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

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.

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.

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

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

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

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

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

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

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

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

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

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

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).

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

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.

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

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

7

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

Thin

membraneglass

Figure 1.3. A glass pH electrode

8

Agwire

Internal filling solution

(0.1 MHCl)

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

10

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.

11

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

12

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.