura, Supramol. Chem., 2010, 22, 420-424.

Craig Alexander

261

94. K. Kimura, S. Yajima, H. Takase, M. Yokoyama and Y. Sakurai, Anal. Chem., 2001, 73, 1605-1609.

95. J. Liu, X. Wu, Z. Zhang, S. Wakida and K. Higashi, Sensor. Actuat., B-Chem, 2000, 66, 216-218.

96. M. Mazloum Ardakani, M. Salavati-Niasari, M. Khayat Kashani and S. M. Ghoreishi, Anal. Bioanal.

Chem., 2004, 378, 1659-1665.

97. M. M. Ardakani, M. K. Kashani, M. Salavati-Niasari and A. A. Ensafi, Sensor. Actuat., B-Chem,

2005, 107, 438-445.

98. M. Arvand, M. Moghimi and M. A. Bagherinia, Electroanal., 2007, 19, 2571-2576.

99. M. A. Zanjanchi, M. Arvand, N. O. Mahmoodi and A. Islamnezhad, Electroanal., 2009, 21, 1816-

1821.

100. A. Islamnezhad and M. Valizadeh, Anal. Bioanal. Electrochem., 2012, 4, 386-398.

101. E. M. G. Santos, A. N. Araújo, C. M. C. M. Couto and M. C. B. S. M. Montenegro, Anal. Bioanal.

Chem., 2006, 384, 867-875.

102. N. Zárate, A. N. Araujo, M. Conceiçao, B. S. M. Montenegro and R. Pérez-Olmos, Am. J. Anal.

Chem., 2011, 2, 491-499.

103. A. Jiménez-Morales, J. C. Galván and P. Aranda, Electrochim. Acta, 2002, 47, 2281-2287.

104. W. Kim, S. Chung, S. B. Park, S. C. Lee, C. Kim and D. D. Sung, Anal. Chem., 1997, 69, 95-98.

105. W. Kim, D. D. Sung, G. S. Cha and S. B. Park, Analyst, 1998, 123, 379-382.

106. B. H. Lee, Y. B. Shim and S. B. Park, Anal. Chem., 2004, 76, 6150-6155.

107. H. Zhou, M. E. Meyerhoff, K.-S. Bi and S. B. Park, J. Korean Electrochem. Soc., 2009, 12, 335-

341.

108. A. Demirel, A. Doğan, E. Canel, S. Memon, M. Yilmaz and E. Kili, Talanta, 2004, 62, 123-129.

109. P. Schulthess, Y. Shijo, H. V. Pham, E. Pretsch, D. Ammann and W. Simon, Anal. Chim. Acta,

1981, 131, 111-116.

110. H.-L. Wu and R.-Q. Yu, Talanta, 1987, 34, 577-579.

111. V. V. Egorov and Y. F. Lushchik, Talanta, 1990, 37, 461-469.

112. R. Yuan, Y.-Q. Chai and R.-Q. Yu, Analyst, 1992, 117, 1891.

113. V. V. Cosofret, T. M. Nahir, E. Lindner and R. P. Buck, J. Electroanal. Chem., 1992, 327, 137-146.

114. E. Lindner, V. V. Cosofret, R. P. Kusy, R. P. Buck, T. Rosatzin, U. Schaller, W. Simon, J. Jeney, K.

Toth and E. Pungor, Talanta, 1993, 40, 957-967.

115. D.-H. Cho, K.-C. Chung and M.-Y. Park, Talanta, 1998, 47, 815-821.

116. V. V. Egorov and Y. V. Sin'kevich, Talanta, 1999, 48, 23-38.

117. X. J. Liu, B. Peng, F. Liu and Y. Qin, Sensor. Actuat., B-Chem, 2007, 125, 656-663.

118. D.-H. Cho, K.-C. Chung, S.-S. Jeong and M.-Y. Park, Talanta, 2000, 51, 761-767.

119. T. Inoue, T. Baba and A. Yuchi, Electroanal., 2011, 23, 536-542.

Craig Alexander

262

120. J. Georges, Clin. Chem., 1979, 25, 1888-1890.

121. M. S. Ghauri and J. D. R. Thomas, Analyst, 1994, 119, 2323.

122. J. Chin, J. Oh, S. Y. Jon, S. H. Park, C. Walsdorff, B. Stranix, A. Ghoussoub, S. J. Lee, H. J. Chung,

S. M. Park and K. Kim, J. Am. Chem. Soc., 2002, 124, 5374-5379.

123. K. Suzuki, D. Siswanta, T. Otsuka, T. Amano, T. Ikeda, H. Hisamoto, R. Yoshihara and S. Ohba,

Anal. Chem., 2000, 72, 2200-2205.

124. S. Sasaki, T. Amano, G. Monma, T. Otsuka, N. Iwasawa, D. Citterio, H. Hisamoto and K. Suzuki,

Anal. Chem., 2002, 74, 4845-4848.

125. H.-S. Kim, H. Y. Park, H. J. Oh, K. K. Young, J.-H. Choi, D.-H. Lee, G. S. Cha and H. Nam, Anal.

Chem., 2000, 72, 4683-4688.

126. H.-S. Kim, D.-H. Kim, K. S. Kim, J.-H. Choi, J. H. Shim, I. S. Jeong, G. S. Cha and H. Nam, J.

Inclusion Phenom. Macrocyclic Chem., 2003, 46, 201-205.

127. H. L. Jin, T. H. Kim, J. Kim, S. S. Lee and J. S. Kim, Bull. Korean Chem. Soc., 2004, 25, 59-62.

128. S. Chandra, R. Buschbeck and H. Lang, Talanta, 2006, 70, 1087-1093.

129. P. D. Beer and P. A. Gale, Angew. Chem., Int. Ed., 2001, 40, 486-516.

130. D. Gao, J. Gu, R.-Q. Yu and G.-D. Zheng, Analyst, 1995, 120, 499.

131. P. Schulthess, D. Ammann, B. Krautler, C. Caderas, R. Stepanek and W. Simon, Anal. Chem., 1985,

57, 1397-1401.

132. R. Stepánek, B. Kräutler, P. Schulthess, B. Lindemann, D. Ammann and W. Simon, Anal. Chim.

Acta, 1986, 182, 83-90.

133. U. Schaller, E. Bakker, U. E. Spichiger and E. Pretsch, Talanta, 1994, 41, 1001-1005.

134. M. Shamsipur, M. Javanbakht, A. R. Hassaninejad, H. Sharghi, M. R. Ganjali and M. F. Mousavi,

Electroanal., 2003, 15, 1251-1259.

135. M. R. Ganjali, M. Rezapour, M. R. Pourjavid and M. Salavati-Niasari, Anal. Sci., 2003, 19, 1127-

1131.

136. M. R. Ganjali, S. Shirvani-Arani, P. Norouzi, M. Rezapour and M. Salavati-Niasari, Microchim.

Acta, 2004, 146, 35-41.

137. W. Wroblewski, Z. Brzozka, D. M. Rudkevich and D. N. Reinhoudt, Sensor. Actuat., B-Chem.,

1996, 37, 151-155.

138. M. Pietrzak and M. E. Meyerhoff, Anal. Chem., 2009, 81, 3637-3644.

139. T. R. L. C. Paixão, J. L. Cardoso and M. Bertotti, Talanta, 2007, 71, 186-191.

140. M. G. Mitrakas, C. A. Alexiades and V. Z. Keramidas, Analyst, 1991, 116, 361.

141. L. Ebdon, J. Braven and N. C. Frampton, Analyst, 1991, 116, 1005.

142. L. Campanella, C. Colapicchioni, G. Crescentini, M. P. Sammartino, Y. Su and M. Tomassetti,

Sensor. Actuat., B-Chem., 1995, 26-27, 329-335.

143. P. Sutton, J. Braven, L. Ebdon and D. Scholefield, Analyst, 1999, 124, 877-882.

Craig Alexander

263

144. A. Asghari, Talanta, 2003, 61, 557-563.

145. A. Asghari, M. K. Amini, H. R. Mansour, M. Salavati-Niasari and M. Rajabi, Anal. Sci., 2003, 19,

1121-1125.

146. J. Ortuno, R. Exposito, C. Sanchezpedreno, M. Albero and A. Espinosa, Anal. Chim. Acta, 2004,

525, 231-237.

147. M. M. Ardakani, M. Salavati-Niasari and M. Jamshidpoor, Sensor. Actuat., B-Chem., 2004, 101,

302-307.

148. H. K. Lee, K. Song, H. R. Seo and S. Jeon, Talanta, 2004, 62, 293-297.

149. P. Kong Thoo Lin, A. N. Araujo, M. C. B. S. M. Montenegro and R. Perez-Olmos, J. Food Chem.,

2005, 53, 211-215.

150. R. Mahajan, R. Kaur, H. Miyake and H. Tsukube, Anal. Chim. Acta, 2007, 584, 89-94.

151. B. Schazmann and D. Diamond, New J. Chem., 2007, 31, 587.

152. A. Watts, V. Gavalas, A. Cammers, P. Andrada, M. Alajarin and L. Bachas, Sensor. Actuat., B-

Chem., 2007, 121, 200-207.

153. Selectophore® Sensoric Applications - Available at http://www.sigmaaldrich.com/analytical-

chromatography/analytical-reagents/sensoric-applications.html (accessed: 23/01/2014)

154. R. V. Harrison, Chemical Sensors: Properties, Performance and Applications, Nova Science

Publishers, Inc., New York, 2010.

155. Cell Constant of Interdigitated Electrodes. Available at http://www.mosaic-

industries.com/embedded-systems/instrumentation/conductivity-meter/microfabricated-planar-

interdigitated-electrodes-cell-constant (accessed: 07/02/2014)

156. A. A. Shul'ga, B. Ahlers and K. Cammann, J. Electroanal. Chem., 1995, 395, 305-308.

157. A. Bratov, N. Abramova, J. Ramón-Azcón, A. Merlos, F. Sánchez-Baeza, M. P. Marco and C.

Domínguez, Electrochem. Commun., 2008, 10, 1621-1624.

158. A. V. Mamishev, A. Sundara-Rajan, F. Yang, Y. Du and M. Zahn, IEEE Sens. J., 2004, 92, 808-

845.

159. N. Jaffrezic-Renault and S. V. Dzyadevych, Sensors, 2008, 8, 2569-2588.

160. K. Cammann, B. Ahlers, D. Henn, C. Dumschat and A. A. Shul'ga, Sensor. Actuat., B-Chem., 1996,

35-36, 26-31.

161. B. Khadro, P. Namour, F. Bessueille, D. Leonard and N. Jaffrezic-Renault, Int. J. Environ. Anal.

Chem., 2009, 89, 11-19.

162. I. D. Nickson, C. Boxall and S. N. Port, IOP C. Ser. Mat. Sci. Eng., 2010, 9, 012044.

163. H. Suzuki, Electroanal., 2000, 12, 703-715.

164. P. Van Gerwen, W. Laureyn, W. Laureys, G. Huyberechts, M. Op De Beeck, K. Baert, J. Suls, W.

Sansen, P. Jacobs, L. Hormans and R. Mertens, Sensor. Actuat., B-Chem., 1998, 49, 73-80.

165. S. Laschi and M. Mascini, Med. Eng. Phys., 2006, 28, 934-943.

Craig Alexander

264

166. K. Norrman, A. Ghanbari-Siahkali and N. B. Larsen, Annu. Rep. Prog. Chem., Sect. C, 2005, 101,

174-201.

167. C. J. Brinker, G. C. Frye, A. J. Hurd and C. S. Ashley, Thin Solid Films, 1991, 201, 97-108.

168. Micro10x™ Robotic Dispenser - Available from http://www.hudsonrobotics.com/products/liquid-

handling/micro10x/ (accessed: 12/09/2014)

169. S. Ostling and P. Virtama, Acta phys. Scandinav., 1946, 11, 289-293.

170. Megaposit SRP220 Material Safety Data Sheet - http://kni.caltech.edu/facilities/msds/SPR220.pdf

(accessed: 10/04/2014)

171. Megaposit MF-26A Material Safety Data Sheet -

http://sindhu.ece.iisc.ernet.in/nanofab/twikii/pub/Main/LaserWriter,Microtech/MF-

26A_Developer__msds.pdf (accessed: 10/04/2014)

172. Megaposit SPR-220 Series Photoresists Data Sheet -

http://microchem.com/PDFs_Dow/SPR%20220%20DATA%20SHEET%20R%26H.pdf (accessed:

06/08/2014)

173. Micrux Technologies - Thin-film interdigitated array microelectrodes -

http://www.micruxfluidic.com/archivos/MicruX_thin-film_IDA1_electrodes.pdf (accessed: 06/08/2014)

174. M. S. Ghauri and J. D. R. Thomas, Anal. Proc., 1994, 31, 181-183.

175. Chemat Scientific Dip Coating. Available at -

http://www.chemat.com/chematscientific/DipCoater.aspx (accessed: 01/09/2014)

176. U. Trebbe, M. Niggemann, K. Cammann, G. Fiaccabrino, M. Koudelka-Hep, S. Dzyadevich and O.

Shulga, Fresenius. J. Anal. Chem., 2001, 371, 734-739.

177. Filmetrics Thin-Film Thickness Measurement. Available at - http://www.filmetrics.com/ (accessed:

12/09/2014)

Craig Alexander

265

10 Appendices

10.1 Molar Equivalent Concentrations of Ions

Ion Concentration (ppm) Concentration (mol/l)

Recommended upper limits for freshwater aquaria

Ammonia (NH3) 0.02 1.18 x 10-6

Nitrite (NO2-) 0.2 4.35 x 10

-6

Nitrate (NO3-) 50 8.0 x 10

-4

Phosphate (PO43-

) 0.05 5.27 x 10-7

Calcium carbonate

(CaCO3) 100–300 1.0 x 10

-3 – 3.0 x 10

-3

Stock solutions

Ammonium (NH4+) 10,000 0.56

Nitrite (NO2-) 10,000 0.22

Nitrate (NO3-) 10,000 0.16

Chloride (Cl-) 10,000 0.28

Potassium (K+) 10,000 0.26

Sodium (Na+) 10,000 0.44

Magnesium (Mg2+

) 10,000 0.41

Calcium (Ca2+

) 10,000 0.25

Table 10.1: Concentration values of species stated in ppm throughout the thesis converted to the

molar equivalent. The upper limits of the key water quality factors are shown, along with the stock

solutions that were prepared of each target and interfering ion

Craig Alexander

266

10.2 Details of Commercial Ionophores Used C

hem

ical

stru

ctu

re

IUP

AC

Nam

e

Tet

radodec

yla

mm

oniu

m n

itra

te

9,1

1,2

0,2

2-T

etra

hydro

tetr

aben

zo[d

,f,k

,m][

1,3

,8,1

0]

tetr

aaza

cycl

ote

trad

ecin

e-10,2

1-d

ithio

ne

Nonac

tin

N,N

-Dio

ctad

ecylm

ethyla

min

e

Cal

ix[4

]-az

a-cr

ow

n

Cyan

oaq

ua-

cobyri

nic

aci

d-h

epta

kis

-(2

-

phen

yle

thyle

ster

)

Com

mercia

l N

am

e

of

Ion

op

hore

N/A

Nit

rate

Ionophore

V

Am

moniu

m I

onophore

I

Hydro

gen

Ionophore

III

Hydro

gen

Ionophore

V

Nit

rite

Ionophore

I

Target

Ion

NO

3-

NO

3-

NH

4+

H+

H+

NO

2-

Table 10.2: Details of the commercial ionophores used in the experimental work described

throughout this thesis. The commercial name of each ionophore is shown, along with its

corresponding target ion, IUPAC name and chemical structure

Craig Alexander

267

10.3 Potentiometric Data Acquisition System

A system was designed to obtain data from potentiometric ISEs. Sixteen PHTX-22

pH/ORP preamplifiers (Omega, Manchester, UK) were arranged in two banks of eight.

Each bank was connected to a connector block (BNC-2110, National Instruments,

Austin, TX, USA), with 1 BNC lead per amplifier, and then to a 16-bit National

Instruments data acquisition card using a shielded cable (SHC68-68-EPN). For

availability reasons two different data acquisition cards were used: a PCIe-6320

and a PCI-6221.

Each 16-bit data acquisition card was operated in referenced single-ended mode, that is,

the measurement was taken between system ground and the preamplifier output voltage.

The reference electrodes were attached to system ground via the BNC connector blocks;

a double banana socket was inserted into each connector block. Therefore, four separate

reference electrodes could be connected, allowing for measurements to be taken from

up to four test samples simultaneously.

Initially, all 16 of the preamplifiers were powered using a linear lab supply, set to

provide 0 and 10 VDC. Unfortunately it was discovered that the amplifier units are

designed to drive the reference electrode to approximately 2.5 V above their negative

supply, which meant that experimentally obtained potentials lower than the reference

potential could not be measured with the reference electrode grounded. This is

illustrated in Figure 10.1, with a calibration graph from a pH electrode, with the

obtained measurements ‘clipping’ near 0 V.

Craig Alexander

268

Figure 10.1: Calibration graph from a pH electrode obtained using a PHTX-22 pH/ORP

preamplifier which was powered using a linear lab supply at 10 V

The offset is amplifier dependent, so groups of amplifiers sharing the same power

supply all have different reference electrode potentials, and hence only one working

electrode and one reference can be used in each sample solution. To use the array as

intended, all the reference voltages needed to be connected together, so a floating power

supply was needed for each preamplifier.

The first attempt to provide a floating supply for each amplifier was to connect two 9 V

batteries to each one; however, the power consumption of the amplifiers was too great

and the batteries required replacement with impractical frequency. A circuit board was

constructed with sixteen isolated DC/DC converters (NMR101C, Murata Power

Solutions, MA, USA) and output filters (a high-frequency inductor, IM02EB470K,

Vishay Dale, Selb, Germany) to supply each amplifier separately.

Temperature measurements were made using four negative temperature coefficient

(NTC) thermistors (B57831M871A3, EPCOS, Munich, Germany), which were

Craig Alexander

269

purchased from Farnell. These were wired in series with an 887 ohm 0.1% resistor and

each circuit was excited with a 5 V supply from a third National Instruments data

acquisition card (12-bit, PCI-6024E), which was also used to measure the voltage across

the thermistors.

The thermistors were calibrated by measuring the resistance at a series of known

temperatures between 25 °C and 50 °C, and fitted to the β parameter equation, shown in

Equation 10.1, where R0 is the resistance at T0 (25 °C).

R = R0 e−β(

1T0

− 1T)

Equation 10.1

The three data acquisition cards were synchronized using the National Instruments

Real-Time System Integration (RTSI) bus, with signals provided by the PCI-6024E

(M Series Synchronization with LabVIEW and NI-DAQmx, National Instruments

(http:// http://www.ni.com/white-paper/3615/en/). The data were acquired from the

cards (two cards for potential measurements and one for temperatures) and saved to a

table in a mySQL database (Oracle Corp., Redwood, CA, USA), under the control of a

program written using LabVIEW (National Instruments). A further program was written

to retrieve, display, and save the data in individual files.

Although the system worked much better to obtain potentiometric data from ISEs once

each amplifiers was provided with its own floating power supply, there were several

issues with obtaining the data from the database, and several times analytical data was

lost or unable to be recalled. Numerous attempts were made to overcome these issues

but the system proved very problematic and due to time constraints, a decision was

made to move to a commercial instrument, and an ELIT 4-channel ion analyser

was purchased.

Craig Alexander

270

Had more time been available to fully optimise the bespoke 16-channel system, it would

have been very beneficial to use it for this project. It was a much more cost-effective

way of producing a multi-channel analysing system than purchasing a commercially

available one. As all the results from the system were outputted to a database, this

would have made it very easy to store and quickly locate large amounts of data. This

system would also have been better suited to long-term monitoring, as all of the data

points between two time-stamps could have been recalled, which would have been

particularly beneficial for deducing the longevity of potentiometric sensors and also for

testing them alongside other sensors in a test sample, such as an aquarium, to

approximate the target ion concentration.

Documents

The Development of Smart Sensors for Aquatic Water Quality