Page 1
ENTIOMETRIC MEMBRANE SENSOR FOR DETERMINATION OFGLUTAMATE BASED ON [4](1)(2,3-DIAZABUTA-l,3-
DIENE)FERROCENOPHANE
NORHAFIZAH BT BAKEMAN
THESIS SUBMITTED IN FULFILLMENT OF THE REQUIREMENT FOR THEDEGREE OF MASTER OF SCIENCE ANALYTICAL CHEMISTRY
(MASTER BY RESEARCH)
FACULTY OF SCIENCE AND MATHEMATICSUNIVERSITI PENDIDIKAN SULTAN IDRIS
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111
ABSTRACT
tric membrane sensor has been developed and optimized based on
abuta-l,3-diene)ferrocenophane as an ionophore for high selectiveof glutamate. The best performance was shown by a membrane withuio of [4](1)(2,3-diazabuta-l,3-diene)ferrocenophane (ionophore): PVCP (plasticizer) at 9: 36: 55 (% w/w). The sensor works well in a linear� 10-5 to 1.0 X 10-1 M glutamate with a Nernstian slope of 57.6±1.0d its detection limit is 7.95 x 10-6 M. The sensor shows working rangeo at temperature 25. O± 1 °C and stable for a period of 3 months without� in potentials with response time equal or less than 30 seconds. Thefficient values determined by mixed solution method, indicate a goodglutamate over a wide variety of other tested anions.
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IV
PENGESAN MEMBRAN POTENSIOMETRI BAGI PENENTUANGLUTAMAT BERASASKAN [4](1)(2,3-DIAZABUTA-l,3-
DIENA)FEROSENOFAN
ABSTRAK
Pengesan membran potensiometri telah dibina dan dioptimum berasaskan [4](1)(2,3-diazabuta-1,3-diena)ferosenofan sebagai ionofor untuk penentuan glutamatberkepilihan tinggi. Keupayaan terbaik telah ditunjukkan oleh membran dengannisbah komposisi [4](1)(2,3-diazabuta-1,3-diena)ferosenofan (inofor): PVC
(pengikat): TEHP (pemplastik) pada 9: 36: 55 (% w/w). Pengesan ini bekerja denganbaik di dalam julat linear 1.0 x 10-5 hingga 1.0 x 10-1 M glutamat dengan kecerunanNernstian 57.6±1.0 mV/dekad dan had pengesanannya ialah 7.95 x 10- M. Pengesanini menunjukkanjulat bekerja diantara pH 6-10 pada suhu 25.0±1 °C dan stabil untuk
jangkamasa 3 bulan tanpa sebarang perubahan keupayaan dengan masa gerak balas 30saat atau kurang. Nilai pekali kepilihan ditentukan dengan kaedah larutan bercampur,menunjukkan kepilihan yang baik terhadap glutarnat mengatasi pelbagai jenis anion
yang telah diuji.
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v
TABLE OF CONTENTS
Page
DECLARATION
APPRECIATION
ABSTRACT
ABSTRAK
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS
I
11
111
IV
v
IX
X
XU
CHAPTER 1 INTRODUCTION
l.1 Electrochemical Sensor 1
l.l.1 Potentiometric Measurements 1
l.l.2 Amperometric Measurements 2
l.l.3 Conductivity Measurements 5
l.2 Ion Selective Electrode (ISE) 6
1.2.1 ISE Classification 7
1.2.1.1 Glass Electrodes 7
a) pH Electrode 7
b) Glass Electrodes for Other Cations 12
l.2.l.2 Liquid Membrane Electrode 13
l.2.l.3 Solid State Electrode 14
l.2.2 ISE Membrane Components 18
l.2.2.1 Polymeric Matrix 18
l.2.2.2Ionophore 19
l.2.2.3 Membrane Solvent (Plasticizer) 19
1.2.2.4 Ionic Additives 20
l.2.3 Membrane Potential 21
1.2.3.1 Ion Exchange Process 22
1.2.3.2 Ion Transport Process 22
l.3 Basic Principles of Potentiometric Measurement 24
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VI
1.3.1 Electrode Potential of an ISE 27
1.3.2 Activity vs Concentration of an Ion 29
1.3.3 Membrane Selectivity Coefficient 31
1.3.3.1 Mixed Solution Method 33
a) Fixed Interference Method 33
b) Fixed Primary Ion Method 34
1.3.3.2 Separate Solution Method 35
a) Separate Solution Method 35
(aA =aB)
b) Separate Solution Method 36
(EA =EB)1.3.3.3 Matched Potential Method 36
1.3.4 Limit of Measurements 37
1.3.5 Response Time 38
CHAPTER 2 LITERATURE REVIEW
2.1. Monosodium Glutamate (MSG) 40
2.1.1. Introduction 40
2.1.2. Physical and Chemical Properties 42
2.1.3. Sources of Glutamate 43
2.1.4. Estimated Intakes 45
2.1.5. Involvement of Glutamate in Neurotoxicity 49
2.2. Analysis of MSG 55
2.2.1. HPLC 56
2.2.2. Amperometric 58
2.2.3. Flow Injection Analysis 61
2.3. Research Objectives 63
CHAPTER 3 METHODOLOGY
3.1 Synthesis of [4](1 )(2,3-diazabuta-l,3-diene)
ferrocenophane3.1.1 Chemicals and Reagents
66
66
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CHAPTER 4
VB
3.1.2 Synthesis of Ferrocenecarboxaldehyde (FcCHO) 67
and Hydrazine (NH2NH2)3.l.3 Characterization of [4](1)(2,3-diazabuta-1,3 67
diene)ferrocenophane3.2 Preparation of Glutamate Ion Selective Electrode 68
3.2.1 Chemicals and Reagents 68
3.2.2 Instruments
3.2.3 Membrane Preparation3.3 Potentiometric Measurement
3.4 pH Effect
3.5 Lifetime
3.6 Selectivity of the Membrane Electrode, K {��3.7 Membrane Characterization
3.7.1 Water Content
3.7.2 Porosity3.7.3 Thickness and SwellingDetermination ofMSG in Real Sample3.8.1 ISE
69
69
70
73
74
3.8
74
75
76
76
77
77
78
783.8.l.1 Glutamate Standard and Sample
Preparation3.8.1.2 Potentiometric Measurement 78
79
79
79
3.8.2 HPLC
3.8.2.1 Reagents3.8.2.2 Glutamate Standard and HPLC Sample
Preparation3.8.2.3 Instrumentation and HPLC Analysis 80
RESULTS AND DISCUSSION
[4](1 )(2,3- diazabuta-1 ,3-diene)ferrocenophane4.1.1 [4] (1)(2,3- diazabuta-1,3-diene)ferrocenophane
FTIR Analysis4.2 Electrode Preparation and Potential Measurements 89
4.1 81
85
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CHAPTERS
. REFERENCES
PUBLICATION
AWARD
Vlll
4.2.1 pH Effect 96
4.2.2 Response Time and Lifetime 97
4.2.3 Selectivity of the Membrane Electrode, K i� 100
4.3 Membrane Characterization
4.4 Analytical Application
103
106
CONCLUSION 112
114
128
129
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IX
LIST OF TABLES
Tables Page
2.1 Glutamate in various proteins 49
4.1 Elemental analysis of carbon, hydrogen and nitrogen for 84
[4](1 )(2,3-diazabuta-1 ,3-diene)ferrocenophane
4.2 Optimized membrane composition of glutamate membrane 90
sensor and their potentiometric responses
4.3 Comparison between the proposed glutamate membrane sensor 95
based on [4](1 )(2,3-diazabuta-1 ,3-diene)ferrocenophane and
other reported techniques
4.4 Selectivity coefficients of glutamate membrane sensor towards 101
various interfering ions by mixed interference method with
fixed primary ion (1.0 x 10-3 M)
4.5 Percent of water content of glutamate membrane sensor 104
4.6 Porosity of glutamate membrane sensor 104
4.7 Percent of swelling of glutamate membrane sensor 105
4.8 Analysis of glutamate in food samples by the proposed glutamate 111
membrane sensor and HPLC method
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x
LIST OF FIGURES
Figures Page
l.1 Schematic diagram of an electrochemical cell for potentiometric 3
measurements
l.2 Glucose electrode: amperometric enzyme electrode 5
l.3 A glass pH electrode 8
l.4 Vacancy defect 15
l.5 Three variations of solid state membrane selective electrode 16
l.6 Development of a potential of an ISE 23
l.7 Membrane based device separate the sample from the internal 26
solution
l.8 Values ofproportionality constant 28
l.9 General idealized pattern of the evaluation of the K��� by the 34
fixed interference method
l.10 Calibration graph for a typical ISE 38
2.1 Chemical structure ofmonosodium glutamate 43
3.1 Casting of the membrane mixture 71
3.2 Preparation ofhomogeneous membrane electrode 72
3.3 Leaking test of a membrane electrode 72
4.1 Molecular structure of [4](1 )(2,3 -diazabuta-l ,3-diene)ferrocenophane 82
4.2 Reaction between FcCHO and NH2NH2 83
4.3 Possible mechanism at the membrane-solution interface 85
4.4 Ferrocene 86
4.5 Ferrocenecarboxaldehyde 86
4.6 [4](1 )(2,3-diazabuta-l,3-diene)ferrocenophane 86
4.7 Infrared spectrum of (a) ferrocene (b) ferrocenecarboxaldehyde and 87
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Xl
(c) [4](1 )(2,3-diazabuta-1 ,3-diene)ferrocenophane
4.8 Potential response of glutamate membrane sensor based on 93
[4](1 )(2,3-diazabuta-1,3-diene)ferrocenophane with various plasticizers
4.9 Effect of pH at 1.0 x 10-3 M and 1.0 x 10-4 M glutamate 97
solutions on the potential responses of membrane no. 5.
4.10 Response time of the glutamate membrane sensor for step change in 98
concentrations of glutamate: (a) 1.0 x 10-5 M, (b) 1.0 X 10-4 M,
(c) 1.0 X 10-3 M, (d) 1.0 x 10-2 M, (e) 1.0 x 10-1 M.
4.11 Lifetime of the glutamate membrane sensor 99
4.12 Emfversus logarithm of the standard glutamate concentration 107
4.13 Chromatograms of standard MSG solutions (a) 16911 ng/mL 108
(b) 1691 ng/mL (c) 169 ng/mL
4.14 Chromatograms of sample foods (a) oyster sauce (b) prawn snack 109
(c) seasoned flour
4.15 Standard curve ofMSG 110
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XlI
LIST OF ABBREVIATIONS
Ecel/ Cell potential
Standard cell potential
E* New constant ofpotential
G
Ag/AgCI
Conductance
Argentum!Argentum chloride
KpotA,B Selectivity coefficient of primary ion, A and interference ion, B
IUPAC International Union of Pure and Applied Chemistry
Matched potential electrodeMPM
SSM
Emf
Separate solution method
Electromotive force
Aluminosilicate ions
ABS Acrylonitrile butadiene styrene
NH2 Amine group
PMMA Poly(methyl methacrylate)
CWE Coated wire electrode
o-NPOE ortho 2-nitrophenyl octyl ether
MSG Monosodium glutamate
FDA Food and Drug Administration
FAOIWHO Food and Agriculture OrganizationIWorld Health Organization
JECFA
NMDA
ALS
NDA
Joint FAOIWHO Expert Committee on Food Additives
N-methyl-D-aspartate receptor
Amyotrophic lateral sclerosis
CBI
Naphthalene-2,3-dicarboxaldehyde
l-cyanibenz[flisoindole
mGluR Metabotropic glutamate receptor
Page 12
AMPA a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
receptor
y-aminobutyric acid
Carbon hydrogen oxygen functional group
Conducting polymer
L-glutamate oxidase
Flow injection analysis
Dichloromethane
Fourier transform Infra Red
Carbon, hydrogen, nitrogen, oxygen and sulphur
Diocthyl phthalate
Disodium hydrogen phosphate
Acetonitrile
Fluorescence detector
Diode array detector
Dioctyl phenylphosphonate
Glutamate dehydrogenase
Phenylglycine aminotransferase
Correlation coefficient
Lysine
Sodium tetraphenyl borate
GABA
CRO
CP
GLOD
FIA
DCM
FTIR
CHNOS
DOP
Na2HP04
ACN
FLD
DAD
DOPP
GDR
PhgAT
r
Lys
NaTPB
Xlll
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CHAPTER 1
INTRODUCTION
1.1 Electrochemical Sensor
Electrochemical sensors are based upon potentiometric, amperometric, or conductivity
measurements. These 3 different principles have their own specific design of
electrochemical cell.
1.1.1 Potentiometric Measurements
Potentiometric measurements are based on the determination of a voltage difference
between two electrodes (reference electrode and working electrode) plunged into a
sample solution with very small current is allowed (Rouessac & Rouessac, 2000).
Each of these electrodes constitutes a half cell. The working electrode is in direct
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2
contact with the analyte solution and the reference electrode is usually separated from
the analyte solution by a salt bridge of various forms (Figure 1.1). The electrode
potential of the working electrode is normally directly proportional to the logarithm of
the activity of the analyte in the solution (Kehlert, 2002; Evans, 1987). The potential
difference is described by Nernst equation:
2.303 RT---log acation
zF(1.1)
2.303 RTE = EO - log aanion
zF(1.2)
Where EO is the standard electrode potential of the sensor electrode, a is an ion
activity, z is the charge of an ion, R is the gas constant (8.314 J KI mol"), T is the
absolute temperature in Kelvin and F is Faraday constant (96500 coulombs). A
common potentiometric sensor for the measurement of electrolytes is ion selective
electrode (ISE). The ISE can be represented in the following way:
internal reference electrode I I internal solution I membrane
1.1.2 Amperometric Measurements
Amperometric electrode is a type of electrochemical sensor, as potentiometric
electrode discussed earlier. All chemical sensors consist of a transducer, which
transforms the response into a signal that can be detected (current) and a chemically
selective layer (Wang, Xu, Zhang, & Li, 2008).
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mV
Analyte solution-- -------------------------
------4-�- - - - - - - - - - - - - - - - - - - - - - --
---------------------------
---------
ISE = Ion Selective Electrode
ERE = external reference electrode
Internal reference
electrode
Internal solution
Membrane
Stirrer
Figure 1.1. Schematic diagram of an electrochemical cell for potentiometricmeasurements
3
Page 16
4
The transducer may be optical (example: fiber optic cable sensor), electrical
(potentiometric, amperometric) and thermal. The signal from amperometric electrode
is linearly dependent upon the activity of the analyte. As certain chemical species are
oxidized or reduced (redox reactions) at inert metal electrodes, electrons are
transferred from the analyte to the working electrode or from the working electrode to
the analyte.
Enzyme electrodes make use of one of the types of amperometric electrodes.
The enzyme is used to convert the species under test into an ion. As enzymes are
specific in their reactions, the analytical process based on them should be highly
selective (Evans, 1987). An example of an amperometric enzyme electrode is the
glucose electrode (Figure 1.2). The enzyme glucose is immobilized in a gel (example,
acrylamide) and coated on the surface of a platinum wire cathode. The gel also
contains a chloride salt and makes contact with Ag/Agel ring to complete the
electrochemical cell. Glucose oxidase enzyme catalyzes the aerobic oxidation of
glucose as follows:
glucose oxidase
Glucose + 02 + H20 ------.. gluconic acid + H202
Glucose and oxygen from the test solution diffuse into the gel where their
reaction is catalyzed to produce H202; part of this diffuses to the platinum cathode
where it is oxidized to give a current on proportion to the glucose concentration. The
remainder eventually diffuses back out of the membrane.
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5
Enzyme gel
Glucose
02
Pt cathode Ag anode ring
Figure 1.2. Glucose electrode: amperometric enzyme electrode
Other examples of amperometric enzyme electrodes based on the measurement
of oxygen or hydrogen peroxide include electrodes for the measurement of galactose
in blood (galactose oxidase enzyme), oxalate in urine (oxalate oxidase) and
cholesterol in blood serum (cholesterol oxidase).
1.1.3 Conductivity Measurements
Conductometric sensors are based on the measurement of electrolyte conductivity.
Conductivity measurements are generally performed with alternate current supply.
The conductivity is a linear function of the ion concentration; therefore, it can be used
for sensor applications. The conductance, G, measured between two electrodes of
area, A, and spacing, d, inserted into a conducting medium is the reciprocal of
resistance, R. For a given ion, the conductance of the solution will vary with the
concentration of the electrolyte. This relationship is linear for very dilute solutions. A
cell to measure the conductivity of electrolyte solutions usually comprises two parallel
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6
electrodes usually face to face made up of two planar and parallel electrodes (Wang,
Xu, Zhang, & Li, 2008).
1.2 Ion Selective Electrode (ISE)
A high percentage of chemical analyses are based on electrochemistry.
Electrochemical methods can be separated into two categories: those that measure
voltages and those that measure currents. The first group uses ion selective electrodes
(ISEs). Based on research conducted by Ganjali, Norouzi and Rezapour (2006) stated
that ISEs have been widely used for more than thirty years and have been used in a
wide variety of applications for determining the concentration of various ions in
aqueous solution such as pollution monitoring (determining fluoride, chloride and
nitrate in effluents and natural waters), agriculture (determining potassium,
ammonium, cyanide and others in soils and fertilizers), food processing (determining
nitrate and nitrogen dioxide in meat preservatives), corrosive effects ofN03 in canned
foods, determining fluoride in drinking waters and other drinks and last but not least
in education and research.
There are many advantages of ISEs. ISEs are relatively inexpensive, simple to
use, fast response, wide range of concentration and wide range of applications
compared to many other analytical techniques. ISEs are also particularly useful in
applications where only an order of concentration is required or it is only necessary to
know that a particular ion is below certain level of concentration. Besides that, ISEs
are particularly useful in medical and biological applications because they measure the
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activity of the ion directly rather than concentration. Since ISEs are one of the
techniques which can measure both positive and negative ions, they are unaffected by
turbidity and color of samples. In applications where interfering ions, pH levels or
high concentrations are a problem, then, many manufacturers can supply a specialized
experimental methods and special reagents to overcome many of these difficulties
(Rundle, 2000).
1.2.1 ISE Classification
Depending on the nature of the membrane material used to impart the desired
selectivity, ISEs can be divided into three groups: glass, liquid or solid electrodes.
Based on research conducted by Wang in 2006, stated that more than three dozen ISEs
are commercially available and are widely used.
1.2.1.1 Glass Electrodes
Glass electrodes are responsive to univalent cations. The selectivity for these cations
is achieved by varying the composition of a thin ion-sensitive glass membrane (Wang,
2006). The most common potentiometric device is the pH electrode (Figure 1.3).
a) pH Electrode
This electrode has been widely used for pH measurements for several decades. Its
success is attributed to its outstanding analytical performance, in extremely high
Page 20
Thin
membraneglass
Figure 1.3. A glass pH electrode
8
Agwire
Internal filling solution
(0.1 MHCl)
Page 21
9
selectivity for hydrogen ions, broad response range, and its fast and stable response
(Rundle, 2000).
Various types of membrane electrodes have been developed in which the
membrane potential is selective toward a given ion, just as the potential of the glass
membrane of a conventional glass electrode is selective toward hydrogen ions. These
electrodes are important in the measurement of ions, especially in small
concentrations. Generally, they are not disturbed by the presence of proteins, as some
other electrodes are, and so they are ideally suited to the measurements in biological
media. None of these electrodes is specific for a given ion, but each will possess
certain selectivity toward a given ion. So they are properly referred to as ion-selective
electrodes.
This glass electrode is specific to H+ ions. Glass in this case does not refer to
the material of the electrode body but to the membrane that ensures contact with the
solution. The membrane is a thin wall glass that has very high sodium content (25%).
In the presence of water, hydration occurs and the membrane's surface becomes
comparable to a gel while its interior corresponds to a solid electrolyte.
On a microscopic scale, the glass consists of a network of orthosilicate
Si(OH)4 whose open structure contains sodium cations that allow the movement of
charges from one side of the membrane to the other. The outside of the membrane is
in contact with the sample solution while the inside is in contact with the internal
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electrolyte, which has constant acidity (pH 7). The membrane is the seat of exchange
between Na+ and W cations as follows:
H+ (solution) + Na+ (glass) '" Na+ (solution) + H+ (glass)
When the concentration of H+ is different on either side of the membrane, a
potential difference is generated, which is related to the activity of H" ions in solution
(i.e. pH). The latter is determined using an electronic milivoltrneter, the pH meter,
which monitors the potential difference between the glass electrode and an internal
reference electrode of Ag/AgCI (currently preferred to the mercurous chloride
electrode for environmental purposes). When an W ion forms a silanol bond, a
sodium ion moves into the solution to preserve electroneutrality. Some of the more
popular glasses have three component compositions of 72% Si02, 22% Na20,
6% CaO or 80% Si02, 10% LiO, 10% CaO (Wang, 2006).
Before using the pH electrode, it should be calibrated using two (or more)
buffers of known pH. Many standard buffers are commercially available, with an
accuracy of ±O.01 pH unit. Calibration must be performed at the same temperature at
which the measurement will be made. The exact procedure depends on the model of
pH meter used. The pH electrode must be stored in an aqueous solution when not in
use, so that the hydrated gel layer of the glass does not dry out. A highly stable
response can thus be obtained over a long time period. After calibration, the
instrument will directly yield the pH of a solution.
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For measurement, only the bulb needs to be submerged. There is an internal
reference electrode and electrolyte (Ag/AgCI/Cr) for making electrical contact with
the glass membrane. Its potential is constant and is set by the concentration ofHCl. A
complete cell is represented as:
reference glass referenceelectrode W (unknown) membrane W (internal) electrode
(external) (internal)
Where H+int is concentration of internal hydrogen ion and H+unk is concentration of
unknown hydrogen ion. The potential of the glass membrane is given by;
2.303 R T aH+ int
Eglass = constant - log--F aH+unk(1.3)
And the voltage of the cell is given by;
2.303 R TEcell = k + Flog aw unk (1.4)
k is a constant which include the potentials of the two reference electrodes, the liquid
junction potential, a potential at the glass membrane due to W (internal) and
asymmetry potential. The asymmetry potential is a small potential across the
membrane that is present even when the solutions on both sides of the membrane are
identical. It is associated with factors such as nonuniform composition of the
membrane, strains within the membrane, mechanical and chemical attack of the
external surface, and the degree of hydration of the membrane. It slowly changes in
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time, especially if the membrane is allowed to dry out. For this reason, a glass pH
electrode should be calibrated from day to day. The asymmetry potential will be
varied from one electrode to another, owing to differences in construction of the
membrane (Christian, 2004).
b) Glass Electrode for Other Cations
Alkaline solutions were noted to display some interference on the pH response for
glass pH electrodes. Deliberate changes in the chemical composition of the glass
membrane (along with replacement of the internal filling solution) have thus led to
electrodes responsive to monovalent cations other than hyderogen, including sodium,
ammonium and potassium (Wang, 2006). This usually involves the addition of B203
or Al203 to sodium silicate glasses to produce anionic sites of appropriate charge and
geometry on the outer layer of the glass surface. Example, sodium selective glasses
have the compositions 11 % Na20, 18% Ah03, 71 % Si02 while ammonium selective
glasses have the compositions of 27% Na20, 4% Ah03, 69% Si02 (Wang,2006).
These compositions are different from sodium silicate glasses which are used for pH
measurements, it is because these sodium aluminosilicate glasses posses what may be
termed AlOSiO- sites with weaker electrostatic field strength and a marked preference
for cations other than protons.