VOT 75149 A NOVEL CAPILLARY … · vot 75149 a novel capillary electrophoretic method for the analysis of dioxins and furans (kaedah novel elektroforesis rerambut untuk analisis dioksin

VOT 75149

A NOVEL CAPILLARY ELECTROPHORETIC METHOD FOR THE ANALYSIS OF DIOXINS AND FURANS

(KAEDAH NOVEL ELEKTROFORESIS RERAMBUT UNTUK ANALISIS DIOKSIN DAN FURAN)

WAN AINI BINTI WAN IBRAHIM

PUSAT PENGURUSAN PENYELIDIKAN UNIVERSITI TEKNOLOGI MALAYSIA

2008

VOT 75149

VOT 75149

A NOVEL CAPILLARY ELECTROPHORETIC METHOD FOR THE ANALYSIS OF DIOXINS AND FURANS

(KAEDAH NOVEL ELEKTROFORESIS RERAMBUT UNTUK ANALISIS DIOKSIN DAN FURAN)

WAN AINI BINTI WAN IBRAHIM

RESEARCH VOTE NO: 75149

Jabatan Kimia Fakulti Sains

Universiti Teknologi Malaysia

2008

ii

ACKNOWLEDGEMENT

Financial assistance from Ministry of Higher Education (MOHE) for the

project number 75149 is greatfully acknowledged.

iii

ABSTRACT

A NOVEL CAPILLARY ELECTROPHORETIC METHOD FOR

THE ANALYSIS OF DIOXINS AND FURANS

(Keywords: Micellar electrokinetic chromatography, capillary elctrophoresis, dioxin, furan)

Micellar electrokinetic chromatography (MEKC) is increasingly popular in the analysis of organic pollutants in the environment due to its high separation efficiency, less solvent usage and shorter analysis time. In this study, MEKC was used for the separation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibenzo-p-furan (TCDF) with a separation buffer consisting of 20 mM sodium cholate, 20 mM sodium tetraborate decahydrate and 5% v/v organic modifier acetonitrile-methanol (3:1 v/v) at a final buffering pH of 9.16-9.22. Separation voltage was at 25 kV with anodic injection and optimum wavelength set at 225 nm. To improve the limit of detection (LOD), five on-line preconcentration techniques were evaluated. The techniques were normal stacking mode (NSM-MEKC), reversed electrode polarity stacking mode (REPSM-MEKC), high conductivity sample stacking mode (HCSSM-MEKC), sweeping and field enhanced sample injection (FESI-MEKC). High conductivity sample stacking method-MEKC (HCSSM-MEKC) that gave an LOD of 46 ppb for TCDF and 18.5 ppb for TCDD was chosen. Solid phase disc extraction (SPDE) was used to further improve the LOD during the extraction of TCDF and TCDD from water thus reducing the LODs by 1000 fold. LODs in the ppt range were achieved for both analytes.

Key researchers:

Assoc. Prof. Dr. Wan Aini Wan Ibrahim (Head) Prof. Dr. Mohd Marsin Sanagi

Ms Sharain Liew Yen Ling

E-mail : [email protected] Tel. No. : 07-5534311 Vote No. : 75149

iv

ABSTRAK

KAEDAH NOVEL ELEKTROFORESIS RERAMBUT UNTUK

ANALISIS DIOKSIN DAN FURAN

(Kata kekunci: Kromatografi rerambut elektrokinetik misel, elektroforesis rerambut, dioksin, furan)

Kromatografi rerambut elektrokinetik misel (MEKC) semakin kerap digunakan dalam analisis bahan pencemar alam sekitar kerana kecekapan pemisahan yang tinggi, penggunaan isipadu pelarut yang kecil dan masa analisis yang singkat. Dalam kajian ini, MEKC telah digunakan untuk memisahkan 2,3,7,8-tetraklorodibenzo-p-dioksin (TCDD) dan 2,3,7,8-tetraklorodibenzo-p-furan (TCDF) dengan menggunakan larutan penimbal 20 mM natrium kolat, 20 mM natrium tetraborat dekahidrat dan campuran pengubahsuai organik 5% asetonitrile-methanol (3:1 v/v) dengan pH 9.16-9.22. Voltan pemisahan adalah 25 kV dan panjang gelombang optimum pada 225 nm. Lima teknik pra-pemekatan talian terus iaitu mod penyusunan normal (NSM-MEKC), penyusunan kekutuban elektrod berbalik (REPSM-MEKC), mod penyusunan sampel kekonduksian tinggi (HCSSM-MEKC), sapuan (S-MEKC) dan suntikan sampel peningkatan medan (FESI-MEKC) telah diuji untuk membaiki had pengesanan analit. Mod penyusunan sampel kekonduksian tinggi (HCSSM-MEKC) telah dipilih dengan had pengesanan 46 ppb untuk TCDF dan 18.5 ppb untuk TCDD. Kaedah pengekstrakan cakera fasa pepejal (SPDE) telah digunakan untuk memperbaiki had pengesanan TCDF dan TCDD dalam sampel air dan berjaya menurunkan had pengesanan sebanyak 1000 kali ganda. Had pengesanan adalah dalam julat ganda bahagian per trillion (ppt).

Penyelidik Utama:

Assoc. Prof. Dr. Wan Aini Wan Ibrahim (Head) Prof. Dr. Mohd Marsin Sanagi

Cik Sharain Liew Yen Ling

E-mail : [email protected] Tel. No. : 07-5534311 Vote No. : 75149

v

TABLE OF CONTENTS

CHAPTER TITLE PAGE

TITLE PAGE i

ACKNOWLEDGEMENT ii

ABSTRACT iii

ABSTRAK iv

TABLE OF CONTENTS v

LIST OF TABLES xi

LIST OF FIGURES xv

LIST OF SYMBOLS xxvii

LIST OF ABBREVIATIONS

LIST OF APPENDICES

xxviii

xxxi

1 INTRODUCTION

1.1 Background 1

1.2 Summary 2

2 LITERATURE STUDY 5

2.1 Organic Pollutants 5

2.2 Chlorinated Dioxins and Dibenzofurans 6

2.2.1 Physical and Chemical Properties Of 2,3,7,8-TCDF

and 2,3,7,8-TCDD

7

vi

2.2.2 Toxicity of PCDDs and PCDFs 8

2.2.3 Potential Routes of PCDDs and PCDFs Exposure In

Wildlife and Humans

9

2.2.3.1 Commercial and Technical Products 9

2.2.3.2 Municipal Incinerators and Hazardous Waste Incinerators 10

2.2.3.3 Food Products 10

2.3 Organophosphorous Pesticides 11

2.3.1 Physical and Chemical Properties 12

2.3.2 Toxicity of OPPs 14

2.4 Sample Extraction and Clean-up Strategies of Organic

Pollutants

14

2.4.1 Methods For The Extraction Organic Pollutants 15

2.4.2 Clean-up Procedures for Organic Pollutants 17

2.5 Identification and Quantification of Organic Pollutants 19

2.5.1 Gas Chromatography 19

2.5.2 Micellar Electrokinetic Chromatography (MEKC) 20

2.5.3 Types of Surfactants 21

2.5.4 Organic Modifier Effect 25

2.5.5 Matrix Effect 26

2.6 On-line Preconcentration Techniques 27

2.6.1 Stacking 28

2.6.2 Sweeping 34

2.7 Problem Statement 35

2.8 Research Objectives 37

2.9 Scope of Work 38

3 EXPERIMENTAL 41

3.1 Introduction 41

3.2 Reagents 41

3.3 Instrumentations 42

3.4 Conditioning of the Capillary 43

3.5 Procedures for MEKC Separation of TCDF and TCDD 44

vii

3.5.1 Preliminary Investigation For The Separation of TCDF and

TCDD

44

3.5.2 Evaluation Of Organic Modifier Effect On The Separation

of TCDF and TCDD

45

3.5.3 Separation Efficiency of TCDF and TCDD Under Different

Sample Matrix

45

3.5.4 Normal Stacking Mode (NSM-MEKC) In The Separation

Of TCDF and TCDD

46

3.5.5 Reversed Electrode Polarity Stacking Mode 47

3.5.6 High Conductivity Sample Stacking Mode (HCSSM) 47

3.5.7 Sweeping 47

3.5.8 Field-Enhanced Sample Injection (FESI-MEKC) 48

3.6 Off-line Preconcentration Using Solid Phase Disc

Extraction (SPDE)

48

3.6.1 Sample Preparation 48

3.6.2 Solid Phase Disc Extraction 49

3.7 Summary of Methodology of Optimization of eparation of

TCDD and TCDF

51

3.8 Procedures for MEKC Separation Of Hydrophilic

Organophosphorous Pesticides (OPPs)

52

3.8.1 Wavelength Optimization In The Separation Of OPPs

Under Acidic Conditions

52

3.8.2 Stacking With Reverse Migrating Micelles (SRMM) In The

Analysis Of OPPs

53

3.9 Off-line Concentration Using Solid Phase Disc

Extraction (SPDE) Of OPPs

53

3.9.1 Sample Preparation 53

3.9.2 Extraction 53

3.9.3 Method Validation Of Extraction Process 54

4 OPTIMIZATION OF SEPARATION PARAMETERS 55

4.1 Introduction 55

viii

4.2 Preliminary Investigation On The Separation of TCDF and

TCDD Using MEKC

55

4.2.1 Confirmation of Individual Peaks 56

4.2.2 Optimization of Analyte Separation By Varying The

Percentage Of Acetontrile In The Running Buffer Solution

57

4.3 Evaluation of Organic Modifier Effect On The Separation

Efficiency of TCDF and TCDD Using MEKC

62

4.3.1 Comparison of Separation Performance Between

Acetonitrile and Methanol

63

4.3.2 Comparison Of Mixed Modifier With Single Organic

Modifier On Separation Performance

68

4.4 Investigation of Sample Matrix Effect On The Separation

Efficiency of TCDF and TCDD

74

4.4.1 Baseline Stability and Unknown Peak Elimination 74

4.4.2 Comparison Of Peak Area, Peak Height

And Separation Efficiency In Different Matrix

77

5 OFF-LINE AND ON-LINE PRECONCENTRATION

TECHNIQUES

80

5.1 Introduction 80

5.2 Normal Mode (NM-MEKC) In Analysis of TCDF and

TCDD

80

5.2.1 Normal Mode MEKC (NM-MEKC) 80

5.2.2 Calibration Lines, Linearity (r2), LODs 81

5.2.3 Repeatability and Efficiency (N) 82

5.3 Normal Stacking Mode (NSM-MEKC) 84

5.3.1 Optimization of Injection Time 85


5.3.3 Repeatability, Reproducibility and Efficiency (N) 89

5.3.4 Stacking Efficiency 92

5.4 Reversed Electrode Polarity Stacking Mode (REPSM) 93

ix

5.4.1 Influence Of Various Stacking Periods 93

5.4.2 Calibration lines, linearity (r2) and LODs 97

5.4.3 Repeatability and Efficiency 98


5.5 High Conductivity Sample Stacking Mode

(HCSSM-MEKC)

99

5.5.1 Optimization of Sodium Chloride Concentration 100

5.5.2 Calibration lines, linearity (r2) and LODs 103

5.5.3 Repeatability, Reproducibility and Efficiency 104


5.6 Sweeping 106

5.7 Field-Enhanced Sample Injection (FESI) 107

5.8 Off-line Preconcentration Technique With Solid Phase Disc

Extraction (SPDE)

109

5.9 Conclusions

113

6 SEPARATION OF HYDROPHILIC

ORGANOPHOSPHOROUS PESTICIDES USING

MICELLAR ELECTROKINETIC CHROMATOGRAPHY

(MEKC) WITH ACIDIC BUFFER

114

6.1 Introduction 114

6.2 Reverse Mode Micellar Electrokinetic Chromatography 115

6.2.1 Wavelength Optimization 115


6.2.3 Repeatabilities and Efficiency (N) 119

6.3 Stacking With Reverse Migrating Micelles 121


6.3.2 Repeatability and Efficiency (N) 124

6.3.3 Stacking Efficiencies (SEF) 125

x

6.4 Off-line Preconcentration Technique With Solid Phase

Disc Extraction (SPDE)

127

7 CONCLUSIONS AND FUTURE DIRECTION 130

7.1 Conclusions 130

7.2 Future Direction

134

REFERENCES 135

APPENDICES

List of Publications

List of Presentations

152

152

153

xi

LIST OF TABLES

TABLE NO. TITLE PAGE

2.1 Physical and chemical properties of 2,3,7,8-TCDF and 2,3,7,8-

TCDD.

7

2.2 Toxicity Equivalent Factors (TEF) for chlorinated

dibenzodioxins and chlorinated dibenzofurans

8

2.3 Functions and physical properties of the three hydrophilic OPPs 12

2.4 Example of various sorbents used in SPE for the analysis of

polychlorinated dibenzodioxins, polychlorinated dibenzofurans,

polychlorinated biphenyls (PCBs) and organophosphorous

pesticides (OPPs) from various matrices.

18

2.5 Some examples of GC analysis used for PCDD/Fs 22

2.6 Some examples of GC analysis used for OPPs 23

2.7 Some examples of determination of organic pollutants using

MEKC.

24

4.1 Repeatability of migration time, peak areas and peak height for

TCDF and TCDD under different fractions of acetonitrile in

running buffer. Separation conditions remain the same as in

Figure 4.1.

61

4.2 A comparison on the effect of different fractions of MeCN on

the peak resolution, Rs for TCDF measured relative to peak X.

62

4.3 A comparison on the effect of different fractions of MeOH on

the peak resolution, Rs for TCDF measured relative to peak X.

63

4.4 A comparison of the effect of 15 % v/v MeCN and 15 % v/v

MeOH on the migration time (tm), resolution (Rs), efficiency

(N) and selectivity (α) of TCDF and TCDD MEKC separation.

65

xii

4.5 Repeatability of migration time, peak area and peak height for

TCDF and TCDD under different fractions of methanol, MeOH

in running buffer. Separation conditions the same as in Figure

4.1.

66

4.6 The effect of different organic modifiers on the relative

standard deviation (% RSD) of migration time, peak area and

peak height for TCDF and TCDD.

73

5.1 Equation of calibration curves, correlation coefficients, r2,

LODs (at S/N = 3) on the basis of calibration curves in Figure

5.1.

82

5.2 Repeatability of migration time, peak area and peak height

(mAU) in the separation of TCDF and TCDD using NM-

MEKC.

82

5.3 Injection volume and plug length as a function of pressure, time

and capillary id. % of sample plug and corresponding % of

remaining length available for separation is also indicated.

86

5.4 Equation of calibration curves, r2, LODs (for S/N = 3)

on the basis of calibration curves in Figure 5.6.

89

5.5 Repeatability of migration time, peak height and peak area for

TCDF and TCDD using NSM.

90

5.6 Reproducibility of migration time (min), peak area (mAUs) and

peak height (mAU) for TCDF and TCDD.

91

5.7 Sensitivity Enhancements Factor (SEF) in NSM-MEKC over

NM-MEKC in the separation of TCDF and TCDD.

92

5.8 Corresponding percentage of original current, % with polarity

switching time,s used in REPSM.

95

5.9 Equation of calibration curves, r2, LODs (for S/N = 3) on the

basis of calibration curves in Figure 5.11 using REPSM.

97

5.10 Repeatability of migration time, peak area and peak height in

the separation of TCDF and TCDD using REPSM.

98

5.11 Sensitivity Enhancement Factors (SEF) in REPSM over NM-

MEKC in the separation of TCDF and TCDD.

99

xiii

5.12 Equation of calibration curves, r2, LODs (at S/N = 3) for TCDF

and TCDD based on calibration curves in Figure 5.15.

103

5.13 Repeatability of migration time (min), peak area (mAUs) and

peak height (mAU) in the separation of TCDF and TCDD.

104

5.14 Reproducibility over four consecutive days with n=5 per day of

migration time (min), peak area (mAUs) and peak height

(mAU) in the separation of TCDF and TCDD using HCSSM.

TCDF is at 2 ppm while TCDD is at 0.3 ppm.

104

5.15 Sensitivity Enhancements Factor (SEF) in HCSSM-MEKC

over NM-MEKC in the separation of TCDF and TCDD.

106

5.16 Recovery and repeatability of extraction of TCDF and TCDD

from spiked water using SPDE.

110

5.17 Concentration of TCDF and TCDD discovered in pulp-mill

treated and untreated effluent. ND: Not detected.

111

6.1 Summary of wavelength optimization for dicrotophos,

monocrotophos and phosphamidon. Separation conditions:

separation buffer contained 20 mM phosphate (pH 2.3), 10 mM

SDS and 10% v/v methanol; samples at 100 ppm each prepared

in methanol; applied potential -25kV; hydrodynamic sample

injection for 10 s at 50 mbar. Total capillary length: 48.5 cm.

Effective length: 40 cm.

116

6.2 Regression equation, r2, LODs (S/N = 3) on the basis of

calibration curves in Figure 6.4.

119

6.3 Intraday % RSD of migration time (min), peak area (mAUs)

and peak height (mAU) in the separation of dicrotophos,

monocrotophos and phosphamidon at three replicates each at

100 ppm.

120

6.4 Regression equation, r2, LODs (for S/N = 3) using SRMM-

MEKC based on calibration curves in Figure 6.8.

123

xiv

6.5 Repeatability of migration time (min), peak area (mAUs) and

peak height (mAU) in the separation of dicrotophos,

monocrotophos and phosphamidon at three replicates each at

10 ppm.

125

6.6 Sensitivity Enhancement Factors in SRMM-MEKC over RM-

MEKC in the separation of dicrotophos, monocrotophos and

phosphamidon. Conditions as in Figure 6.10.

126

6.7 Recovery and repeatability of extraction. 128

7.1 LODs (for S/N = 3) obtained using the three online

preconcentration techniques as compared to normal mode (NM)

for TCDF and TCDD.

131

7.2 Repeatabilities for migration time, peak area and peak height

for both analytes using the three online preconcentration

techniques as compared to normal mode (NM) for TCDF and

TCDD. Sweeping and FESI were not successful in enhancing

the detection of both analytes.

132

xv

LIST OF FIGURES

FIGURE NO. TITLE PAGE

2.1 General structures of A) polychlorinated dibenzo-p-dioxins,

PCDDs and B) polychlorinated dibenzofurans, PCDFs.

6

2.2 General structure of organophosphorous pesticides. 12

2.3 The chemical structures of (A) phosphamidon, (B)

dicrotophos and (C) monocrotophos.

13

2.4 Schematic diagram of the principle of sample stacking in

MEKC. SB is the stacking boundary. EOF is the

electrooosmotic flow. (Quirino and Terabe, 1997a).

29

2.5 Schematic diagram of stacking of analytes during REPSM.

(A) Before stacking; (B) micelles enter the capillary and

carry with it neutral analytes a, k(ax)>k(ay)>k(az); (C)

micelles and neutral analytes stack at the concentration

boundary (B2) and (B1) and polarity is switched later to

positive; (D) separation and later detection of zones.

Retention factor is referred with the symbol k. ax, ay and az

refer to the stacking boundary for analyte x, analyte y and

analyte z.(Quirino and Terabe, 1997b).

31

xvi

2.6 Schematic diagram of stacking of neutral analytes in

FESI-MEKC. (A) initial situation (water plug, unshaded;

BGS shaded); (B) micelles enter the capillary and carry

with them neutral analytes emanating from the cathodic

vial, k(x)>k(y)>k(z); (C) micelles and neutral analytes

stacked at the concentration boundary, voltage is cut and

sample vial replaced with BGS vial when the measured

current is approximately 97-99% of the predetermined

current, voltage is then applied at positivie polarity; (D)

separation of zones occur. k refers to the retention factor of

analyte x, y and z

(Quirino and Terabe, 1998b).

33

2.7 Schematic diagram of sweeping of analytes in MEKC under

low electroosmotic flow. (a) Starting situation, injection of

S prepared in a matrix having a conductivity similar to that

of the BGS; (b) application of voltage at negative polarity,

micelles emanating from the cathodic side sweeping analyte

molecules; (c) the injected analyte zone is assumed

completely swept (Shao and Tseng, 2005).

35

2.8 Flow chart of research methodology of separation TCDD

and TCDF.

39

2.9 Flow chart of research methodology of separation of the

three hydrophilic OPPs viz. phosphamidon, dicrotophos and

monocrotophos.

40

3.1 Chemical structure for TCDF and TCDD. 42

3.2 SPE disc extraction setup in the extraction of analytes from

water samples.

50

3.3 Summary of the methodology of MEKC separation

involved in the separation of TCDF and TCDD.

51

xvii

4.1 Electropherogram of TCDD and TCDF at 40 ppm each in

1,4-dioxane. Run buffer: 20 mM di-sodium tetraborate

decahydrate, 20 mM sodium cholate and 5% (v/v)

acetonitrile at a final running buffer pH: 9.16-9.22. Injection

time: 1 s at 50 mbar. Wavelength: 225 nm. Separation

voltage: 25 kV. Total capillary length: 48.5 cm, effective

length: 40 cm.

56

4.2 The effect of acetonitrile percentage on the migration

window for the MEKC separation of TCDF and TCDD

(n=3).

57

4.3 Electropherogram of TCDF with different percentages of

acetonitrile (MeCN) in BGS at 0 % v/v, % v/v, 10 % v/v, 15

% v/v and 20 % v/v. At 20 % v/v, the TCDF peak appeared

before peak X. Separation conditions are the same as in

Figure 4.1.

58

4.4 Effect of acetonitrile fraction on migration time of TCDF

and TCDD at 0% v/v MeCN, 5% v/v MeCN, 10% v/v

MeCN, 15% v/v MeCN and 20% v/v MeCN. Separation

conditions remain the same as in Figure 4.1.

59

4.5 Effect of different percentages of acetonitrile in running

buffer on (A) migration time and (B) efficiency on the

separation of -TCDF and TCDD.

60

4.6 Electropherogram of TCDF and peak X at 15% v/v

methanol. Running buffer conditions: 20 mM sodium

cholate, 20 mM sodium tetraborate-decahydrate at a final

buffer pH: 9.16-9.22. Wavelength: 225 nm. Both test

analytes at 100 ppm each in 1,4-dioxane. Hydrodynamic

injection at 50 mbar for 1s. Separation voltage, 25 kV;

temperature, 25ºC; total capillary length, 48.5 cm; effective

length, 40 cm.

64

xviii

4.7 Peak shouldering for TCDD at 9.147 min (circle) at 20%

v/v methanol. MEKC conditions as in Figure 4.5.

64

4.8 Electropherogram of the effect of the % of MeOH on the

separation of TCDF and TCDD. Separation conditions

remain the same as in Figure 4.5. The peaks between TCDF

and TCDD are suspected to be from the 1,4-dioxane.

67

4.9 Peak resolution for TCDF (measured relative to peak X

mentioned in section 4.2.2) at 15% v/v MeCN, 15% v/v

MeOH and various percentages of mixed modifier.

Separation conditions similar to Figure 4.1.

67

4.10 Effect of (A) 15% v/v MeCN (B) 15% v/v MeOH (C) 5%

v/v MeCN-MeOH (1:1), (D) 5% v/v MeCN-MeOH (3:1)

and (E) 5% v/v MeCN-MeOH (1:3) on the separation of

TCDD. MEKC separation conditions remain

identical as in Figure 4.1.

69

4.11 Effect of (A) 15% v/v MeCN (B) 15% v/v MeOH (C) 5%

v/v MeCN-MeOH (1:1), (D) 5% v/v MeCN-MeOH (3:1)

and (E) 5% v/v MeCN-MeOH (1:3) on the separation

efficiency of TCDF (enlarged). Peak X refers to an

unknown peak due to the 1,4-dioxane used. MEKC

separation conditions similar to Figure 4.10.

70

4.12 Effect of modifier on (A) migration time, (B) peak area, (C)

peak heights and (D) efficiency of TCDF and TCDD.

72

4.13 Electropherograms of TCDF and TCDD at 4 ppm each in

different media used. (A) ethanol, (B) water and (C) buffer.

(B) and (C) are analytes dissolved in aqueous matrix while

(A) is analyte dissolved in non-aqueous matrix. Running

buffer conditions as in Figure 4.5 but with 5% v/v MeCN-

MeOH (3:1).

75

xix

4.14 Electropherogram of TCDF in the presence of (A) ethanol,

(B) water and (C) buffer matrix. TCDF: 4 ppm; running

buffer conditions similar to Fig 4.13.

76

4.15 Comparison of sample matrix effect based on (A) separation

efficiency, (B) peak area and (D) peak height. (C) shows the

enlarged peak areas as circled in (B) for TCDF. Error bars

are in standard errors.

78

5.1 Calibration curves based on peak height for (A) TCDF and

(B) TCDD using NM-MEKC. Separation buffer: 20 mM

sodium cholate, 20 mM sodium tetraborate-decahydrate and

5% v/v MeCN/MeOH (3:1) at a final buffer pH 9.16-9.22.

Separation wavelength: 225 nm. Separation voltage: 25 kV.

Hydrodynamicinjection for 1s at 50 mbar. Capillary total

length: 48.5 cm. Effective length: 40 cm.

81

5.2 Electropherogram of three replicated runs for the separation

of TCDF (40 ppm) and TCDD (15 ppm) in NM-MEKC

under the same conditions as in Figure 5.1.

83

5.3 Variations in N for (A) TCDF and (B) TCDD in the

concentration used in the calibration curve in NM-MEKC.

84

5.4 Electropherograms showing the effect of different injection

times on peak shapes of TCDD and TCDF. Separation

buffer: 20 mM sodium cholate, 20 mM sodium tetraborate-

decahydrate and 5% v/v MeCN-MeOH (3:1) at a final

buffer pH 9.16-9.22. Separation wavelength, 225 nm;

separation voltage, 25 kV; hydrodynamic injection of

samples varied from 1 s, 4 s, 10 s, 20 s, 30 s, 40 s and 50 s.

Capillary total length: 48.5 cm; effective length: 40 cm.

87

xx

5.5 Influence of injection time on (A1) peak area, (A2) enlarged

for TCDF peak area, (B) peak height and (C) efficiency on

the separation of TCDF and TCDD. Hydrodynamic

injection pressure constant at 50 mbar.

88

5.6 Calibration curves based on peak height for (A) TCDF and

(B) TCDD using NSM-MEKC.Injection time at 50 mbar for

10 s. Separation conditions remain similar to Figure 5.4.

89

5.7 Electropherogram of three successive replicated runs for the

separation of TCDF and TCDD in NSM-MEKC under same

conditions as in Figure 5.4 but with an injection time of 10

s.

90

5.8 Variations in N in the concentration range for (A) TCDF

and (B) TCDD used in the calibration curve in NSM-

MEKC.

92

5.9 Effect of polarity switching time on (A1) peak area and

(B1) peak height for both analytes. (A2) enlarged line graph

of peak area for TCDF. (B2) enlarged line graph of peak

height for TCDF.

94

5.10 Electropherogram of REPSM with different polarity

switching times. TCDF at 4 ppm (circled peak) while

TCDD at 1.5 ppm. Separation buffer: 20 mM sodium

cholate, 20 mM sodium tetraborate-decahydrate and 5% v/v

MeCN/MeOH (3:1) at a final buffer pH 9.16-9.22.

Separation wavelength, 225 nm; separation voltage, 25 kV.

Hydrodynamic injection of sample at 50 mbar for 100 s

followed by electrokinetic injection of buffers at -25 kV for

20 s. Total capillary length: 48.5 cm. Effective length: 40.0

cm.

96

5.11 Calibration curves based on peak height for (a) TCDF and

(b) TCDD using REPSM. Injection time set at 50 mbar for

100 s. Electrokinetic injection at -25 kV for 20 s. Separation

conditions similar to Figure 5.10.

97

xxi

5.12 Variations in N with the concentration for (A) TCDF and

(B) TCDD used in the calibration curve in REPSM-MEKC.

Conditions remain the same as in Figure 5.10.

98

5.13 Effect of different concentrations of NaCl (0-300 mM) on

(A) peak area, (B) peak height and (C) efficiency for both

TCDF and TCDF. Separation buffer: 20 mM sodium


(3:1 acetonitrile-methanol) at a final buffer pH 9.16-9.22.

Separation wavelength: 225 nm. Separation voltage: 25 kV.

Hydrodynamic injection of sample at 50 mbar for 10 s.

Total capillary length: 48.5 cm. Effective capillary length:

40 cm.

101

5.14 HCSSM-MEKC electropherograms with different

concentration of NaCl. TCDF at 2 ppm while TCDD at 0.3

ppm. Peak for TCDF is shown by the circle drawn around

the peak. Separation buffer: 20 mM sodium cholate, 20

mM sodium tetraborate-decahydrate and 5% v/v MeCN-

MeOH (3:1) at a final buffer pH 9.16-9.22. Separation

wavelength: 225 nm. Separation voltage: 25 kV.



40 cm.

102

5.15 Calibration curves based on peak height for (a) TCDF and

(b) TCDD using HCSSM. Injection time set at 50 mbar for

10 s. NaCl concentration in sample matrix at 200 mM.


103

5.16 Variations in efficiency, N in the concentration range for

(A) TCDF and (B) TCDD used in the calibration curve in

HCSSM-MEKC. Separation conditions similar to Figure

5.14.

105

xxii

5.17 Electropherogram of sweeping with different concentration

of sodium cholate in sample matrix at (A) 5 mM, (B) 10

mM and (C) 20 mM. Separation buffer: 20 mM sodium


MeCN-MeOH (3:1) at a final buffer pH 9.16-9.22.


Hydrodynamic injection of sample at 50 mbar for 5 s. Total

capillary length: 48.5 cm. Effective length: 40.0 cm. TCDF

at 2 ppm while TCDD at 0.5 ppm.

107

5.18 Electropherogram of TCDF (2ppm) and TCDD (0.5 ppm)

under FESI-MEKC conditions. Analytes were mixed with

20 mM sodium cholate and deionized water. Samples were

electrokinetically injected at -20 kV, 100 s after

hydrodynamic injection of water plug for 5 s at 50 mbar.

Separation conditions remain the same as in Figure 5.18.

108

5.19 Electropherograms of SPDE-HCSSM of TCDF and TCDD

spiked with 50 ng. Separation buffer: 20 mM sodium


MeCN-MeOH (3:1) at a final buffer pH 9.16-9.22.




40 cm.

111

5.20 Electropherograms of pulp mill effluent water obtained as

tabulated in Table 5.17. (A) treated A, (B) treated B, (C)

untreated A and (D) untreated B.

112

xxiii

6.1 Comparison of intensity of (1) dicrotophos, (2)

monocrotophos and (3) phosphamidon at different detection

wavelengths which are 225 nm and 195 nm under the same

running buffer conditions. Separation buffer: 20 mM

phosphate at buffer pH 2.3, 10 mM SDS and 10% v/v

methanol. Separation wavelength, 225 nm; separation

voltage, -25 kV; hydrodynamic injection for 50 s at 50

mbar. Capillary total length, 48.5 cm; effective length, 40

cm.

116

6.2 The influence of different wavelengths on peak area

response for dicrotophos, monocrotophos and

phosphamidon. Separation conditions is similar Figure 6.1.

117

6.3 The influence of different wavelengths on peak height

intensity for dicrotophos, monocrotophos and

phosphamidon. Separation conditions is similar to Figure

6.2.

118

6.4 Calibration curves based on (A) peak areas and (B) peak

heights for the separation of hydrophilic OPPs in RM-

MEKC. Separation conditions remain the same as in Figure

6.3.

119

6.5 Electropherogram of (1) dicrotophos, (2) monocrotophos

and (3) phosphamidon at 100 ppm each when injected in

triplicates. Separation conditions remain the same as in

Figure 6.4.

120

6.6 Variations in efficiency, N in the concentration range for

dicrotophos, monocrotophos and phosphamidon using RM-

MEKC. Separation conditions of Figure 6.5.

121

6.7 Schematic diagram of the priniciple of stacking with reverse

migrating micelles in MEKC. EOF, electroosmotic flow;

SB, stacking boundary.

122

xxiv

6.8 Calibration curves based on (A) peak areas and (B) peak

heights for the separation of hydrophilic OPPs in SRMM-

MEKC. Separation buffer: 20 mM phosphate at buffer pH

2.3, 10 mM SDS and 10% v/v methanol. Separation

wavelength: 225 nm. Separation voltage: -25 kV.

Hydrodynamic injection for 50 s at 50 mbar. Capillary total

length: 48.5 cm. Effective length: 40 cm.

123

6.9 Separation of hydrophilic OPPs using SRMM-MEKC at

mixture at (A) 10 ppm, (B) 20 ppm and (C) 30 ppm for

(1)dicrotophos, (2) monocrotophos and (3) phosphamidon.

Separation conditions remain the same as in Figure 6.8.

124

6.10 Variations in N in the concentration range for dicrotophos,

monocrotophos and phosphamidon using SRMM-MEKC.


125

6.11 Electropherograms of extracted blank pond water (A) and of

dicrotophos (1), monocrotophos (2) and phosphamidon (3)

at (B) 0.5 µg and (C) 1 µg. Separation buffer: 20 mM

phosphate at buffer pH 2.3, 10 mM SDS and 10% v/v

methanol. Separation wavelength, 225 nm; separation

voltage, -25 kV; hydrodynamic injection for 50 s at 50

mbar. Capillary total length, 48.5 cm and effective length,

40 cm.

129

xxvii

LIST OF SYMBOLS

D, d - Diameter (µm)

Ip - Sample plug length

L - Length (cm)

N - Efficiency

P - Pressure (mbar)

r2 - Correlation coefficient

Rs - Peak resolution

T - Temperature (˚C)

tm - Migration time

Vp - Volume of sample loaded

α - Selectivity

η - Viscosity

xxviii

LIST OF ABBREVIATIONS

ACh - Acetylcholine

AChE - Acetylcholinesterase

ASE - Accelerated solvent extraction

BGE - Background electrolyte

BGS - Background solution

CD - Cyclodextrin

CD-MEKC - Cyclodextrin assisted MEKC

CHES - 2-(N-Cyclohexylamino)ethane sulfonic acid

CMC - Critical micelle concentration

CZE - Capillary zone electrophoresis

DAD - Diode-array detection

DMSO - Dimethyl sulphoxide

EPA - Environmental Protection Agency

EOF - Electroosmotic flow

FESI - Field enhanced sample injection

HCSSM - High conductivity sample stacking mode

HRGC - High resolution gas chromatography

HRMS - High resolution mass spectrometry

HSW - Hazardous solid waste

ID - Isotope dilution

i.d. - Internal diameter

LD - Detection limit

LLE - Liquid-liquid extraction

LMT - N-lauroyl-N-methyltaurate

LOD - Limit of detection

MASE - Microwave-assisted solvent extraction

xxix

MeCN - Acetonitrile

MEKC - Micellar electrokinetic chromatography

MeOH - Methanol

MSPD - Matrix solid phase dispersion

MSW - Municipal solid waste incinerator

NACE - Non-aqueous capillary electrophoresis

NaCl - Sodium chloride

NM - Normal mode

NSM - Normal stacking mode

OPPs - Organophosphorous Pesticides

PBDE - Polybrominated diphenyl ether

PCB - Polychlorinated biphenyls

PCDD - Polychlorinated dibenzodioxins

PCDF - Polychlorinated dibenzofurans

PLE - Pressurized liquid extraction

poly-SUS - Polysodium undecyl sulfate

REPSM - Reversed electrode polarity stacking mode

RSD - Residual standard deviation

SC - Sodium cholate

SDS - Sodium dodecyl sulphate

SEF - Sensitivity enhancement factor

SPE - Solid phase extraction

SPDE - Solid phase disc extraction

SPME - Solid phase microextraction

SDME - Single drop microextraction

SPMD - Semi permeable membrane devices

SRM - Standard Reference Material

SRMM - Stacking with reverse migrating micelles

SRW - Stacking with reverse migrating micelles and a water

plug

TEF - Toxic Equivalent Factors

TEQ - Toxic Equivalent Concentrations

TCDD - Tetrachlorodibenzodioxins

- 2,3,7,8-tetracholordibenzo-p-dioxin

xxx

TCDF - Tetrachlorodibenzofurans

- 2,3,7,8-tetrachlorodibenzo-p-furan

UV - Ultraviolet

xxxi

LIST OF APPENDICES

List of Publications 152

List of Presentations 153

1

CHAPTER I

INTRODUCTION

1.1 Background

Since its introduction in the late 1980s, micellar electrokinetic

chromatography (MEKC) has been widely used in the pharmaceutical industry and in

environmental analysis. MEKC is a mode of capillary electrophoresis able to

separate both ionic and neutral analytes with the usage of charged micelles in a single

run. Separation by electrophoresis is obtained via differential migration of solutes of

charged species in an electric field performed in narrow-bore capillaries with inner

diameter (i.d) of 25-75 µm filled only with buffer. Its advantages lie in its flexibility

in manipulating various parameters on-column in order to obtain the best separation

and to improve sensitivity. Furthermore, separation time is faster compared to

conventional methods such as gas chromatography and high performance liquid

chromatography with very little sample and solvent requirement.

2

1.2 Summary

This study was conducted into two parts as two different separation

conditions were used. The first part was the study on separating polychlorinated

dibenzo-p-dioxins (2,3,7,8-tetrachlorodibenzo-p-dioxin) and polychlorinated

dibenzo-p-furans (2,3,7,8-tetrachlorodibenzo-p-furan) which were conducted under

basic conditions. The second part was the study of three hydrophilic

organophosphorous pesticides (OPPs) under acidic buffer conditions. The three

hydrophilic organophosphorous pesticides were monocrotophos, dicrotophos and

phosphamidon. This chapter summarizes every chapter covered in this work.

Chapter 2 introduces the background behind this work. It explains in detail

the physical and chemical properties of the test analytes which are the

polychlorinated compounds (PCDDs and PCDFs) and the hydrophilic pesticides.

The potential routes of PCDDs and PCDFs in humans and wildlife are also

discussed. While for the OPPs, we touched on the toxicity of the pesticides. Various

extraction and detection methods are also discussed for both class of analytes in this

chapter. On-line preconcentration methods using MEKC are also discussed in detail.

The objectives of this study and the problem statements are also covered.

Chapter 3 discusses the experimental methods used in this work. The

instrumental aspect covers the capillary electrophoresis system used for our analysis

and the extraction set-up used. Conditioning of the capillary was also discussed in

detail for both polychlorinated compounds and pesticides analysis. For both

polychlorinated compounds and the pesticides, the methodology used in optimizing

parameters and online preconcentration techniques used are found in Chapter 3.

Sample preparation and extraction methods for both analytes are covered in Chapter

3.

Chapter 4 covers the optimization of parameters used in the MEKC analysis

of TCDF and TCDD. The parameters optimized are the fraction of organic modifier

used in the running buffer in order to improve peak resolution. Single modifiers used

were methanol and acetonitrile at various percentages in the running buffer. These

3

were then compared with mixed modifier mode at different fractions of acetonitrile

and methanol in the running buffer. The second parameter involved the type of

sample matrix used. In aqueous mode, the analytes were diluted in water and in the

same fraction as the running buffer. While for non-aqueous mode, analytes were

diluted in the organic solvent which was ethanol. Different sample matrixes were

investigated to study the effect of the sample matrix on separation efficiency.

Chapter 5 explores the various on-line preconcentration techniques used to

reduce the detection limit for both TCDF and TCDD. Five types of on-line

preconcentration techniques were used which were normal stacking mode (NSM),

reversed electrode polarity stacking mode (REPSM), high conductivity sample

stacking mode (HCSSM), sweeping and field-enhanced sample injection (FESI).

Out of the five, the on-line preconcentration technique which gives the best limit of

detection (LOD) would be chosen to be applied to the real sample. HCSSM was

chosen as the optimized stacking method. This is then followed by off-line

preconcentration technique which is solid phase disc extraction (SPDE) to further

reduce the existing limit of detection (LOD) by offering sample enrichment. SPDE

is also able to do sample clean-up by removing interference in the matrix that would

interfere with the analysis. The combined HCSSM-SPDE was then applied to real

sample analysis of spiked deionised water and effluent water obtained from a pulp

mill at Temerloh, Pahang.

Chapter 6 involves the analysis of three hydrophilic OPPs which are

dicrotophos, monocrotophos and phosphamidon. In this chapter, the analysis using

MEKC was conducted under acidic conditions. This section discusses the

wavelength optimization for analyzing the three analytes using diode-array detection

(DAD). The wavelength that offers the best peak area and peak height was used in

the stacking method. Stacking with reverse migrating micelles (SRMM) was used to

reduce the detection limit (LODs) of the three analytes. This is followed by solid

phase disc extraction (SPDE) in order to reduce the LODs further by offering sample

enrichment and clean-up. The combined SRMM-SPDE was then applied to

analyzing spiked pond water obtained from the UTM lake.

4

Chapter 7 presents the conclusions and suggestions for further studies. This

chapter summarizes the results obtained throughout the study such as the optimized

conditions and recovery studies from the extraction method. Suggestions are

presented and discussed for further improvement of the study for future usage.

5

CHAPTER II

LITERATURE STUDY

2.1 Organic Pollutants

Persistent organic pollutants (POPs) are prone to long range transport as they

can be transported via the atmosphere and through water as leachate therefore being

carried to the upper levels of the atmosphere and deposited in remote areas. Even as

remote as the polar regions of the earth. Some of these chemicals have toxic

properties and have the ability to accumulate in biota due to high lipophicity and

resistance towards biodegradation such as dioxins.

Pesticides are another class of pollutants which covers a wide range of

substances such as insecticides, acaricides, fungicides, mollusicides, nematocides,

rodenticides and herbicides (Hamilton and Crossley, 2004). Organophosphorous

(OPP) pesticides have gradually replaced organochlorine compounds in the past 15-

20 years in agriculture, horticulture etc. as they are generally less toxic compared to

organochlorine compounds. But due to their high acute toxicity, they are hazardous

to workers who use them.

6

2.2 Chlorinated Dioxins and Dibenzofurans

“Dioxins” is a generic name given to 75 polychlorinated dibenzo-p-dioxins

(PCDDs) and 135 polychlorinated dibenzofurans (PCDFs). The most toxic PCDD

congener is 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) while another

closely related compound is 2,3,7,8-tetrachlorodibenzo-p-furan (2,3,7,8-TCDF)

(Choudhary et al., 1983). The toxicity of PCDDs and PCDFs is dependent on the

chemical structure of the compound. Dioxins and dibenzofurans chlorinated in the 2,

3, 7, and 8 positions have been demonstrated to be carcinogens. The general

structures of PCDDs and PCDFs are shown in Figure 2.1. The isomers with the

highest acute toxicities have 4-6 chlorine atoms and they have their four lateral

positions substituted for chlorine with an LD50 values in the range 1-100 µg/kg for

the most sensitive animal species (Choudhary et al., 1983). Therefore, the

Environmental Protection Agency (EPA) has made dioxin as one of the most toxic

chemicals regulated by EPA to be the subject of a series of agency-assessment dating

back to the 1980s (U.S. EPA 613, 1984, U.S. EPA 1613, 1994 and U.S. EPA 8280A,

1996). Its extreme toxicity requires very sensitive and highly specific analytical

techniques due to different isomers having different biological and toxicological

properties.

Figure 2.1: General structures of A) polychlorinated dibenzo-p-dioxins, PCDDs and B) polychlorinated dibenzofurans, PCDFs.

A) B)

7

2.2.1 Physical and Chemical Properties of 2,3,7,8-TCDF and 2,3,7,8-TCDD

The physical and chemical properties of 2,3,7,8-TCDF and 2,3,7,8-TCDD are

shown in Table 2.1. The extremely low water solubilities and vapor pressures

contribute to the difficulty in determining these and related physico-chemical

properties (e.g., Kow and Henry’s law constant) of these compounds. In general, the

melting point increases and the vapor pressures and water solubilities of PCDDs and

PCDFs decreases with the increase in number of chlorine substitutes. These

hydrophobic compounds are generally colorless solids and are soluble in organic

solvents. Generally they are thermally stable but decompose only at extreme heating

around 700°C. log Kow refers to the logarithm of the partition coefficients

octanol/water and it is sometimes known as log P. Due to their high log Kow, PCDDs

and PCDFs would partition between sediment or biota and water in a concentration

ratio between 106 to 107 (Choudhary et al., 1983). This characteristic gives them the

potential to accumulate in food chains due to their resistance towards metabolism or

chemical degradation.

Table 2.1: Physical and chemical properties of 2,3,7,8-TCDF and 2,3,7,8-TCDD.

Properties Compound

2,3,7,8-TCDF 2,3,7,8-TCDD

Molecular Weight 305.96 322

Melting Point, °C 219-221 305-306

Vapor Pressure, mm Hg at

25°C

9.21 x 10-7 7.4 x 10-10

Water Solubility 0.42 µgL-1 19.3 ngL-1

Thermal Decomposition No Data 700°C

log Kow 5.82 7.02

Henry’s Law Constant

(atm-m3/mol)

1.48 x 10-5 1.62 x 10-5

8

2.2.2 Toxicity of PCDDs and PCDFs

PCDDs and PCDFs have known toxicity and 2,3,7,8-tetrachlorodibenzo-p-

dioxin (TCDD) is the most toxic congener. Risk evaluation posed by their toxicity is

done using toxic equivalent factors (TEFs) (Danielsson et al., 2005). It was adopted

for the purpose of assessing cancer risk. The isomer TCDD appears to be the most

potent therefore in the TEF scheme it was assigned a TEF of 1.0. Table 2.2 shows

the TEFs for chlorinated dibenzodioxins and chlorinated dibenzofurans. Meanwhile

the toxic equivalent concentrations or TEQ values are defined as the summed

products of congener concentrations and corresponding TEFs and represent the

TCDD toxic potencies of the samples (Korytár et al., 2004).

Table 2.2: Toxicity Equivalent Factors (TEF) for chlorinated dibenzodioxins and chlorinated dibenzofurans (U.S EPA 8290A, 1998).

Analyte TEF

2,3,7,8- TCDD 1.00 1,2,3,7,8- PeCDD 0.50 1,2,3,6,7,8-HxCDD 0.10 1,2,3,7,8,9-HxCDD 0.10 1,2,3,4,7,8-HxCDD 0.10 1,2,3,4,6,7,8-HpCDD 0.01 1,2,3,4,6,7,8,9-OCDD 0.001 2,3,7,8-TCDF 0.1 1,2,3,7,8-PeCDF 0.05 2,3,4,7,8-PeCDF 0.5 1,2,3,6,7,8-HxCDF 0.1 1,2,3,7,8,9-HxCDF 0.1 1,2,3,4,7,8-HxCDF 0.1 2,3,4,6,7,8-HxCDF 0.1 1,2,3,4,6,7,8-HpCDF 0.01 1,2,3,4,7,8,9-HpCDF 0.01 1,2,3,4,6,7,8,9-OCDF 0.001

This compounds proceed through the binding to a soluble intracellular

protein, the aryl hydrocarbon receptor (AhR) which then deregulates expression of

key genes under the Ah receptor (Reiner et al., 2006). This then elicits Ah receptor-

9

mediated biochemical and toxic responses causing enzyme induction,

immunotoxicity, tumor promotion, developmental toxicity, chloracne, etc. (Behnisch

et al., 2001). The major pathway of human exposure to dioxins is food with a daily

intake ranging from 2 to 10 pg TEQ kg-1 body weight and per day for a 60 kg adult in

industrialized countries (Baeyens et al., 2004). Consequently, the separation,

identification and quantification of the most toxic isomers become important.

2.2.3 Potential Routes of PCDDs and PCDFs Exposure In Wildlife and

Humans

There are various ways PCDDs and PCDFs can enter the food chain and into

the biological systems of wildlife and humans. A majority of these routes are via

industrial processes.

2.2.3.1 Commercial and Technical Products

During the 1960s and 1970s there was evidence of phenoxy herbicides such

as 2,4,5-T (2,4,5-trichlorophenol) and other chlorophenoxy acids, pentachlorophenol

and other chlorinated phenols and PCBs (Kamrin and Rodgers, 1985 and

Baeyens et al., 2001) referred to as technical products being contaminated with

PCDD and PCDF. The major contaminant in 2,4,5-T is TCDD which was formed

from 2,4,5-trichlorophenol during the manufacturing of herbicides which made

significant amounts of TCDD being released into the atmosphere through herbicide

usage. Pentachlorophenol is mainly used as wood preservatives in which numerous

congeners of PCDDs and PCDFs were found with the chlorine numbers bigger than

6 (Nakamata and Ohi, 2003 and Lacorte et al., 2003). Commercial polychlorinated

biphenyl (PCB) mixtures contain PCDF in ppm amounts whereas PCDDs weren’t

detected causing PCBs to be phased out in the 1980s as a dielectric fluid in

capacitors and transformers.

10

2.2.3.2 Municipal Incinerators and Hazardous Waste Incinerators

The occurrence of PCDFs and PCDDs beside other chlorinated compounds in

fly ash from municipal solid waste (MSW) incinerators and hazardous waste

incinerators (HSW) has been well documented (Gough, 1986; Fiedler, 1996;

Kim et al., 2001 and Ferré-Huguet et al., 2005). With respect to the formation of

PCDDs and PCDFs, chloroethenes and chloroethines played the role as precursors.

The formation of PCDDs and PCDFs from chloroethenes and chloroethines could

only occur under pyrolytic conditions at temperatures above 400°C which matches

the environment in an incinerator. It was also discovered that complex thermal

reactions as the result of poor combustion could also be the cause of PCDDs and

PCDFs (Pandelova et al., 2006). Another potential source of PCDDs and PCDFs in

MSWs are from the wet scrubbers found in the flue gas cleaning systems in MSWs

and HSWs (Kreisz et al., 1996 and Hübner et al., 2005). This was due to plastics

(polypropylene and butyl rubber) applied in the area of the wet scrubbing system

(packed rubber, coating material). This material can be considered as hazardous

waste as it is strongly contaminated with PCDD/F. Therefore, the efficiency of the

incinerator is important in reducing the emission of these compounds.

2.2.3.3 Food Products

It is generally accepted that a majority of human exposure to PCDDs and

PCDFs is through the diet. This is because the mixture of dioxins and furans emitted

from combustion sources are in both the gaseous and particulate phase therefore

being adsorbed on airborne particulate or in sediments before being ingested by

humans. Most studies report fish and seafood being the main route of exposure

(>95%) (Gómara et al., 2005; Oh et al., 2005 and Turyk et al., 2006) followed by

oils and fats (Bocio and Domingo, 2005). In humans, children receive higher doses

of PCDD/Fs from food on body weight basis compared to adults (Charnley and

Kimbrough, 2005) such as in infants through contaminated breast milk

11

(How-Ran et al., 2005) and in baby food contaminated by ball clay (Hayward and

Bolger, 2005) although children have the ability to eliminate dioxins more rapidly

compared to adults. Following several episodic dioxin contamination incidents such

as during the Belgian animal feed-and-food crisis in 1999 (Behnisch et al., 2001) the

European Community (EC) has set maximum residue limits (MRLs) for dioxins in

various foods which were adopted on October 24, 2001 (Baeyens et al., 2004).

MRLs for foodstuffs are on a fat basis expressed in pg PCDD/F TEQ g-1 fat and have

been set to 1 for pork, 2 for poultry, mixed animal fat and fish oil for human

consumption while 3 for ruminants. For liver it was at 6, 3 for milk and eggs and

0.75 for vegetable oil. In order to reduce the presence of dioxins in feed and food, a

revision is planned to set lower levels by the year 2006.

2.3 Organophosphorous Pesticides (OPPs)

Organophosphorous (OPPs) pesticides have gradually replaced

organochlorine compounds in the past 15-20 years in agriculture, horticulture etc.

They are widely used in agriculture and animal production for the control of various

insects. They have higher acute toxicity than chlorinated pesticides but have the

advantage of being more rapidly degraded in the environment compared to

organochlorine compounds. There are 2 classes of OPPs depending on their

hydrophobicity. Hydrophilic OPPs include phosphamidon, dicrotophos and

monocrotophos. These class of OPPs were chosen as they are not easily detectable

in biological samples and aqueous conditions due to the alkylating activity of

organophosphate esters as they are very reactive. In modern agricultural practices,

excessive application of pesticides has caused serious environmental problems. The

leaching of pesticides into surface and groundwater is through bypass flow either in

solution or suspended in colloidal matter. This can cause poor pest control, crop

injury and increased loss of pesticides or accumulation of pesticides in the soil.

12

2.3.1 Physical and Chemical Properties

The functions and physical properties of the hydrophilic OPPs which are

phosphamidon, dicrotophos and monocrotophos are shown in Table 2.3. Referring

to Table 2.3, the log Kow refers to the logarithmic of octanol/water partition

coefficient which indicates the hydrophobicity of the analytes. Higher Kow refers to

increasing hydrophobicity. Phosphamidon is the most hydrophobic with a Kow of

0.795 while the most hydrophilic is monocrotophos with Kow of -1.97. OPPs are

generally esters, amides or thiol derivatives of phosphoric, phosphonic,

phosphorothioic or phosphonothioic acids (Lucio et al., 1986). The general

structural formula is shown in Figure 2.2 while the chemical structures for

phosphamidon, dicrotophos and monocrotophos are shown in Figure 2.3.

Table 2.3: Functions and physical properties of the three hydrophilic OPPs

Name

log Kow Function H2O Solubility, (g/L) (Zhu et al., 2005)

Ref.

Phosphamidon, 0.795 insecticides nematicides

299.69 Rabindranathan et al., 2003

Dicrotophos

-0.50 insecticides 237.20 Maul et al., 2005

Monocrotophos

-1.97 acaricides insecticides

223.16 Vijay et al., 2005

PR1

R2

O(S)

X

Figure 2.2: General structure of organophosphorous pesticides.

13

Figure 2.3: The chemical structures of (A) phosphamidon, (B) dicrotophos and (C) monocrotophos.

R1 and R2 are usually simple alkyl or aryl groups both of which may be

bonded directly to the phosphorous atom (phosphinates) or linked via –O-, or –S-

(phosphates) or R1 bonded directly and R2 via one of the above groups

(phosphonates). In phosphoroamidates, carbon is linked to phosphorous through an

–NH group. X can be any of a wide variety of substituted and branched aliphatic,

aromatic or heterocyclic groups, linked to phosphorous via a bond of some labiality

(usually –O- or –S-) known as the leaving group. The double bonded atom may be

oxygen or sulfur, and related compounds would be called phosphates or

phosphorothioates. The P=O form of a thioate ester known as the oxon is often

incorporated into the trivial name.

14

2.3.2 Toxicity of OPPs

OPPs work by inhibiting the acetychlolinesterase enzyme (AChE) in the

nervous system and subsequent accumulation of toxic levels of endogenous

acetylcholine (ACh) in nervous tissue and effector organs in both insects and

mammals (Aprea et al., 2002). Acetylcholinesterase is essential for acetylcholine.

In mammals, ACh is the chemical transmitter of nerve impulses at endings of post

ganglionic nerve fibers, somatic motor nerves to skeletal muscle and certain synapses

in the central nervous system. Therefore the accumulation of ACh causes signs that

mimic nicotinic, muscarinic and central nervous system actions of ACh. In fatal

cases this leads to disruption of nerve functions, convulsions, respiratory failure

which untreated would lead to asphyxiation. Muscarinic and nicotinic symptoms

include diarrhea, urination, miosis, bronchospasm, emesis, lacrimation and salivation

(Tarbah et al., 2001).

The alkylating activity of organophosphate esters explain why it is very

difficult to detect OPPs in food, in animal or human tissue or in blood; as they are

very reactive. Their instability in aqueous solutions especially in blood is due to the

presence of esterases. For example, phosphamidon is easily degraded into dimethyl

phosphate in the presence of blood or urine. Therefore, a highly sensitive, simple

and rapid method for quantification of unchanged OPPs in aqueous and biological

samples is required.

2.4 Sample Extraction and Clean-Up Strategies of Organic Pollutants

Extraction and clean-up strategies are needed for environmental samples

containing organic pollutants due to the presence of matrix interference which could

reduce the detection sensitivity of the method. Furthermore, extraction offers sample

enrichment which are able to concentrate the organic pollutants in the sample by

various folds as the analytes are usually at trace level.

15

2.4.1 Methods For The Extraction Of Organic Pollutants

Once the sample has been collected, extraction is needed to extract the

analyte concerned from environmental samples such as water samples, lipids etc. in a

volume that is sufficient for analysis. Several extraction techniques have been

introduced and the most popularly used due to its economical set up is Soxhlet

extraction. It was developed by Professor von Soxhlet in 1879. Soxhlet extraction

has been used in the extraction of OPPs from solid matter such as soil or grass before

being analysed with gas chromatography (GC-NPD) achieving a limit of detection

(LOD) of 10 µgkg-1 (Andreu and Pico, 2004). Analysis of PCDD/Fs has been done

on sediments (U.S. EPA 613, 1984; U.S. EPA 1613, 1994; U.S. EPA 8280A, 1996

and Suarez et al., 2006), water (Wenning et al., 1999), fly ash (Byung-Hoon et al.,

2005) and biological samples such as blood and serum (Focant and Pauw, 2002). Its

drawbacks are that it is very time consuming with one extraction taking up to 48

hours and require large amounts of organic solvents such as toluene or benzene. An

alternative would be to use accelerated solvent extraction (ASE) which uses

conventional liquid solvents at elevated pressures (1500-2000 psi) and temperatures

(50-200ºC) with extraction time reduced to 10-20 minutes per sample with only 10-

20 mL of solvent (Byung-Hoon et al., 2005). An even faster extraction method

would be to use a microwave oven giving the name microwave-assisted solvent

extraction (MASE) with an extraction time of 30 s for each seven cycles and at

recoveries exceeding 95% (Liem, 1999). MASE has the advantage of high sample

throughput and relatively small extraction times needed. Shorter extraction times

also prevent problems of degradation.

Pressurized liquid extraction (PLE) is similar to liquid-liquid extraction

(LLE) and uses increased temperatures and pressure and works according to static

extraction with superheated liquids. It was originally launched by Dionex Inc. in

1995 and called accelerated solvent extraction (ASETM) and is heavily used in the

extraction of dioxins and furans from environmental samples (Richter et al., 1997;

Saito et al., 2003; Focant et al., 2004 and Todaka et al., 2007). In practice the

stainless steel extraction cell containing the sample can be heated up to 200ºC and

pressurized to 3000 psi and the whole extraction process takes 10 minutes. Another

16

method besides ASE is PLE which is also commonly used in environmental analysis

especially in polychlorinated compounds (Kiguchi et al., 2006; Kiguchi et al., 2007

and Wiberg et al., 2007). Although PLE offers a higher degree of automation, yet it

is more expensive and still requires sample clean-up after extraction (Pirard and

Pauw, 2005), especially in the lipid rich biological samples that require large

amounts for dioxin analysis, PLE is not suitable to accommodate such a large

amount of sample as in Soxhlet extraction. In the extraction of OPPs, 9 part

acetonitrile and 1 part methanol was used as the extraction solvent before being

analysed with gas chromatography/mass spectrometry (GC/MS) (Menzinger et al.,

2000). PLE was combined with dimethyl sulfoxide/acetonitrile/hexane partitioning

for dioxin analysis in lipid-rich biological matrices under 2000 psi and ≥ 180ºC thus

reducing the amount of lipids generated making sulfuric acid treatment unnecessary

(Kitamura et al., 2004).

Solid phase extraction (SPE) is a widely used sample-preparation technique

for the isolation of selected analytes in a mobile phase which are transferred to the

solid phase and are retained for the duration of the sampling process. SPE is

commonly used in extraction of pesticides (Nordmeyer, K. and Their, H., 1999;

Schenck and Donoghue, 2000 and Wong, et al., 2003). Elution with solvents is then

carried out to extract the analyte from the solid phase (Poole, 2003). Some of the

various sorbents that have been used for extraction purposes from different matrices

for PCDDs, PCDFs, OPPs and PCBs are shown in Table 2.4. Based on Table2.4, the

most commonly used catridge is the C18 catridge which is suitable for organic

pollutants. SPE is easier and faster compared to conventional LLE and requires less

solvent. Furthermore, it easily automated and coupled with other clean-up system

(Focant et al., 2004) which cuts costs and reduces sample loss. Another similar

version to SPE is semipermeable membrane devices (SPMD) which was used to

extract PCDD/Fs from pulp mill effluents in the form of polyethylene tubes

containing triolein to trap these analytes from sludge (Koistinen et al., 1998).

Solid phase microextraction (SPME) a miniaturized technique is a new, fast

and simple sample preparation technique that uses coated fused-silica fibers to

extract analytes from aqueous or gaseous samples. SPME has been developed

17

rapidly for pesticide residues and dioxin analysis detection in soils (Cubas et al.,

2004), gases from thermal pyrolisis (Chia et al., 2004) and vegetables etc. A

recently developed approach in the detection of OPPs residues using SPME coupled

with SnO2 gas sensor has increased the detection sensitivity of the SPME fibre

(Xingjiu et al., 2004). A 100 µm poly(dimethylsiloxane) fiber was used in the

detection of OPPs in wines with a limit of detection below 5 ppb (Lόpez-Blanco et

al., 2005). Besides SPME, there are other similar types such as single drop

microextraction (SDME) which uses a microdrop of the solvent that is suspended

directly at the tip of the microsyringe needle that is immersed in a stirred aqueous

sample solution (Ahmadi et al., 2006). This approach has been able to solve the

limited lifetime of the SPME fibre and sample carry over. Matrix solid phase

dispersion (MSPD) that homogenized the vegetables in C18 sorbent and elution with

ethyl acetate was used in determination of fungicide residues which was simple and

less labor intensive (Kristenseon et al., 2004).

2.4.2 Clean-up Procedures for Organic Pollutants

During the extraction step, many interfering organic components are co-

extracted from environmental soil samples together with analytes. In the case of

dioxin analysis in biological samples, the most important task would be to remove

large quantities of lipids in the sample. As dioxin levels are extremely low, accurate

analysis of dioxins requires large amount of samples and the removal of lipids. In

order to remove lipids, acetone/hexane is commonly used followed by sulfuric acid

treatment to remove lipids. Yet this process is lengthy and conducive to emulsion

formulation that results in dioxin loss (Focant et al., 2004). Kitamura et al. used

tandem multi-layer silica gel-activated carbon (MLS-AC) column chromatography as

a rapid clean-up measure and has the potential to greatly reduce the amount of lipid

extracted by 1/100 causing sulfuric acid treatment unnecessary (Kitamura et al.,

2004). Silver nitrate silica column has been able to separate polybrominated

diphenyl ethers (PBDEs) from PCDDs and PCDFs which can’t be achieved with

merely a multi-layer silica column (Liu et al, 2006).

18

Tab

le 2

.4:

Exam

ple

of v

ario

us s

orbe

nts

used

in

SPE

for

the

anal

ysis

of

poly

chlo

rinat

ed d

iben

zodi

oxin

s, po

lych

lorin

ated

dib

enzo

fura

ns,

poly

chlo

rinat

ed b

iphe

nyls

(PC

Bs)

and

org

anop

hosp

horo

us p

estic

ides

(OPP

s) fr

om v

ario

us m

atric

es.

Ana

lyte

s M

atri

x So

rben

t E

lutin

g

Solv

ent

Ana

lytic

al

met

hod

LO

D

Ref

.

PCD

D/F

M

ilk

Non

-end

capp

ed

C18

bon

ded

silic

a.

hexa

ne

GC

-HR

MS

0.10

p

g/g

Foca

nt e

t al

., 20

04

So

il Si

lica

gel,

N

a 2SO

4 5%

2-p

ropa

nol

in h

exan

e (3

mL)

H

RG

C-M

S N

S G

awlik

et

al. ,

2000

Wat

er

SPE

Spee

disk

s Et

hano

l/tol

uene

(7

0:30

) H

PLC

0.

009

µg/g

M

artín

ez-

Cor

ed e

t al

., 20

00

Ef

fluen

t -C

18 b

onde

d si

lica.

-C

18 S

peed

isks

Et

hano

l/tol

uene

(7

0:30

) G

C-H

RM

S N

S 4.

2 pg

/L

Tayl

or e

t al

. , 19

95

and

Puja

das e

t al

. , 20

01

PCB

W

ater

C

18 E

mpo

re d

iscs

n-

pent

ane

GC

-µEC

D

NS

Wes

tbom

et

al.,

200

4

Hum

an

seru

m

Endc

appe

d

C18

bon

ded

silic

a.

n-he

xane

/DC

M

(1:1

v/v

) G

C-µ

ECD

0.

0012

- 0.

0026

ngm

L-1

Čon

ka e

t al

., 20

05

So

ils

and

sedi

men

ts

Ph+C

18+A

l D

iol+

C18

+Al

Ace

toni

trile

G

C-M

S-SI

M

NS

Dąb

row

ska

et a

l., 2

003

OPP

s So

il M

olec

ular

ly im

prin

ted

poly

mer

s (M

IP)

Dei

onis

ed

wat

er

LC-M

S N

S M

oulle

c et

al

., 20

06

W

ater

, po

tato

es a

nd g

rain

M

odifi

ed st

yren

e-di

viny

lben

zene

po

lym

er

Ace

tone

C

E-M

EKC

7-

150

ngm

L-1

Pére

z-R

uiz

et a

l., 2

005

W

ater

C

18 b

onde

d si

lica.

M

etha

nol

CE-

MEK

C

0.01

-0.1

ngm

L-1Sü

sse

et

al. ,

1996

*N

ote:

DC

M:d

ichl

orom

etha

ne. N

S: N

ot st

ated

.

19

Although silica column chromatography is popularly used, there have been

alternative sorbents such as XAD-2 resin (Suarez et al., 2006) and selective elution

from an AX-21activated charcoal/Silica gel chromatographic column based on

molecular relative planarity (Wenning et al, 1999). Florisil fractionation

chromatography was used to separate PCBs from PCDDs and PCDFs as activated

carbon chromatography gave inconsistent results (Liu et al., 2006). With human

blood samples, cleanup involved gel permeation chromatography and carbon-fiber

chromatography prior to HRGC/HRMS analysis (Kim et al., 2005).

2.5 Identification and Quantification of Organic Pollutants

In this section we are going to discuss the various detection methods used to

detect polychlorinated compounds and organophosphorous pesticides and their limits

of detection.

2.5.1 Gas Chromatography

Nowadays, gas chromatography (GC) plays an important role in

identification and quantification of organic pollutants in the environment. GC has

gone through many stages of developments from the classical packed column GC, to

capillary-GC columns and the need for multi-dimensional GC techniques (GC × GC)

(Santos and Galceran, 2002). Various methods of GC analysis for some examples of

PCDD/Fs from selected matrix are shown in Table 2.5 while Table 2.6 shows the GC

analysis of some examples of OPPs in selected matrix. From Table 2.5, HRGC-

HRMS has played an important role in detection of PCDD/Fs. HRGC-HRMS is

commonly used compared to other methods due to the very high detection sensitivity

achieving as low as pg level (Focant et al., 2006). For the OPPs, either GC-ECD or

GC-NPD has been used in the detection of pesticides in environmental samples.

Detection limits using GC are also satisfactory for pesticides achieving to as low as

0.2 µgL-1 (Ahmadi et al., 2006).

20

For the identification and quantification purpose, mass spectrometry (MS) is

the best suited and most widely used detection method. For greater sensitivity beside

low resolution MS (LRMS), high resolution MS (HRMS) and tandem mass

spectrometry (MS-MS) have also been used in the detection of OPPs and PCDD/Fs

in environmental samples. Limits of detection have been improved from the sub part

per million (ppm) level down to as low as part per trillion (ppt) or part per

quadrillion (ppq) level (Singh and Kulshrestha, 1997). Gas chromatography (GC)

coupled to 13C-based isotope dilution (ID) high-resolution (HR) mass spectrometry

(MS) is the hyphenated instrumentation of choice to measure ultra-trace levels till as

low as sub-picogram levels and it is also known as GC-IDHRMS (Focant et al.,

2006).

Yet GC analysis has its limitations in which it has a limited polarity range

that is not suitable for polar compounds commonly found in pesticides. For example

in GC-IDHRMS, the extracts must be free of any matrix interferences responsible for

ion-suppression. Furthermore, it requires a large amount of organic solvent that is

not environmentally friendly and a large amount of sample in GC analysis.

Therefore, alternative analytical methods are needed.

2.5.2 Micellar Electrokinetic Chromatography

Separation by electrophoresis is obtained via differential migration of solutes

of charged species in an electric field performed in narrow-bore capillaries with inner

diameter (i.d) of 25-75 µm filled only with buffer (Pyell, 2001). Micellar

electrokinetic chromatography (MEKC) is a hybrid of electrophoresis and

chromatography which can separate both neutral and ionic substance on the basis of

the chromatographic principle of partitioning the solute between the micelle and the

buffer solution giving differential migration of the two phases which was developed

by Terabe (Terabe, 1989). MEKC is becoming an advantageous tool for

determination of organic compounds such as shown for certain samples in Table 2.7.

Its flexibility in manipulating various parameters on-column in order to obtain the

21

best separation and to improve sensitivity makes it promising to be used for further

analysis of organic pollutants. Furthermore, separation time is faster compared to

conventional methods such as gas chromatography and high performance liquid

chromatography with very little sample and solvent is required.

2.5.3 Types of Surfactants

The separation of neutral analytes by MEKC is driven by the use of

surfactants at a level in the running buffer above its’ critical micelle formation

(CMC). The CMC is the concentration above which micelles start to form and its

value decreases with increasing concentration of the electrolyte counter ion (Fuguet

et al., 2005). This is due to the effect of the electrolyte neutralizing the charge at the

micelle surface reducing the electrostatic repulsion between them, thus improving

micellization. Knowing the CMC of surfactant at the buffering concentration to be

used is important as surfactant concentration below the CMC would prevent the

formation of micelles. These micelles are important to provide the micellar phase in

order for separation of analytes to occur. Sodium dodecyl sulphate (SDS), a

commonly used surfactant in MEKC has a CMC of 8.1 mM at 25°C in pure water

which differs in different electrolyte and a low Kraft point of 16°C in pure water

(Muijiselaar, et al., 1997). This characteristic enables it to be commonly used in

MEKC and furthermore it is cheaper compared to other surfactants. The Kraft point

refers to the temperature at which the solubility of the surfactant is lower than the

CMC.

22

Tab

le 2

.5: S

ome

exam

ples

of G

C a

naly

sis u

sed

for P

CD

D/F

s.

A

naly

tes

Mat

rix

Det

ecto

r L

OD

R

ef.

PCD

D/F

M

ilk

GC

-HR

MS

(GC

x G

C) -

µEC

D

0.10

pg/

g fa

t 70

-90

fg

Foca

nt a

nd P

auw

, 200

2 K

oryt

ár e

t al.,

200

4

Efflu

ent

GC

-HR

MS

GC

-MS

HPL

C

GC

-MS

GC

-MS

NS

NS

0.1

pg

NS

NS

43 n

g/L

Wes

tbom

et a

l., 2

004

Čon

ka e

t al.,

200

5 N

akam

ata

and

Ohi

, 200

3 M

artín

ez-C

ored

et a

l.,

1999

K

oist

inen

, et a

l., 1

999

Góm

ez e

t al.,

200

7

Sedi

men

ts

HR

GC

/HR

MS

HR

GC

/HR

MS

GC

-MS

GC

-MS

HR

GC

/HR

MS

GC

-MS

HR

GC

/HR

MS

NS

NS

NS

NS

NS

2.2-

77.2

ng/

g N

S

Gar

dina

li et

al.,

199

6 Li

u et

al.,

200

6 H

ashi

mot

o et

al.,

199

5 G

awlik

et a

l., 2

000

Suar

ez e

t al.,

200

6 C

hia

et a

l., 2

004

Pand

elov

a et

al.,

200

6

A

ir H

RG

C/H

RM

S H

RG

C/H

RM

S H

RG

C/H

RM

S

NS

NS

NS

Ass

uncã

o et

al.,

200

5 H

übne

r et a

l., 2

005

Byu

ng-H

oon

et a

l., 2

005

H

uman

seru

m

GC

-µEC

D

GC

-ID

HR

MS

0.00

12-

0.00

26 n

gmL-1

2 pg

/L

Čon

ka e

t al.,

200

5 Fo

cant

et a

l., 2

006

Shel

lfish

and

seaf

ood

GC

-µEC

D

NS

Góm

ara

et a

l., 2

005

*Not

e: N

ot st

ated

(NS)

.

23

Tab

le 2

.6: S

ome

exam

ples

of G

C a

naly

sis u

sed

for O

PPs.

A

naly

tes

Mat

rix

Det

ecto

r L

OD

R

ef.

OPP

s O

il R

apid

GC

-FPD

0.

003-

0.

0015

mg/

kg

Dug

o et

al.,

200

5

Sp

ices

G

C-E

CD

-FID

NS

Dan

is e

t al.,

200

2

B

iolo

gica

l sam

ples

G

C/P

ND

0.01

mgL

-1

Tarb

ah e

t al.,

200

1

W

ater

G

C-N

PD

0.

2-5.

0 µg

L-1

Ahm

adi e

t al.,

200

6

Fr

uits

G

C-F

TD

GC

-NPD

4-

20 µ

gkg-1

N

S B

oual

d et

al.,

200

0 B

arek

et a

l., 2

003

GC

-MS

0.

03-0

.06

mgL

-1G

itahi

et a

l., 2

002

Se

dim

ents

G

C-E

CD

N

S Y

ou e

t al.,

200

4

G

C-E

CD

1 µg

kg-1

A

ybar

-Muñ

oz e

t al.,

200

5

V

eget

able

s G

C-µ

ECD

, G

C-(

EI)M

S

5.7

µgkg

-1

Abo

u-A

rab

et a

l., 2

001

*Not

e: N

ot st

ated

(NS)

.

24

Tab

le 2

.7: S

ome

exam

ples

of d

eter

min

atio

n of

org

anic

pol

luta

nts u

sing

MEK

C.

C

ompo

und

Run

ning

buf

fer

LO

D

Ref

. PC

DD

/F

100

mM

SD

S-40

mM

γ-C

D, 5

M u

rea

solu

tion

(pH

9.0

).

0.1

ppm

O

tsuk

a et

al.,

199

9

Poly

chlo

rinat

ed

biph

enyl

s 0.

090

M C

HES

(pH

10.

0), 2

M u

rea,

0.1

1 M

SD

S, 0

.079

M β

-CD

an

d 0.

024

M γ

-CD

. 0.

08 M

CH

ES (p

H 1

0.0)

, 2 M

ure

a, 0

.09

M S

DS,

0.0

72 M

β-C

D

and

0.02

5 M

γ-C

D.

7.5

mM

bor

ate,

40%

(v/v

) ace

toni

trile

buf

fere

d at

pH

9.2

usi

ng

0.5%

(w/v

) pol

ysod

ium

und

ecyl

sulfa

te (p

oly-

SUS)

as t

he m

icel

le

.

40 m

gL-1

10

0-50

0 m

gL-1

N

S

Mar

ina

et a

l., 1

996

Ben

ito e

t al.,

199

7 Ed

war

ds a

nd S

ham

si, 2

000

OPP

s 20

mM

pho

spha

te b

uffe

r (pH

7.5

), 75

mM

SD

S SD

S, so

dium

cho

late

, sod

ium

deo

xych

olat

e an

d El

vaci

te 2

669

as

mic

elle

s at v

ario

us c

once

ntra

tions

with

eith

er p

hosp

hate

or b

orat

e bu

ffer

. 20

mM

sodi

um te

trabo

rate

(pH

9.1

), 10

mM

β-c

yclo

dext

rin, 1

20

mM

SD

S.

7-15

0 ng

L-1

NS

4-52

0 ng

L-1

Pére

z-R

uiz

et a

l., 2

005

Bag

heri

et a

l., 2

004

Agu

ilar e

t al.,

199

7

Phen

olic

co

mpo

unds

25

mM

SD

S in

25

mM

pho

spha

te-5

0 m

M b

orat

e bu

ffer

(pH

7.0

). 0.

04-0

.08

µgL-1

Wat

anab

e et

al.,

199

8

Ani

line

de

rivat

ives

50

mM

sodi

um la

uryl

sulfo

acet

ate

(LSA

),0.0

2 M

bor

ate-

phos

phat

e (p

H 7

.0).

NS

Take

da e

t al.,

199

8

*Not

e: N

ot st

ated

(NS)

. SD

S: so

dium

dod

ecyl

sulp

hate

, CH

ES: 2

-(N

-Cyc

lohe

xyla

min

o)et

hane

sulfo

nic

acid

.

25

Alkanesulfonate surfactants which are industrial products such as sodium N-

lauroyl-N-methyltaurate (LMT) are generally more stable than SDS at extreme pH

values but are not commonly used due to lower purity. A newer surfactant, sodium

lauroylsulfoacetate proved to be suitable as a micelle in the separation of aniline

derivatives (Takeda et al., 1998). Macromolecular surfactants such as poly (methyl

methacrylate-ethyl acrylate-methacrylic) acid (Elvacite 2669) (Watanabe et al., 1998)

and polysodium undecyl sulfate (poly-SUS) (Edwards and Shamsi, 2000) proved to

be ideal for moderate to highly hydrophobic compounds. They have the advantage

of a zero CMC thus precise micellar concentrations can be prepared irrespective of

temperature and buffer compositions. Bile salts such as sodium cholate (SC) and

sodium deoxycholate are a group of natural and chiral surfactants possessing

steroidal structures. They can separate neutral enantiomers and mixtures containing

both chiral and achiral compounds (Fuguet et al., 2002). They however cannot be

utilized in acidic conditions.

A relatively new area is the use of mixed micelles. The addition of either an

ionic or a non-ionic surfactant can alter the hydrophilic surface of the micelles even

at low concentration. This could introduce additional interactions which could

improve selectivity. It was reported of high separation efficiency in the separation of

eight aromatic compounds without an increase in current and Joule Heating with the

use of an ionic surfactant (SDS) with a non-ionic surfactant (Tween 20) which also

improved detection limits (Wang et al., 2004). Shang et al. utilized CD-MEKC with

mixed micelle separation involving SDS-SC in the separation of five

hydroxyanthraquinoids, thus creating a three pseudostationary phase system (Shang

and Yuan, 2003).

2.5.4 Organic Modifier Effect

The resolution, Rs between two peaks at times are less than 1.5 which is more

prevalent in hydrophobic compounds as they would completely solubilise in the

micelle (Bretnall and Clarke, 1995). In order to achieve baseline separation and to

26

enhance selectivity, organic modifiers can also be added to alter the partition constant

between the pseudostationary phase and the running buffer (Bretnall and Clarke,

1995) by enhancing the solubility in the aqueous buffer (Szökő et al., 1996). This is

done by reducing the retention factors of strongly bound solutes to the micelles, to

lengthen the migration time window and to enhance selectivities (Thorsteinsdóttir et

al., 1999). Addition of organic modifiers reduces the zeta potential, ζ at the capillary

wall-mobile phase interface which reduces the electroosmotic flow (EOF) resulting

in higher resolution at the expense of a longer analysis time. Examples of organic

modifier used in enhancing resolution includes methanol (Wan Ibrahim et al., 2005),

acetonitrile (Persson-Stubberud and Ǻström, 1998 and Weinberger and Lurie, 1999 ),

acetone, dimethyl sulphoxide (DMSO) and 2-propanol (Bretnall and Clarke, 1995).

Acetonitrile gave better peak resolution and peak shape in the separation of insulin as

stated by Deng et al., 2002. In addition acetonitrile improved the solubility of

running buffer components preventing the formation of air bubble peaks as compared

to other solvents.

2.5.5 Matrix Effect

Sample matrix has practical implications on both resolution and quantization

which may either improve or worsen the separation depending upon the CE

conditions used. Matrix effects are more profound in larger than small sample

injections. If the samples to be injected were to be dissolved in an adequate and

suitable sample matrix, narrower peaks would be produced, allowing greater volumes

of sample to be injected (Acedo-Valenzuela et al., 2004). In MEKC, most

hydrophobic compounds are not very soluble in water therefore most samples are

prepared in fully organic solvent such as in methanol or in a mixture of two solvent

systems (Otsuka et al., 1999) to aqueous rich media (Riekkola, et al., 2000). The

addition of organic solvent necessitates the solubilization which could effect the

separation. Due to convenience, most samples were preferred to be diluted in

aqueous rich media provided they are miscible in water. But with aqueous media,

due to the higher thermal conductivity there is a lower resistance towards heat

27

transfer compared to non-aqueous media (Quirino et al., 1999). Due to this, there is

an evidence of higher Joule heating in using aqueous media. Therefore, a very small

capillary internal diameter (i.d) is required to effectively dissipate the heat.

Examples of non-aqueous media include highly polar organic solvent such as

acetonitrile (Persson-Stubberud and Ǻström, 1998) to reduce this problem. Low heat

production leads to enhanced separation efficiency owing to reduced longitudinal

diffusion and uniform temperature distribution. The preparation of samples in

different media would give conductivity differences with respect to the separation

buffer which would influence the intensity of the peak area, peak height and

separation efficiency.

The type of media used in preparing the samples is important especially in

online concentration techniques whereby the type of media used is defined in terms

of conductivity differences in reference to the separating buffer. If the sample is

dissolved in a solution that has a lower conductivity (low ionic strength) than of the

background solution (BGS) such as in water or in the buffer matrix (20 or 100 times

lower concentration compared to the running buffer), it resembles the stacking

phenomenon which improves peak intensity and separation efficiency (Kim and

Terabe, 2003). Most people choose to prepare sample simply in organic solvents as

analysis can be performed easily and may remain stable for certain time. If the

sample were to be prepared in buffer matrix, each run requires fresh buffer with the

sample as the sample would degrade or destabilize in the buffer. Therefore a few

micro liters of sample needs to be prepared before hand which is a severe constraint

if the amount of real sample is small.

2.6 On-line Preconcentration Techniques in MEKC

The advantage of using CE is that only a few nanolitres of sample per

injection is required and CE is an energy efficient and more environmentally method

as it uses less organic and/or aqueous solvent. However detection sensitivity as

compared to gas chromatography has not been satisfactory due to the low UV

28

sensitivity contributed by its short path-length and minute injection volumes in order

to maintain high efficiency (Jiang and Lucy, 2002). A solution to this problem

would be to use on-column preconcentration techniques such as stacking and

sweeping. Stacking methods include normal stacking mode (NSM), reversed

electrode polarity stacking mode (REPSM), high conductivity sample stacking mode

(HCSSM) and field-enhanced sample injection (FESI)

2.6.1 Stacking

Stacking involves dissolving the neutral analytes in a solution that has a lower

conductivity than that of the BGS and injected as a long plug than that in normal

injection into the capillary which has been previously conditioned with anionic

micellar BGS (Quirino and Terabe, 1997a). The stacking method for MEKC is

illustrated in Figure 2.4. In the method, the sample is dissolved in a lower

conductivity solution in comparison to the BGS and injected as a long plug than in

normal injection. Sample stacking occurs as analytes cross the stacking that

separates the high field sample zone and low field BGS zone. Due to the boundary

difference in electric field, analytes in the sample zone would then move quickly then

slow down when they reach the low field BGS zone. Therefore, ions are stacked at

the boundary of the two zones (SB) (Quirino and Terabe, 1997a).

The simplest mode of stacking would be normal stacking mode (NSM) which

can be carried out under basic or acidic conditions that involves injecting a long plug

of low conductivity solution containing sample into the capillary previously filled

with high conductivity separation solution or background solution (BGS) (Quirino

and Terabe, 1997b). The stacking mechanisms in NSM are similar to that as

illustrated in Figure 2.4 under basic conditions. If it were to be under acidic

conditions, the polarity would be reversed and the samples would migrate from

negative to positive polarity. Quirino and Terabe managed to achieve almost an

order of magnitude improvement in detection limit for resorcinol (Quirino and

29

Terabe, 1997b). It has also been used to detect pesticides in foods achieving a LOD

of 60-70 ppb (Hernández-Borges et al., 2005).

Figure 2.4: Schematic diagram of the principle of sample stacking in MEKC. SB is the stacking boundary. EOF is the electrooosmotic flow. (Quirino and Terabe, 1997a).

Besides NSM, reversed electrode polarity stacking mode (REPSM) can also

be used under basic conditions. Figure 2.5 is the schematic diagram of REPSM

stacking mechanism of analytes in MEKC. Firstly, (Fig 2.5A), long hydrodynamic

injection of sample in low conductivity matrix is injected after conditioning the

capillary with high conductivity BGS. Followed by (Fig 2.5B), application of

voltage at negative polarity with both inlet and outlet vials containing the BGS. Fig

2.5C and Fig 2.5D is the switching of polarity from negative to positive when current

reaches 97-99% of the actual value. Voltage is maintained until all the analytes have

been detected. The conditions remain the same as in NSM but with electrokinetic

injection of micelles from the BGS at negative polarity to facilitate stacking of

analytes and removal of the sample matrix. Once the current reaches 97-99% of the

predetermined current, polarity is switched to positive to enable separation and

detection of stacked zones (Carabias-Martínez, 2003). Although REPSM gave much

greater sensitivity enhancement around 85, yet if polarity was switched later, a loss

of the more polar analytes would be observed as these compounds are being pumped

30

out from the capillary by the EOF and they are not retained in the micelle (Núñez,

2001; Süsse and Müller, 1996; Nevado et al., 2002 and Quirino and Terabe, 1998a).

This is because stacking efficiency depends on analyte retention factors. Analytes

with higher retention factors, k which are usually more hydrophobic are more

efficiently stacked. These analytes are able to withstand the EOF while for a more

polar analyte they would have been removed along with the EOF from the capillary,

thus causing sample loss. In CZE, REPSM is also known as large volume sample

stacking (Albert et al., 1997; Smyth et al., 1997; Núñez et al., 2001; Leung et al.,

2002 and Laamanen et al., 2006).

High-conductivity sample stacking mode (HCSSM) uses a high-conductivity

sample matrix to transfer field amplification from sample zone to the separation

buffer promoting stacking. This can be carried out by adding salt such as sodium

chloride to the sample matrix giving LODs in the nanomolar range (Palmer and

Landers, 2000). The critical factors for stacking to occur are that the sample anion

has a larger charge-to-mass ratio than the run buffer micelle and the ionic strength of

the sample matrix is higher than the run buffer (Palmer, 2004). The sample matrix

must contain a co-ion with a higher intrinsic electrophoretic mobility than the micelle

to guarantee the formation of a pseudo-steady-state boundary between the micelle

and co-ion component in the sample region (Jiang and Lucy, 2002). This stacking

method was able to analyze nitroaromatic explosives in seawater achieving detection

limits of 0.1 ppm (Giordano et al., 2006). High salt stacking was also used in the

determination of pesticides in fruits and natural waters achieving limits of detections

(LODs) within the µg/L range (Molina and Silva, 2000).

31

Figure 2.5: Schematic diagram of stacking of analytes during REPSM. (A) Before stacking; (B) micelles enter the capillary and carry with it neutral analytes a, k(ax)>k(ay)>k(az); (C) micelles and neutral analytes stack at the concentration boundary (B2) and (B1) and polarity is switched later to positive; (D) separation and later detection of zones. Retention factor is referred with the symbol k. ax, ay and az refer to the stacking boundary for analyte x, analyte y and analyte z. (Quirino and Terabe, 1997b)

Field enhanced sample injection (FESI) is another stacking method with the

sample being prepared in a low-conductivity micellar matrix at neutral pH which is

electrokinetically injected at negative polarity (Quirino and Terabe, 1998b). The

mechanisms of FESI-MEKC are illustrated in Figure 2.6. Before sample injection, a

water plug is introduced at the inlet end of the capillary filled with BGS as shown in

Figure 2.6A. Sample prepared in micellar matrix is placed in the inlet position and

polarity applied in negative mode. Due to the enhanced field of the water plug,

which generates electrophoretic velocities that are greater than the bulk EOF flow,

micelles and neutral analytes solubilized in them thus entering the capillary as shown

in Figure 2.6B. Once the current has reached 97-99%, the voltage is shut off and the

sample vial replaced with the BGS vial in Figure 2.6C. Separation voltage is then

applied at positive polarity for separation (Figure 2.6D). FESI was coupled with

reverse migrating micelles in MEKC (FESI-RMM) in acidic conditions in the

32

analysis of flavonoids found in Epimedium brevicornum Maxim, a Chinese medicinal

herb achieving 360 fold improvement in detection sensitivity (Wang et al., 2003).

FESI-RMM has also been used in the determination of saponins in ginseng achieving

an LOD of 1.7-6.3 µg/mL (Wang et al., 2006). FESI was also used in determination

of pesticides in food and water samples with LODs from 0.02-0.08 mg/L under

electrokinetic injection at 10 kV for 35 s (Hernández-Borges et al., 2005).

Stacking in acidic conditions offer greater choice compared to basic

conditions which requires the suppression of the EOF. Large volume stacking with

polarity switching by Albert et al. in the analysis of anions involves hydrodynamic

introduction of a large sample plug followed by application of negative voltage at the

injection extremity (Albert et al., 1997). The large solvent plug were then

electroosmotically pushed out of the capillary while the anions stack at the boundary

between the sample zone and the background electrolyte. This was then followed by

separation voltage at positive polarity. A 20-fold increased in sensitivity has been

achieved. This stacking method carried out in aqueous conditions whereby the

buffers are prepared in fully aqueous media but Blanco et al. have demonstrated that

non-aqueous CE (NACE) whereby buffers were prepared in methanol is also possible

which performed superior to aqueous conditions (Blanco et al., 2005).

Stacking with reverse migrating micelles (SRMM) is performed by preparing

the sample in a low conductivity matrix and introduced hydrodynamically at the

cathodic end and then separation voltage is applied at negative polarity at the

injection end (Quirino et al., 2000; Chou et al., 2005). The concept is similar to NSM

but as for NSM the stacking is conducted under basic conditions. It is simpler

compared to REPSM as no polarity switching is required. SRMM was applied to the

trace analysis of multi-residue pesticides in water and vegetables with off-line solid-

phase extraction achieving LOD of 0.1 µg/L (Silva et al., 2003). SRMM has been

able to reduce the LOD of melatonin to 30ng/mL and was applied to the analysis of

this hormone in human serum (Musijowski et al., 2006).

33

Figure 2.6: Schematic diagram of stacking of neutral analytes in FESI-MEKC. (A) initial situation (water plug, unshaded; BGS shaded); (B) micelles enter the capillary and carry with them neutral analytes emanating from the cathodic vial, k(x)>k(y)>k(z); (C) micelles and neutral analytes stacked at the concentration boundary, voltage is cut and sample vial replaced with BGS vial when the measured current is approximately 97-99% of the predetermined current, voltage is then applied at positive polarity; (D) separation of zones occur. k refers to the retention factor of analyte x, y and z. (Quirino and Terabe, 1998b).

Stacking using reverse migrating micelles and a water plug (SRW) requires

samples to be prepared in low conductivity matrix with the surfactant added at a

concentration slightly higher than its CMC. Firstly, a long water plug is injected into

the capillary at acidic pH after conditioning with BGS followed by injection of a long

sample plug. SRW is more suitable for hydrophobic compounds with large retention

factors as demonstrated by Otsuka et al. in the analysis of PCDDs and PCDFs

achieving an enhancement of 200 fold (Otsuka et al., 1999).

A

B

C

D

34

2.6.2 Sweeping

Sweeping is where analytes are picked up and concentrated by the micelle

that penetrates the sample zone devoid of micelle. This process is explained in

Figure 2.7. This technique generally uses very low pH buffers to suppress the EOF

achieving an enhancement of 500 times (Huang and Lin, 2005). The analytes are

prepared in a matrix with the same conductivity as the background solution (BGS)

which can either be neutral or charged analytes (Huang et al., 2006). The injected

length of a neutral analyte zone is theoretically narrowed by a factor equal to 1/(1+k)

(k, retention factor) and the concentration can be increased approximately by a factor,

1+k (Quirino and Terabe, 1999 and Quirino et al., 2000). These were under

conditions of relatively constant electric fields, throughout the capillary, negligible

electroosmotic flow, and neutral analytes prepared in a matrix void of the micelle.

Sweeping is dependent on the retention factor of the analyte, therefore it is suitable

for strongly retained analytes. Sweeping is basically used for highly hydrophobic

analytes which have high k values. Drugs such as flunitrazepam (Huang et al., 2006),

steroids (Jen et al., 2003) and ketamine (Chen et al., 2004) have been detected using

sweeping MEKC in biological fluids achieving sensitivity enhancements ranging

from 110 to as high as 800 times. Sweeping has also been used in the detection of

trace amounts of pesticides in real samples (Silva et al., 2003 and Otsuka et al.,

2003). Sweeping was able to determine of steroids in plasma and urine samples with

good sensitivity within the ngmL-1 range (Chen et al., 2004b).

35

Figure 2.7: Schematic diagram of sweeping of analytes in MEKC under low electroosmotic flow. (a) Starting situation, injection of S prepared in a matrix having conductivity similar to that of the BGS; (b) application of voltage at negative polarity, micelles emanating from the cathodic side sweeping analyte molecules; (c) the injected analyte zone is assumed completely swept (Shao and Tseng, 2005).

2.7 Problem Statement

Analysis of PCDDs and PCDFs has usually been carried out using capillary

gas chromatography (cGC) such as cGC-MS and cGC-ECD (Singh and Kulshrestha,

1997). cGC-MS provides high resolution and high sensitivity can be attained.

However, this method requires rather large amount of sample, and hence it requires

high cost and time consuming pre-treatment of the sample. By applying MEKC to

the analysis of PCDDs, we can expect to reduce the sample amount as well as obtain

a simple method that requires less energy, less organic solvents or aqueous solvent

and environmentaly friendly method. 2,3,7,8-TCDD is the most commonly analyzed

isomer as it is the most toxic congener. Analysis has also been carried out on other

tetraCDD/CDFs, other isomers chlorinated in the 2,3,7 and 8 positions,

pentaCDD/CDFs, hexaCDD/CDFs, heptaCDD/CDFs and octaCDD/CDF (Gómara et

al., 2005). The studies done using MEKC involved 1247-/1248-TCDD isomer pair

mixture (Grainger et al., 1996). Another study by Otsuka involved dibenzofuran,

dibenzo-p-dioxin, 2,3- and 2,7-dichlorodibenzo-p-dioxins and 2,3,7-

trichlorodibenzo-p-dioxin (Otsuka et al., 1999). This study compared normal CD-

MEKC and SRW-CD-MEKC. The LOD (S/N=3) obtained were around 20 ppm with

normal CD-MEKC. The LOD reduced greatly to 0.1 ppm with SRW-CD-MEKC.

36

This creates opportunity to further reduce the existing LOD as this sort of level is

insufficient for real sample analysis. Thus far, no study has been conducted on

2,3,7,8-TCDD and 2,3,7,8-TCDF using MEKC. Therefore this study was carried out

to see whether MEKC is feasible in separating both compounds under minimal

analysis time as compared to conventional methods such as GC in addition to

reducing the limit of detection further by coupling on-line and off-line concentration

techniques. The off-line concentration method to be used would be solid phase

extraction. The optimized method would then be applied to analyzing effluent from

industrial wastewater obtained from a pulp mill.

Despite its extremely high separation efficiency, short analysis time and wide

application range, yet CE-MEKC cannot achieve detection limits as low as in GC or

HPLC which can reach to as low as part per trillion (ppt) or part per quadrillion

(ppq). This is due to the short optical path length of the UV detection cell which is

equal to the capillary diameter. Furthermore, poor concentration sensitivity is also

due to minute injection volumes needed to maintain high efficiency. Therefore,

further method development is required in order to improve detection sensitivity.

The usage of different detectors such as laser-induced fluorescence, electrochemical

or amperometric detection and installation of capillaries with extended detection path

length (Z-shaped or bubble cell) is costly and involve complex hardware and

maintenance. Therefore method development using online preconcentration

techniques in order to improve the detection limit is important in MEKC. However,

as preconcentration techniques have their limitations, off-line preconcentration

techniques such as solid phase extraction (SPE) are needed. The coupling of off-line

and on-line concentration techniques, can reduce the limit of detection greatly.

As for the hydrophilic OPPs, they are one of the most commonly used

pesticides in industrialized countries and are an important source of environmental

contamination especially in water bodies. The wide introduction of synthetic organic

pesticides posed to chemists many new analytical tasks associated with the

determination of these pesticides and the products of their decomposition which are

frequently more toxic compared to the original compounds. Therefore, the analysis

of pesticide residues requires methods capable of determining the analytes at ppb, ppt

37

or even lower levels. Furthermore as they are highly soluble in water, they are easily

moved from agricultural lands and get into drinking water by runoff into surface

water or by leaching into groundwater. Thus they pose a great environmental hazard

to humans as well as flora and fauna making greater detection sensitivity is needed in

order to control the content of these pollutants in our environment. Currently only

hydrophilic OPPs dicrotophos and monocrotophos have been studied using MEKC

(Alam, 2005). Most of the studies focused more on the hydrophobic OPPs and the

three hydrophilic OPPs have not been studied as a group.

2.8 Research Objectives

This research objectives were to carry out and evaluate four different on-line

preconcentration techniques viz. normal stacking mode (NSM), reversed electrode

polarity stacking mode (REPSM), high-conductivity sample stacking mode

(HCSSM), field enhanced sample injection (FESI) and sweeping to increase

detection sensitivity and choosing the best on-line preconcentration technique.

Performance evaluation and method validation would be based on calibration graph

linearity and reproducibility of peak area, peak height and migration time

(repeatability and reproducibility). Efficiency of extraction would be carried out by

spiking a known amount of analyte into the real sample. In order to reduce the

existing detection limit, preconcentration techniques with solid phase disc extraction,

stacking with reverse migrating micelles (SRMM) would be utilized in the study

involving the OPPs. The optimized MEKC conditions would be applied to the

analysis of water samples for both TCDD and TCDF and the OPPs.

38

2.9 Scope of Work

The overall study would focus on MEKC with diode array detection (DAD)

in separation of selected organic pollutants which are TCDD and TCDF and selected

hydrophilic organophosphorous pesticides (OPPs) viz. phosphamidon,

monocrotophos and dicrotophos. The study on TCDD and TCDF focus on the

optimization of separation conditions using MEKC with diode-array detection and

using the optimized condition to perform on-line preconcentration techniques. The

on-line preconcentration techniques include normal stacking mode (NSM), reversed

electrode polarity stacking mode (REPSM), high-conductivity sample stacking mode

(HCSSM), field enhanced sample injection (FESI) and sweeping. The on-line

preconcentration techniques giving the best LOD for both analytes will be selected

for the analysis of real sample (water sample). Off-line SPDE will also be performed

to reduce the LOD to a level useful for environmental purpose such as in water

sample analysis. In the analysis of the selected hydrophilic OPPs, we would be using

diode-array detection in optimizing the wavelength for the three analytes. The

buffer, organic modifier and surfactant concentration have already been optimized in

a previous study and therefore would be used in this study. We would then proceed

to stacking with reverse migrating micelles (SRMM) to reduce the limit of detection.

Off-line SPDE would also be used in order for the method to be applied to water

sample analysis. The flow chart for the research methodology of TCDF and TCDD

are shown in Figure 2.8 while for the three hydrophilic OPPs the flow chart is shown

in Figure 2.9.

39

Optimization of Separation Parameters:Organic modifier content and sample matrix effect

On-line preconcentration techniques:NSM, REPSM, HCSSM, FESI and sweeping

Data analysis:Repeatability, Linearity and LOD

Off-line SPDE coupled with on-line preconcentration techniques

Real sample analysis

Optimization of Separation Parameters:Organic modifier content and sample matrix effect

On-line preconcentration techniques:NSM, REPSM, HCSSM, FESI and sweeping

Data analysis:Repeatability, Linearity and LOD

Off-line SPDE coupled with on-line preconcentration techniques


Figure 2.8: Flow chart of research methodology of separation TCDD and TCDF.

40

`

Optimisation conditions from previous study (Alam, 2005):Separation buffer: 20 mM phosphate (pH 2.3), 10

mM SDS and 10% v/v methanol, Separation voltage: -25 kVHydrodynamic sample injection for 10 s at 50 mbar

Wavelength Optimisation:195, 205, 220, 225 and 230 nm

Stacking With Reverse Migrating Micelles (SRMM)

Off-line SPDE coupled with on-line preconcentration techniques.


Optimisation conditions from previous study (Alam, 2005):Separation buffer: 20 mM phosphate (pH 2.3), 10

mM SDS and 10% v/v methanol, Separation voltage: -25 kVHydrodynamic sample injection for 10 s at 50 mbar

Wavelength Optimisation:195, 205, 220, 225 and 230 nm

Stacking With Reverse Migrating Micelles (SRMM)

Off-line SPDE coupled with on-line preconcentration techniques.


Figure 2.9: Flow chart of research methodology of separation of the three hydrophilic OPPs viz. phosphamidon, dicrotophos and monocrotophos.

41

CHAPTER III

EXPERIMENTAL

3.1 Introduction

In this chapter, the reagents, instrumentations and experimental designs used

in this research are discussed in detail.

3.2 Reagents

Analytical reagent (AR) grade sodium cholate was from Wako, Japan while

AR grade Di-sodium tetraborate decahydrate, Na2B4O7.10H2O and AR grade

anhydrous sodium sulphate, was obtained from Fisher Chemicals (UK). Sodium

dodecyl sulphate (SDS) was from Fischer Scientific (Loughborough, UK). Di-

sodiumhydrogen phosphate and sodium hydroxide pellets were from Riedel-de Haen

(Seelze, Germany). Hydrochloric acid (0.1 M) was from Sigma (St. Louis, MO,

USA). HPLC grade acetonitrile and HPLC grade methanol were obtained from J.T.

Baker (California, USA). ACS grade toluene, dicloromethane and concentrated

sulphuric acid were obtained from J. T. Baker Inc. (New Jersey, USA) while

denatured absolute ethanol was obtained from HmbG Chemicals (Germany). The

polychlorinated dioxins (PCDDs) and polychlorinated furans (PCDFs) which were

2,3,7,8-tetrachlorodibenzo-p-furan (TCDF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin

42

(TCDD) as test analytes were from AccuStandard (New Haven, Connecticut, USA).

Stock solutions of TCDF at 800 ppm and TCDD at 670

ppm were prepared in HPLC grade 1,4-dioxane obtained from BDH Laboratory

Reagents, UK. AR grade hydrophilic organophosphorous pesticides chosen were

phosphamidon, dicrotophos and monocrotophos, obtained from Dr. Ehrenstorfers

GmbH laboratory (Augsburg, Germany). Stock solutions of these pesticides were

prepared in methanol at 1000 ppm. Deionised water was obtained from Milipore

UltraPure Water System purified up to 18 MΩ. Figure 3.1 shows the chemical

structure of TCDF and TCDD.

Figure 3.1: Chemical structure for TCDF and TCDD.

TCDF TCDD

O

Cl

Cl

Cl

Cl

O

OCl

Cl

Cl

Cl

3.3 Instrumentations

MEKC separations of TCDF, TCDD and the three hydrophilic

organophosphorous pesticides (OPPs) were conducted on a Agilent capillary

electrophoresis system (220V Agilent Capillary Electrophoresis System, Hanover,

Germany) equipped with a diode array detector with the optimum wavelength set at

225 nm. Data acquisition and system control was carried out by the 3D-CE

ChemStation Software by Agilent Technologies. Standard bare fused silica

capillaries from Agilent Technologies (Hanover, Germany) with 48.5 cm total

length, 40 cm effective length and 50 µm i.d. were utilized to perform the separation.

Injection offset was set at 4 mm with a constant temperature within 25ºC ± 1ºC.

Polypropylene vials (1 mL) from Agilent Technologies (Hanover, Germany) were

used to place samples, buffers and other solutions in the electrophoretic systems.

43

All samples and buffer solutions were filtered through a 0.45 µm nylon filter

disc from Whatman (New Jersey, USA). The final buffering solution pH was

measured by a Cyberscan 500 pH meter from Eutech Instruments Pte. Ltd.

(Singapore). Samples were introduced hydrodynamically at a constant pressure of

50 mbar at different time span and are mentioned as appropriate. Separation voltage

remained constant at 25 kV at positive polarity for the analysis of the two

polychlorinated compounds under basic condition while for the OPPs, separation

voltage was at -25 kV under acidic conditions. Conductivities were measured with a

Horiba ES 12 conductivity meter (Kyoto, Japan).

Envi-DiskTM octadecyl (C18) disks (47 mm i.d., 0.5 mm thick) were

supplied by Supelco (Bellefonte, PA, USA). These C18 disks were to be used in the

solid phase extraction of TCDF and TCDD and OPPs from water samples. Samples

were extracted with a Sigma Aldrich vacuum manifold system hooked up to the A-

3S Aspirator supplied by Eyena, (Tokyo Rikakikai Co. Ltd). Filtration was carried

out twice using a 47 mm diameter, 0.45 µm cellulose acetate membrane filter

provided by Whatman (New Jersey, USA) for the water samples.

3.4 Conditioning of the Capillary

At the beginning of each day, the capillary was flushed for 10 min with 1N

NaOH to activate the silanol groups of the capillary followed by 20 min of deionized

water, and 5 min of running buffer. Before each sample injection, the capillary was

rinsed for 5 min with 0.1N NaOH, followed by 5 min with the running buffer. In

between runs, high pressure water was passed through the capillary for 10 min to

flush out any impurities and to ensure a longer capillary life followed by 3 min of 1N

NaOH, and 3 min of the running buffer to ensure reproducibility. At the end of the

day, the capillary was flushed with 20 min of water followed by 20 min of air. Both

ends of the capillary were dipped in deionized water before shutting down for the

day.

44

For the analysis of OPPs, the capillary was flushed for 10 min with 0.1 M

NaOH to activate the silanol groups of the capillary followed by 10 min of deionized

water followed by 10 min of running buffer at the beginning of the day. Before each

sample injection, the capillary was rinsed for 5 min with 0.1M NaOH, followed by 5

min with deionised water before flushing with 5 min of running buffer. With

separations in acidic buffers, continuous flushing with deionised water for

approximately 15 min in between runs was necessary to clean the capillary properly

with the usual EOF characteristics. To end the day, the capillary was flushed with

water for 20 min followed by air for 20 min. Both ends of the capillary were dipped

in deionized water before shutting down for the day. Peak identification was

performed by injection of single solution of the respective standard under similar

conditions and comparison based on migration times.

3.5 Procedures for MEKC Separation of TCDF and TCDD.

The MEKC separation of TCDF and TCDD such as optimization of

parameters and online concentration techniques are discussed in detail in this section.

3.5.1 Preliminary Investigation For The Separation of TCDF and TCDD

MEKC separations of the two analytes were performed with 20 mM Di-

sodium tetraborate decahydrate buffer containing 20 mM of sodium cholate (SC) as

the chiral surfactant. The effect of various percentages of acetonitrile (MeCN) on the

migration time and separation efficiency were investigated using 5-20% MeCN. The

final buffer pH range after combining all components was 9.16-9.22. Standard

samples of TCDD (40 ppm) and TCDF (40 ppm) were injected individually to obtain

their individual migration time. This is then followed by a mixture of the two

analytes (100 ppm each) for the optimization step. Hydrodynamic injections were

made by applying a pressure at 50 mbar to the sample vial for 1 s. Separations were

45

carried out with positive polarity (anodic injection) at 25 kV at a constant

temperature of 25°C.

3.5.2 Evaluation of Organic Modifier Effect on The Separation of TCDF and

TCDD

Normal mode MEKC separations of the two analytes were performed with

the running buffer described in Section 3.5.1. The effects of various percentages of

methanol (MeOH) on migration time and separation efficiency were investigated

using 5-20% v/v of each organic modifier in separate running buffer vials. This was

followed by investigation using mixed modifiers, namely 5% v/v of MeCN-MeOH

(1:1 v/v), 5% v/v of MeCN-MeOH (3:1 v/v) and 5% v/v of MeCN-MeOH (1:3 v/v).

The addition of 5% v/v mixed modifier to the background solution was chosen as it

gave reasonable baseline separation of less than 8 min. The final buffer pH range

after combining all components was 9.16-9.3. A mixture of TCDD and TCDF (100

ppm each) was injected into the system after determination of their individual

migration times. The concentration of each test solution used throughout this

investigation was 100 ppm unless noted otherwise. Hydrodynamic injections were

made by applying pressure at 50 mbar to the sample vial for 1 s. Separation runs

were carried out with positive polarity (anodic injection) at 25 kV at a constant

temperature of 25°C. Performance was evaluated based on peak resolution,

separation efficiency, migration time, peak height and peak area intensity.

3.5.3 Separation Efficiency of TCDF and TCDD Under Different Sample

Matrix

Normal mode MEKC separations of the two analytes were performed with

the running buffer described in section 3.5.2 but with added 5% v/v mixed modifier

consisting MeOH-MeCN (1:3 v/v). The final buffer after combining all components

had a pH range of 9.16-9.3. To study the sample matrix effect on separation, 4 ppm

46

of TCDD and TCDF mixture were prepared in three different media: ethanol, water

and buffer. From the stock solution, the test analytes were diluted with water to give

a 50 ppm solution. From the 50 ppm solution, 4 ppm of the test analytes were

prepared in various matrices which are water, buffer and ethanol. For the preparation

of test analytes in ethanol, the test analytes were diluted in ethanol directly from the

stock solution. This was to provide a non-aqueous environment. For samples

prepared in buffer matrix they were prepared in buffer and modifier in proportions

similar to those used in the running buffer. Each set of conditions was carried out in

triplicates.

3.5.4 Normal Stacking Mode in the Separation of TCDF and TCDD

Running buffer conditions and pH remain the same as in Section 3.5.3. We

did not use SDS as we did not obtain any separation during our preliminary

investigation. This maybe due to the analytes higher hydrophobicity compared to the

polychlorinated dioxins used in Otsuka’s study (Otsuka et al., 1999). Firstly, we

optimized the injection time under hydrodynamic injection at constant pressure of

50 mbar to initiate stacking. In order to study the effect of injection time on peak

sensitivity and separation efficiency, 1 ppm of TCDD and 2 ppm of TCDF (due to its

lower absorbance) compared to TCDD were prepared as mixture in water under

normal stacking (NS) mode MEKC (NSM-MEKC). The injection times used in the

study were 1 s, 4 s, 10 s, 20 s, 30 s, 40 s and 50 s. From the optimized injection time

obtained, calibration graphs of both TCDF and TCDD were constructed using

external standard method and the LOD was calculated using the 3Sy/m formula

where Sy refers to the relative standard deviation obtained from the calibration curve

through least square fit (scatter of measured values around the regression line) while

m refers to the slope of the calibration curve. The LODs obtained from NSM-MEKC

was then compared with normal mode (NM-MEKC). Normal mode refers to an

injection time of 50 mbar for 1 s.

47

3.5.5 Reversed Electrode Polarity Stacking Mode

The stacking procedure for reversed electrode polarity stacking mode

(REPSM) is as follows: the capillary was filled with the background electrolyte

(BGE) and then a long plug of sample, prepared in water (low conductivity matrix)

was introduced via hydrodynamic injection at 50 mbar for 100 s (plug length; 50

mm). A high voltage at negative polarity (-25 kV) was then applied to remove the

sample matrix from the capillary. Current decreases due to the electrical resistance

caused by the lower conductivity sample plug. The current increased again once the

sample plug was removed from the capillary. Polarity was switched to normal mode

(25 kV) once current reached 95-99% of the original value.

3.5.6 High Conductivity Sample Stacking Mode

In high conductivity sample stacking mode (HCSSM), the samples were

prepared in sodium chloride solution and injected hydrodynamically at 50 mbar for

10 s. The concentration of sodium chloride in the sample was optimized in the range

of 0-300 mM NaCl. The optimal concentration would be the one giving the

maximum sensitivity with the least peak broadening.

3.5.7 Sweeping

Samples were prepared in a non-micellar matrix without the surfactant and

pH maintained at the same pH as the separating buffer. The conductivity of the

matrix was maintained at the same value as the separating buffer and adjusted with a

few drops of borate from the stock solution.

48

3.5.8 Field-Enhanced Sample Injection

In field-enhanced sample injection MEKC (FESI-MEKC), a long water plug

was hydrodynamically injected after conditioning the capillary with buffer. This was

followed by electrokinetic injection (EKI) performed at negative polarity. The

capillary was flushed at high pressure with water for long periods. The injection end

of the capillary was placed on a micellar sample solution before electrokinetic

injection. When the current flowing through the capillary when both ends of the

capillary filled with BGS reaches 97-99% of the current under normal separation, the

voltage was turned off and the inlet vial containing the sample prepared in a micellar

solution was replaced by the BGS. Voltage was then applied with positive polarity

for separation to take place.

3.6 Off-line Preconcentration Using Solid Phase Disc Extraction

Off-line preconcentration using solid phase disc extraction (SPDE) was

carried out on water samples spiked with TCDF and TCDD with the aim of lowering

the LOD by coupling off-line SPDE and on-line preconcentration technique. The

optimized extraction step was then applied to analyzing effluent sample from a pulp

mill at Temerloh, Pahang.

3.6.1 Sample Preparation

Before extracting the analytes, 1 L deionized water was spiked with 50 ng of

both analytes in separate 1 L glass flasks in order to investigate the recovery of the

method. Procedure blanks were prepared with deionized water in 1 L glass flasks.

The sample was then acidified to pH 3 with 50% v/v H2SO4 and 5 mL of methanol

was added to the sample before making up the volume to 1 L. The samples were

then left to equilibrate for 30 minutes before loading unto the SPDE. The final

49

concentration of both analytes after diluting to 1 L were 0.05 ppb respectively. For

the real sample analysis, effluent samples were obtained from a pulp mill at

Temerloh, Pahang and the whole procedure is repeated in the same way.

3.6.2 Solid Phase Disc Extraction

Solid phase disc extraction, SPDE is another extraction method whereby

discs are used as the extraction phase instead of cartridges to accommodate for larger

sample volumes. Firstly, the reservoir and SPE disc were prerinsed with 70:30%

(v/v) ethanol-toluene and placed under vacuum for 5 min to remove any impurities

that may cause analyte loss during extraction. These impurities contribute to analyte

loss when the analytes adsorb strongly to these impurities. Thus during extraction

there would be substantial loss. To the disc, 25 mL methanol was added and left in

contact with the disc for 3 min to condition its hydrophobic surface. Vacuum was

then applied and the methanol was drawn through. Before the disc went dry, 50 mL

of deionized water was added to the reservoir. This critical step ensured that the

surface of the disc is not dry before sampling. Before all the water was drawn

through the disc, the sample was added. After the entire sample had been drawn

through, the disc was air-dried for 5 min. A test tube was placed in the filter flask for

sample extract collection. 30 mL of elution solvent consisting of ethanol-toluene

(70:30 v/v) were used to elute the PCDDs and PCDFs from the disc. The elution

step was repeated twice with 30 mL of the elution solvent. This combination allows

for the action of a strong solvent like toluene to elute/extract PCDD and PCDF and

ethanol to address the residual water left on the SPE disc. The first rinse remained in

contact with the surface of the disk for 20 min, to achieve acceptable extraction of

PCDDs and PCDFs from the particulate matter. For the second rinse, a dropper was

used to rinse the sides of the reservoir before the solvent was sucked through the

disc. The total solvent volume used to elute the disc is 60 mL. Sodium sulphate was

then used to dry the extract before the extract was transferred to a round bottom flask

and the solvent evaporated to 1 mL with a rotary evaporator before being

concentrated under nitrogen stream to dryness. The concentrated extract was then

made up to a final volume of 1 mL. The total volume consisted of 0.8 mL of

50

deionized water and 0.2 mL of sodium chloride solution for analysis with HCSSM-

MEKC. This procedure was also applied to the effluent samples but at 475 mL of

sample was provided. The extraction apparatus setup is shown in Figure 3.2. SRM

of TCDF and TCDD were not used due to financial constraints.

Figure 3.2: SPE disc extraction setup in the extraction of analytes from water samples.

Reservoir

Disc

Waste

51

3.7 Summary of Methodology of Optimization of Separation of TCDD and

TCDF

This section summarizes the methodology used in the optimization and

analysis of TCDD and TCDF as in Figure 3.3.

Optimization ofseparation parameters.

Modifier Effect

Single ModifierMeOH

Mixed ModifierMeOH-MeCN

Single ModifierMeCN

Sample Matrix Effect

Ethanol BufferWater

Various injection time,s(50 mbar)

On-line preconcentrationusing optimized conditions.

NSM, REPSM, HCSSM,Sweeping, FESI

SPDE

Optimization ofseparation parameters.

Modifier Effect

Single ModifierMeOH

Mixed ModifierMeOH-MeCN

Single ModifierMeCN

Sample Matrix Effect

Ethanol BufferWater

Various injection time,s(50 mbar)

On-line preconcentrationusing optimized conditions.

NSM, REPSM, HCSSM,Sweeping, FESI

SPDE

Figure 3.3: Summary of the methodology of MEKC separation involved in the separation of TCDF and TCDD.

52

The flow chart in Figure 3.3 summarizes the methodology of the separation

of both analytes from the optimization of the parameters down to the online

concentration techniques used. The optimized on-line preconcentration technique

used would then be combined with off-line preconcentration which is solid phase

disc extraction (SPDE).

3.8 Procedures for MEKC Separation of Hydrophilic Organophosphorous

Pesticides

The following section would discuss on the MEKC separation and

optimization carried out in the detection of three selected hydrophilic

organophosphorous pesticides.

3.8.1 Wavelength Optimisation in the Separation of OPPs Under Acidic

Conditions

As diode-array detection was used, the OPPs were monitored under five

different wavelengths simultaneously in order to find the optimum wavelength for

respective analytes. Separate solutions (100 ppm) of each standard was injected

separately under identical conditions and detected simultaneously at five different

wavelengths which were 195, 205, 220, 225 and 230 nm, while determining the

migration times of the analytes. This was followed by injecting the standards which

were 100 ppm each as a mixture and analyzed at five different wavelengths to

determine whether their absorption would be influenced by the presence of the other

OPPs. Method performance was evaluated based on peak area and peak height

intensity.

53

3.8.2 Stacking With Reverse Migrating Micelles in the Analysis of OPPs

Samples were prepared in water with concentrations 10 times lower

compared to RM-MEKC and injected at 50 mbar for 50 s. The optimized conditions

have been described in a previous study (Alam, 2005). The calibration standards

used were in the range of 10, 20 and 30 ppm.

3.9 Off-line Preconcentration Using Solid Phase Disc Extraction of OPPs

Off-line preconcentration using solid phase disc extraction was carried out to

reduce the limit of detection for the OPPs. SPDE was chosen as it gave faster

analysis time and reduced plugging for large volumes of water due to the larger

surface to volume ratio.

3.9.1 Sample Preparation

Before extraction, 250 mL of UTM pond water sample was spiked with 1µg

and 0.5 µg of a mixture of pesticides in order to investigate the recovery of the

method. Procedure blanks were also prepared with unspiked pond water. The final

concentrations of the OPPs in pond water were 4 ppb and 2 ppb respectively.

3.9.2 Extraction

The reservoir and SPE disc were pre-rinsed with 20 mL dichloromethane to

remove residues of environmental matrices and impurities from the manufacturing

processes and then placed under vacuum for 5 min. To the disc, 25 mL methanol

was added and left in contact with the disc for 3 min to condition its hydrophobic

54

surface. Vacuum was then applied to draw the methanol through. Before the disc

went dry, 50 mL of deionized water was added to the reservoir. This critical step

was to ensure that the surface of the disc did not go dry. Before all the water was

drawn through the disc, the spiked pond water (250 mL) was added. After the entire

sample had been drawn through, the disc was air-dried for 5 min. A test tube was

placed in the filter flask for sample extract collection. The elution solvent used was

20 mL of methanol at only one step. Sodium sulphate was then used to dry the

extract from any moisture before the extract was transferred to a round bottom flask

and the solvent evaporated to 1 mL with a rotary evaporator before being

concentrated under nitrogen stream to dryness. The concentrated extract was then

reconstituted to a final volume of 1 mL with deionised water.

3.9.3 Method Validation of Extraction Process

The method using Envi-DiskTM octadecyl (C18) disks in the extraction step

was validated with 250 mL pond water spiked with 1 µg of a mixture containing

dicrotophos, monocrotophos and phosphamidon while another batch contained

0.5 µg of the same compounds as in the previous mixture. Thus, after dilution in

250 mL of water the actual concentration would be 4 ppb and 2 ppb for the three

OPPs. Accuracy expressed as recovery (%) and precision (repeatability, % RSD)

were obtained from three replicates of the analysis in different series of the samples.

For recovery studies, it is advisable to use standard reference material (SRM).

However, SRM of the three pesticides is available commercially, thus the use of

spiked samples justify its usage here for the method validation process. Limits of

detection (LODs) of the three OPPs and the linearity of the method were determined.

55

CHAPTER IV

OPTIMIZATION OF SEPARATION PARAMETERS IN MICELLAR

ELECTROKINETIC CHROMATOGRAPHY

4.1 Introduction

In this chapter, MEKC separations of TCDF and TCDD through optimizing

various separation parameters which include fraction of organic modifier added to

the running buffer and the effect of different sample matrix on the separation of these

two test compounds are discussed.

4.2 Preliminary Investigation on the Separation Of TCDF and TCDD Using

MEKC

A preliminary investigation was carried out to determine the migration time

of both analytes. This was then followed by carrying out the necessary optimization

steps to enhance separation.

56

4.2.1 Confirmation of Individual Peaks

Prior to optimization of analyte separation, solutions of TCDD and TCDF

were injected individually at 40 ppm each to determine their respective migration

times before injecting them as a mixture. Both analytes had a slower migration time

compared to the solvent peak (1,4-dioxane) which appeared before TCDF and TCDD

(Figure 4.1). This was due to 1,4-dioxane having a smaller molecular weight

compared to TCDF and TCDD and a higher polarity and solubility in water.

Therefore, the analytes partition more in the aqueous phase than in the micelle giving

a higher mobility compared to TCDD and TCDF. TCDF has a smaller molecular

weight compared to TCDD and the polarity is higher which explains its higher

electrophoretic mobility compared to TCDD. TCDD has a higher hydrophobicity and

therefore it is more incorporated into the micelle giving it a longer migration time

compared to TCDF. 1,4-dioxane appeared as a very sharp and intense peak at 2.762

min. Both analytes were separated within 8 min with TCDF detected at 3.123 min

while TCDD was detected at 7.867 min.

Figure 4.1: Electropherogram of TCDD and TCDF at 40 ppm each in 1,4-dioxane. Run buffer: 20 mM di-sodium tetraborate decahydrate, 20 mM sodium cholate and 5% (v/v) acetonitrile at a final running buffer pH: 9.16-9.22. Injection time: 1 s at 50 mbar. Wavelength: 225 nm. Separation voltage: 25 kV. Total capillary length: 48.5 cm, effective length: 40 cm.

1,4-dioxane

TCDF

TCDD

57

4.2.2 Optimization of Analyte Separation by Varying the Percentage of

Acetonitrile in the Running Buffer Solution

The criteria used for the optimization of the separation of the two analytes are

the baseline resolution of each individual analyte together with no peak splitting in

the shortest possible time. Despite both analytes have been well separated

(Figure 4.1), yet it would be interesting to investigate the effect of various organic

modifier percentage on the separation. The concentration of acetonitrile as the

organic modifier was varied from 0% to 20% (v/v) with an increment of 5% (v/v).

Initially, acetonitrile (MeCN) was chosen as the organic modifier as it was

commonly used in other studies (Bretnall et al., 1995; Szökő et al., 1996;

Thorsteinsdόttir et al., 1999; Wan Ibrahim et al., 2005). Each analysis was carried

out in triplicates.

Migration window refers to the migration time difference between the first

analyte (TCDF) and the last analyte to appear (TCDD). The migration time

increased greatly approximately 68% with increase of MeCN from 5% to 20% v/v

(Figure 4.2). The migration window gives a measure of the total time taken to

separate the analyte mixture in the electropherogram (Bretnall et al., 1995). Baseline

separation was achieved for the two analytes, TCDF and TCDD using (0-20% v/v)

acetonitrile in the running buffer.

Migration window vs %v/v acetonitrile

012345678

0 5 10 15 20 25% v/v acetonitrile

Mig

ratio

n w

indo

w(m

in)

Figure 4.2: The effect of acetonitrile percentage on the migration window for the MEKC separation of TCDF and TCDD (n=3).

58

The peaks for TCDF with different percentages of MeCN are shown in Figure

4.3. For TCDF a third peak was detected before the TCDF peak at percentages of

5% v/v acetonitrile onwards (Figure 4.3). It was suspected that the extra peak was

due to other tetraCDFs isomers. Evidence of the unknown peak as peak X is

illustrated in Figure 4.3. With 20 % v/v MeCN, there was a change in selectivity in

which the TCDF peak appeared before peak X. The presence of peak X would be

explained in further sections in this chapter.

Figure 4.3: Electropherogram of TCDF with different percentages of acetonitrile (MeCN) in BGS at 0%, 5%, 10%, 15% and 20%. At 20%, the TCDF peak appeared before peak X. Separation conditions are the same as in Figure 4.1.

The effect of acetonitrile fraction on TCDF is enlarged and shown in Figure

4.3 while Figure 4.4 shows the electropherogram of both TCDF and TCDD. In

59

general, the migration time of both analytes increases with increasing fraction of

acetonitrile in the running buffer as illustrated in Figure 4.5A. Generally, the effect

of increasing the fraction of acetonitrile is more significant for TCDD, the more

hydrophobic component compared to TCDF. Acetonitrile has greater effect on the

solubility of TCDD which is more hydrophobic compared to TCDF in the aqueous

buffer.

Figure 4.4: Effect of acetonitrile fraction on migration time of TCDF andTCDD at 0% v/v MeCN, 5% v/v MeCN, 10% v/v MeCN, 15% v/v MeCN and 20% v/v MeCN. Separation conditions as in Figure 4.1.

The organic modifier greatly increased the migration time of both analytes

from 10% v/v acetonitrile to 20% v/v acetonitrile as illustrated in Figure 4.5A.

While for TCDF, the change in migration time is less significant. The separation

efficiency of TCDF improved greatly from 15% to 20% v/v acetonitrile. While for

60

TCDD, the separation efficiency decreased from 5% to 20% v/v acetonitrile. At 20%

v/v, the symmetry of the TCDD peak deteriorated and peak broadening was obvious

(Figure 4.4). Figure 4.5 shows that TCDD achieved the lowest efficiency at 20%

v/v.

0

2

4

6

8

10

12

0 5 10 15 20 25

%v/v acetonitrile

Mig

ratio

n, m

in

TCDF TCDD

0100002000030000400005000060000700008000090000

0 5 10 15 20

%v/v acetonitrile

N(ef

ficie

ncy

in p

late

num

bers

)

TCDF TCDD

Figure 4.5: Effect of different percentages of acetonitrile in running buffer on (A) migration time and (B) efficiency on the separation of TCDF and TCDD.

If one were to comment on the best fraction of acetonitrile suitable for a

mixture of these two analytes, then 5 % v/v acetonitrile would be best as it gave the

best efficiency and repeatability for both analytes as shown by Table 4.1. The

efficiency obtained was 20,200 for TCDF and 85,600 for TCDD.

A

B

61

Table 4.1: Repeatability of migration time, peak areas and peak height for TCDF and TCDD under different fractions of acetonitrile in running buffer. Separation conditions as in Figure 4.1.

% RSD (n=3) % MeCN Parameters TCDF TCDD

0 Migration time 0.15 1.04 Peak area 19.91 21.18 Peak height 4.81 23.56

5 Migration time 0.73 1.71

Peak area 10.24 3.13 Peak height 1.83 6.28







The use of organic modifiers is often avoided due to reproducibility problems

as evaporation of the organic solvent from the electrolyte (Bretnall et al., 1995)

buffer can occur especially during the generation of heat as high voltage is applied

during separation. Adsorption onto the capillary during between runs can also cause

repeatability problems which can be reduced by flushing the capillary with running

buffer for a longer time (Weinberger and Lurie, 1995). This experiment was

repeated with longer flushing time of the capillary with the run buffer compared to

the previous experiment and the repeatability for migration time and peak area and

height improved. The repeatability for both migration time, peak area and peak

height are shown in Table 4.1 for both analytes. The highest RSDs for both peak

area and peak height were obtained with 20% v/v acetonitrile. Therefore for

quantification purposes, it is not recommended to use high concentration of

acetonitrile above 20% v/v. At higher concentrations of acetonitrile, destabilization

of the micelle may occur as its critical micelle concentration is altered (Weinberger

62

and Lurie, 1995). In general, 5% v/v acetonitrile gave the best reproducibility

compared to other percentages of acetonitrile (Thorsteinsdóttir et al., 1999).

Concentrations below 20% v/v provided analysis time of less than 8 min. At

20% v/v, the analysis time was approximately 10 min. Based on the best peak

resolution between TCDF peak and peak X, 15% v/v acetonitrile was the optimum

percentage (Figure 4.3). As peak resolution is more important, 15% v/v acetonitrile

is chosen for further optimization. Using 15% v/v MeCN as the best fraction, the

efficiency of both analytes was still high with N=16,017 for TCDF while N=67,004

for TCDD. The resolution achieved between TCDF and peak X is 0.823 as tabulated

in Table 4.2.

Table 4.2: A comparison on the effect of different fractions of MeCN on the peak resolution, Rs for TCDF measured relative to peak X.

Resolution, Rs % MeCN v/v TCDF

0 0.000 5 0.813 10 0.820 15 0.823 20 0.817

4.3 Evaluation of Organic Modifier Effect on the Separation Efficiency of

TCDF and TCDD Using MEKC

This section discusses the effect of various organic modifier which includes

acetonitrile and methanol on the separation efficiency of TCDF and TCDD. The

effects of single and mixed modifier on the separation efficiency of both analytes

were investigated.

63

4.3.1 Comparison of Separation Performance Between Acetonitrile and

Methanol

Methanol and acetonitrile were used by many researchers in enhancing

separation in MEKC. Both organic modifiers were investigated at concentration

ranging from 5% to 20% v/v in the running buffer and the criteria used for the

optimization was based on peak resolution between the TCDF peak and another

unknown peak referred to as peak X as in section 4.2.2 that migrated closely with it.

The behavior of acetonitrile (Section 4.2.2) with increasing concentration increased

the migration time window giving a better resolution but at the expense of longer

analysis time as has been reported in another study (Szökő et al., 1996).

For methanol, no peak resolution between TCDF and peak X was achieved

using 0 to 5% v/v. Peak resolution then improved for 5-15% v/v but decreased

slightly at 20% v/v as shown in Table 4.3. The optimum concentration of methanol

was at 15% v/v in the running buffer with a migration window of 5.02 min and RS

1.03 (Figure 4.6) based on the best peak resolution value between TCDF and peak X.

In this study, acetonitrile gave a better baseline and peak shape especially for TCDD

compared to methanol which gave an obvious peak shouldering for TCDD (Figure

4.7). The appearance of the three peaks between TCDF and TCDD was suspected to

be due to the solvent used which was 1,4-dioxane that would be confirmed in Section

4.4, page 77.

Table 4.3: A comparison on the effect of different fractions of MeOH on the peak resolution, Rs for TCDF measured relative to peak X.

Resolution, Rs % MeOH v/v TCDF

0 0.000 5 0.000 10 0.680 15 1.030 20 0.965

64

time/min

Figure 4.6: Electropherogram of TCDF and peak X at 15% v/v methanol. Running buffer conditions: 20 mM sodium cholate, 20 mM sodium tetraborate-decahydrate at a final buffer pH: 9.16-9.22. Wavelength: 225 nm. Both test analytes at 100 ppm each in 1,4-dioxane. Hydrodynamic injection at 50 mbar for 1s. Separation voltage, 25 kV; temperature, 25ºC; total capillary length, 48.5 cm, effective length, 40 cm.

time/min

Figure 4.7: Peak shouldering for TCDD at 9.147 min (circle) with 20% v/v methanol. MEKC conditions as in Figure 4.5.

A comparison was made between acetonitrile and methanol in terms of

migration time, resolution, efficiency and selectivity (Table 4.4). Acetonitrile gave a

shorter migration time for both analytes. Acetonitrile gave better peak resolution,

efficiency and selectivity for TCDD while for TCDF methanol gave better peak

resolution, efficiency and selectivity. Therefore the usage of mixed modifier might

be able to give better performances for both analytes as both analytes behaved

differently in different organic modifier.

TCDF

X

1,4-dioxane

TCDF

TCDD

Peak shouldering

65

Table 4.4: A comparison of the effect of 15% v/v MeCN and 15% v/v MeOH on the migration time (tm), resolution (Rs), efficiency (N) and selectivity (α) of TCDF and TCDD MEKC separation.

Organic Modifier Parameters TCDF TCDD 15% v/v MeCN Migration time

(tm),s 3.122 7.815

Resolution (Rs) 0.823 5.673 Efficiency (N) 16017 67004 Selectivity (α) 1.043 1.090 15% v/v MeOH Migration time

(tm),s 3.541 9.147

Resolution (Rs) 1.03 2.753 Efficiency (N) 40191 21477 Selectivity (α) 1.067 1.047

The relative standard deviation, RSD of TCDF and TCDD for both migration

time, peak area and peak height is shown in Table 4.5. The highest RSD for both

peak area and peak height was obtained with 10% v/v methanol for TCDD which

implies that there is a destability with interaction between the micelle and the organic

modifier (Szökő et al., 1996). The reproducibility improved from 15 to 20% v/v

methanol. At higher concentrations of methanol, destabilization of the micelle may

occur as its critical micelle concentration is altered which occurred at 20% v/v

methanol (Bretnall et al., 1995). In general, 15% v/v acetonitrile gave the best RSD

in terms of peak height and migration time for TCDF and TCDD while for peak area,

15% v/v methanol gave the best RSD for TCDD while for TCDF it was 5% v/v

methanol. The optimum percentage of methanol was chosen at 15% v/v as it gave

the best peak resolution between TCDF and peak X at Rs 1.03 (Table 4.3). 15% v/v

methanol also gave the minimum %RSD for migration time for TCDF (0.21%) and

TCDD (1.16%) and peak heights for TCDF (0.053%) while TCDD with 2.46%

(Table 4.5). %RSD obtained for TCDD was also reasonable with 1.15% but not for

TCDF which achieved 32.56%.

66

Table 4.5: Repeatability of migration time, peak area and peak height for TCDF and TCDD under different fractions of methanol, MeOH in running buffer. Separation conditions the same as in Figure 4.1.

% RSD (n=3) % MeOH Parameters TCDF TCDD





Figure 4.8 shows the effect of different methanol percentage on the separation

of TCDD and TCDF. Based on the electropherogram, the peak shouldering at TCDD

is obvious at 15% v/v MeOH and at 20% v/v MeOH there was serious peak

broadening for TCDD. Based on the electropherogram, 15% v/v gave the best peak

resolution between TCDF and peak X as compared to other fractions. This further

strengthens our choice of 15% v/v as the optimum methanol fraction. It was also

shown that increasing the percentage of methanol from 15% to 20% v/v worsened the

peak tailing in TCDD which maybe due to the mismatch between the buffering

system and the hydrophobicity of the analyte (Wan Ibrahim et al., 2005). At 20%

v/v, the second peak, peak X that appeared after TCDF, was not detected. This

shows that in terms of resolution, 15% v/v methanol performed better than other

fractions in the background solution (BGS).

67

Figure 4.8: Electropherogram of the effect of the % of MeOH on the separation of TCDF and TCDD. Separation conditions remain the same as in Figure 4.5. The peaks between TCDF and TCDD are suspected to be from the 1,4-dioxane.

68

4.3.2 Comparison Of Mixed Modifier With Single Organic Modifier On

Separation Performance

Both 15% v/v acetonitrile and 15% v/v methanol which gave reasonable

separations with the best reproducibility and peak resolution were then compared

with mixed modifier of acetonitrile (MeCN) and methanol (MeOH) which were

5% v/v MeCN-MeOH (1:1), 5% v/v MeCN-MeOH (3:1) and 5% v/v MeCN-

MeOH(1:3). 5% v/v was chosen as a preliminary value to investigate the effect of

mixed modifier. If the 5% proved to be sufficient, further increments would not be

necessary. This combination was chosen to investigate the effect of mixed modifier

on the separation performance as compared to single modifier. Comparisons were

done based on peak resolution, baseline stability, peak height, peak area, migration

time and peak efficiency. Mixed modifier was used as TCDF gave a better response

in methanol while TCDD in acetonitrile. Therefore, the usage of mixed modifier

might be able to compensate for both differences. 15% v/v methanol gave the best

peak resolution for the TCDF peak (Figure 4.9). The addition of methanol gave the

best peak resolution of TCDF as compared to acetonitrile alone. The addition of

mixed modifier consisting of 5% v/v MeCN-MeOH (3:1) and 5% v/v MeCN-MeOH

(1:3) also gave better separation compared to acetonitrile.

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

Modifier composition

RS

15% v/v MeCN15% v/v MeOH5% v/v MeCN-MeOH (1:1)5% v/v MeCN-MeOH (3:1)5% v/v MeOH-MeCN (3:1)

Figure 4.9: Peak resolution for TCDF (measured relative to peak X mentioned in section 4.2.2) at 15% v/v MeCN, 15% v/v MeOH and various percentages of mixed modifier. Separation conditions as in to Figure 4.1.

69

In terms of baseline stability, mixed modifiers gave a much better stability

compared to 15% v/v MeCN and 15% v/v MeOH. In addition, the mixed modifier

also gave better peak shapes with higher intensity for TCDD peak as shown in Figure

4.10 that shows the enlarged section for TCDD peak. Peak tailing for TCDD

worsened with the usage of methanol in mixed modifier but lessened when there was

a higher percentage of MeCN. MeCN-MeOH (1:1) and MeCN-MeOH (3:1) gave

considerably better baselines, peak shapes and a higher stability compared to MeCN-

MeOH (1:3) which destabilized after each run which can prove to be impractical

when time is a concern.

Figure 4.10: Effect of (A) 15% v/v MeCN (B) 15% v/v MeOH (C) 5% v/v MeCN-MeOH (1:1), (D) 5% v/v MeCN-MeOH (3:1) and (E) 5% v/v MeCN-MeOH (1:3) on the separation of TCDD. MEKC separation conditions as in Figure 4.1.

70

Figure 4.11 shows part of an electropherogram with the enlarged section for

TCDF for 15 % MeCN, 15 % MeOH and the mixed modifiers. Based on Figure

4.11, the resolution between TCDF and peak X improved with the usage of mixed

modifiers such as MeCN-MeOH (1:1), MeCN-MeOH (3:1) and MeCN-MeOH (1:3).

Quantitatively, MeCN-MeOH (1:3) gave the best resolution according to Figure 4.9

but it was very unstable and therefore, 5% v/v MeCN-MeOH (3:1) was chosen as the

optimum mixed modifier.

min Figure 4.11: Effect of (A) 15% v/v MeCN (B) 15% v/v MeOH (C) 5% v/v MeCN-MeOH (1:1), (D) 5% v/v MeCN-MeOH (3:1) and (E) 5% v/v MeCN-MeOH (1:3) on the separation efficiency of TCDF (enlarged). Peak X refers to an unknown peak due to the 1,4-dioxane used. MEKC separation conditions as in to Figure 4.10.

71

Comparison of efficiency, peak heights, peak area and migration time were

made for 15% v/v MeCN, 15% v/v MeOH and mixed modifier as in 5% v/v MeCN-

MeOH (1:1), 5% v/v MeCN-MeOH (3:1)and 5% v/v MeCN-MeOH (1:3) as shown

in Figure 4.12. For migration time, peak area, peak height and efficiency there was

only a slight difference for the TCDF peak as compared to the TCDD peak for pure

acetonitrile, pure methanol and mixed modifiers. This maybe due to the TCDF

having a lower hydrophobicity compared to TCDD giving it a lesser interaction with

the micelle and easily incorporated into the running buffer. The addition of

acetonitrile to the running buffer lengthens the migration time for TCDD while

increasing the fraction of methanol in mixed modifier as in MeCN-MeOH (1:3) gave

TCDD the shortest migration time. This might be due to the increased fraction of

methanol decreases the association of TCDD with the micelles. Peak area, peak

height and efficiency was highest for TCDD at 5% v/v MeCN-MeOH (1:3)

compared to other fractions. This shows that increasing the amount of methanol in

the running buffer increases the peak height, peak area and efficiency of TCDF and

TCDD. But it also gave lower stability while increasing the amount of methanol in

the running buffer. As in MeCN-MeOH (1:3), the buffer destabilized after the first

run shown by a sudden current drop which is impractical and not economical in the

long run. Due to this reason, further comparisons were judged based on repeatability

of migration time, peak area and peak height which are important in quantitative

analysis.

The repeatability was obtained by injecting the sample under constant

experimental conditions for 3 times. The effect of different organic modifiers on the

relative standard deviation (%RSD) on migration time, peak area and peak height on

the separation of TCDF and TCDD are shown in Table 4.6.

72

Figure 4.12: Effect of modifier on (A) migration time, (B) peak area, (C) peak heights and (D) efficiency of TCDF and TCDD.

73

Table 4.6: The effect of different organic modifiers on the relative standard deviation (% RSD) of migration time, peak area and peak height for TCDF and TCDD.

% RSD (n=3)

Fraction, % v/v Parameters TCDF TCDD 15% MeCN Migration time 0.551 0.064


15% MeOH Migration time 0.213 1.163 Peak area 32.563 1.150 Peak height 0.053 2.463

5% MeCN-MeOH (1:1) Migration time 0.067 0.303 Peak area 28.860 2.637 Peak height 16.189 0.908



The mixed modifier that gave the lowest % RSD for migration time was

MeCN-MeOH (1:1) with % RSD of 0.07% for TCDF and 0.30% for TCDD. The %

RSD of migration time for both TCDF and TCDD for all mixed modifiers are less

than 1% except for TCDF with 5% v/v MeCN-MeOH (1:3) which is about 2.4%

RSD and for TCDD with 5% v/v MeCN-MeOH (1:3) which is at least 5% RSD. The

% RSD of peak height is much better compared to the RSD of peak area for both

analytes at all mixed modifier composition used with TCDD sharing a % RSD of

peak height less than 10%. The lowest peak height % RSD for TCDD is with 5% v/v

MeCN-MeOH (1:1) and the higher % RSD is with 5% v/v MeCN-MeOH (1:3) i.e

3.8% while the highest peak height % RSD for TCDF is with 5% v/v MeCN-MeOH

(1:1) i.e. 16%. The % RSD of peak area in mixed modifier for TCDD is much better

(1-10%) compared to the % RSD of peak area for TCDF (17-28%) which has a lower

UV absorption compared to TCDD at the same concentration. The poor repeatability

for peak area, might be due to the inconsistent evaporation of organic modifier from

the open run buffer (Thorsteinsdόttir et al., 1999). The high % RSD obtained for

TCDF maybe due to the smaller absorption obtained for this analyte, thus it is more

vulnerable to any slight errors. This would give it a higher % RSD compared to

74

TCDD. Therefore, for further study, the composition selected was 5% v/v MeCN-

MeOH (3:1).

4.4 Investigation of Sample Matrix Effect on the Separation Efficiency

Sample matrix has been shown to affect the separation efficiency of analytes.

Thus, this study compares the separation performance between aqueous and non-

aqueous media.

4.4.1 Baseline Stability and Unknown Peak Elimination

Sample matrix has practical implications on both resolution and quantization

which may either improve or worsen the separation depending upon the capillary

electrophoresis conditions used (Riekkola et al., 2000). Therefore, in this work, the

analytes were dissolved in selected media (ethanol, water and buffer) to find the most

suitable sample matrix (solvent) for our analytes based on peak area and peak height

intensity, separation efficiency and baseline stability. Furthermore, it is hoped that

the three peaks that appear between TCDF and TCDD and peak X (section 4.3.1)

could be eliminated and their sources verified.

Figure 4.13 shows an electropherogram for the separation of TCDF and

TCDD prepared under different media but used under identical experimental

conditions. The deviations in the migration time of (C) were due to the experiment

for (C) media being carried on a different day. Both (B) and (C) gave satisfactory

baseline but analytes dissolved in water gave higher sensitivity (B) due to the

stacking effect. In (A), the downward peak before TCDF was due to the ethanol

peak.

75

Figure 4.13: Electropherograms of TCDF and TCDD at 4 ppm each in different media used. (A) ethanol, (B) water and (C) buffer. (B) and (C) are analytes dissolved in aqueous matrix while (A) is analyte dissolved in non-aqueous matrix. Running buffer conditions as in Figure 4.5 but with 5% v/v MeCN-MeOH (3:1).

The use of a non-aqueous matrix ethanol gave an unknown peak that co-

migrated before the TCDF peak as shown in Figure 4.14. The same phenomenon

76

was also observed when we dissolved the analyte in 1,4-dioxane as stated in Section

4.3.1. In aqueous matrix TCDF appeared as a single peak. We suspect this maybe

due to unmatched buffer and sample zone when the sample was prepared in organic

solvent (Wan Ibrahim et al., 2005). This finding corroborated with the claim made

by the manufacturer of TCDF (Accustandards) that the standard provided is pure.

Figure 4.14: Electropherogram of TCDF in the presence of (A) ethanol, (B) water and (C) buffer matrix. TCDF: 4 ppm; running buffer conditions similar to Fig 4.13.

77

4.4.2 Comparison of Peak Area, Peak Height and Separation Efficiency In

Different Sample Matrix

Comparison of the sample matrix effect was done based on peak area, peak

height and separation efficiency. Figure 4.15 shows the effect of the different sample

matrices on separation efficiency, peak area and peak height.

TCDF and TCDD prepared in water matrix gave the highest peak area (Fig

4.15B) and peak height (Fig 4.15C) with considerable efficiency (Fig 4.15A).

Analytes prepared in buffer matrix gave the highest peak efficiency which is seen in

Figure 4.15 (A) with very sharp peaks especially for TCDD. The intense peak height

and area obtained for water matrix reflects an increase in plate number compared to

analyte prepared in organic solvent. This phenomenon is also known as normal

stacking mode which is the enhancement of the sample zone field by preparing the

neutral analytes in water giving stacking via on-column micellization (Wan Ibrahim

et al., 2005).

As both TCDF and TCDD are very hydrophobic and are non-polar

compounds, they require solubilization in organic solvents such as 1,4-dioxane and

ethanol. Yet the addition of ethanol (Figure 4.15 A-C) to the sample greatly reduced

the peak area, peak height and efficiency considerably in comparison to water and

buffer matrix except for the peak areas of TCDF. This was consistent with a reported

work by Shihabi and Hinsdale, 1995. This was due to alteration of the analyte’s

distribution between the micelle and aqueous phase. This can be seen by the

absorbance of t0 which refers to the intense downward peak of ethanol which implies

that a large portion of the analytes remain in the aqueous phase unsolubilized by the

micelle as can be seen in Figure 4.14.

78

Figure 4.15: Comparison of sample matrix effect based on (A) separation efficiency, (B) peak area and (D) peak height. (C) shows the enlarged peak areas as circled in (B) for TCDF. Error bars are in standard errors.

Sharper peaks are an indication of improved separation efficiency. Analytes

prepared in buffer matrix gave better efficiency giving sharper peaks compared to

79

water and organic solvent matrix although with a lower intensity compared to water.

This is due to the buffer matrix having the same concentration as the separating

buffer. Therefore, there was no difference in conductivity between the sample zone

and the separation zone which are needed to initiate stacking (Shihabi and Hinsdale,

1995).

Therefore, it can be concluded that for further optimization, mixed modifier

consisting of 5% v/v MeCN-MeOH (3:1) should be used in the running buffer. The

analytes were to be prepared in water as the sample matrix as it gave the best

compared to other matrices. This is used for subsequent studies on on-line

preconcentration on the separation of these two analytes.

80

CHAPTER V

ON-LINE AND OFF-LINE PRECONCENTRATION TECHNIQUES FOR

ANALYSIS OF TCDF AND TCDD

5.1 Introduction

This chapter discusses the on-line and off-line concentration techniques used

in improving the detection limit of TCDF and TCDD. The optimum offline and

online concentration techniques were combined in the analysis of effluent water

samples obtained from a pulp-mill factory from Temerloh, Pahang. The offline

preconcentration technique to be used would be solid phase disc extraction (SPDE).

5.2 Normal Mode MEKC

Normal mode MEKC (NM-MEKC) has been used in the preliminary

determination of limit of detection of both analytes under basic conditions.

5.2.1 Normal Mode MEKC (NM-MEKC)

NM-MEKC in basic buffer, the injection time was for 1 s at 50 mbar. Both

TCDF and TCDD were baseline separated using the optimized conditions obtained

81

earlier which were 20 mM sodium cholate, 20 mM borate buffer and mixed modifier

5% v/v MeCN-MeOH (3:1). The final buffer pH was set at 9.16-9.23. However for

2 ppm TCDF, the injection time was not sufficient to detect it. This was because

TCDF has a lower UV absorbtivity compared to TCDD which was detectable even at

1 ppm.

5.2.2 Calibration Lines, Linearity (r2), LODs

The LODs are calculated on the basis of peak heights since the linearity with

peak areas is not good. Calibration curves obtained for TCDF and TCDD were

analysed with four different concentrations of standard mixtures; 10, 20, 30 and 40

ppm for TCDF and 2.5, 5.0, 10.0 and 15.0 ppm for TCDD. The calibration curves

obtained for both TCDF and TCDD are shown in Figure 5.1 (A) and Figure 5.1 (B)

respectively. The linear, equations and LODs for both analytes are presented in

Table 5.1.

Figure 5.1: Calibration curves based on peak height for (A) TCDF and (B) TCDD using NM-MEKC. Separation buffer: 20 mM sodium cholate, 20 mM sodium tetraborate-decahydrate and 5% v/v MeCN-MeOH (3:1) with final buffer pH 9.16-9.22. Separation wavelength: 225 nm. Separation voltage: 25 kV. Hydrodynamic injection for 1s at 50 mbar. Capillary total length: 48.5 cm. Effective length: 40 cm.

82

The LOD for TCDD is lower compared to the LOD for TCDF. A more

intense peak was obtained for TCDD compared to TCDF. Both analytes show a

linear relationship between peak height and concentration in the range 10-40 ppm for

TCDF and 2.5-15 ppm for TCDD. The r2 values are all above 0.99 with the LOD for

TCDF being 2.20 ppm and 1.71 ppm for TCDD at a S/N=3.

Table 5.1: Equation of calibration curves, correlation coefficients, r2, LODs (at S/N = 3) using NM-MEKC on the basis of calibration curves in Figure 5.1.

ANALYTES EQUATION r2 LOD (S/N = 3), ppm TCDF y = 0.0295x + 0.1644 0.9979 2.20

TCDD y = 0.3841x – 0.1509 0.9930 1.71

5.2.3 Repeatability and Efficiency (N)

Repeatabilities of migration time, peak height and peak area for each analyte

are given in Table 5.2. Migration times of both TCDF and TCDD peaks are uniquely

reproduced with RSDs below 1 % while RSDs for peak heights and peak areas are in

the acceptable range of 3-10 %. The electropherogram of three intraday replicated

runs are shown in Figure 5.2. The two small peaks appearing between TCDF and

TCDD are from the 1,4-dioxane used in preparing the stock solutions.

Table 5.2: Repeatability of migration time , peak area and peak height (mAU) in the separation of TCDF and TCDD using NM-MEKC. ANALYTES % RSD, n=3

Migration time Peak Height Peak Area TCDF 0.16 3.70 10.08 TCDD 0.58 4.36 3.08

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Figure 5.2: Electropherogram of three replicated runs for the separation of TCDF (40 ppm) and TCDD (15 ppm) in NM-MEKC under identical conditions as in Figure 5.1.

84

The peak efficiency, N changed for both TCDF and TCDD over the range of

different concentrations used as seen in Figure 5.3. For TCDD, the variation in N

remained above 100 000 throughout with 5 ppm giving the highest efficiency. While

for TCDF, 10 ppm gave the lowest efficiency at 61 180. The N value of TCDD was

largely unaffected from 5 to 15 ppm. The N value of TCDF was affected throughout

the concentration ranges used as it incorporates less with the micelle (Qurino et

al.,1997a). This may be attributed to variances in the temperature throught the

analysis which causes changes in efficiency (Pyell, 2001).

Figure 5.3: Variations in N for (A) TCDF and (B) TCDD in the concentration used in the calibration curve in NM-MEKC.

5.3 Normal Stacking Mode (NSM-MEKC)

Normal stacking mode (NSM-MEKC) is also carried out in basic buffer.

Stacking was carried out by injecting sample solutions for a much longer time

compared to the usual hydrodynamic injection as performed by Qurino et al. (1997a).

NSM was chosen as it is the simplest stacking method and requires little sample

preparation. Samples are diluted by a factor of 10 times in comparison to NM-

MEKC with water. Firstly, the injection time was optimized to obtain the optimized

injection time that would give good peak intensity and separation efficiency. From

the optimized injection time, 10 s injection time (ten times higher than NM-MEKC)

is chosen as the optimum injection time.

85

5.3.1 Optimization of Injection Time

Only minute volumes of sample are loaded into the capillary in order to

maintain high efficiency. With respect to sample overloading, the injection plug

length determined by the injection time is critical. Therefore, in optimizing the

injection time, performances are based on peak area and peak height intensity and

also on peak efficiency.

The injection used was 1, 4, 10, 20, 30, 40 and 50 s. Based on the injection

time, the plug length, Ip and volume of sample loaded, Vp were calculated and

compared with the length left for separation. The volume was calculated using the

Hagen-Poiseuille equation as shown in equation 5.1:

Vp = ∆Pd4πt (equation 5.1) 128ηL

∆P = pressure difference across the capillary

d = capillary inner diameter (µm)

t = time (s)

η = buffer viscosity (1 at 25˚C)

L = total capillary length (cm)

The limit of detection is proportional to the injected sample zone. The

optimum injection time should also allow high separation efficiency because in real

sample analysis numerous unknown matrix effects would lead to the appearance of

unidentified peaks when on-line concentration techniques are used (Huang and Lin,

2005).

An increase in injection time from normal sample injection at 50 mbar for 1 s

resulted in an increase in peak area and peak height for both TCDF (2 ppm) and

TCDD (1 ppm). The same observation was also reported by Süsse and Müller

(1996). At normal injection time of 1 s, 2 ppm of TCDF was detected at a very low

intensity (Figure 5.4). There is evidence of peak broadening for both analytes with

increasing injection time till it reached 50 s whereby the peak height for TCDD and

86

TCDF broadened. The calculated volume of sample loaded, Vp, injected plug

length, Ip, % of sample length and % of length left for separation is indicated in

Table 5.3. Based on Table 5.3, the % of remaining length for separation decreases

with increasing plug length. This can explain for the reduction in migration time

when the run is carried out till 50 s. The longer the injection plug length, the shorter

the remaining length for separation to take place and thus the shorter the migration

time (Figure 5.4). Although, the % of remaining length for separation seems high,

due to the short migration time of both analytes, it is not advisable to increase the

injection plug length further to avoid peak overlapping.

Table 5.3: Injection volume and plug length as a function of pressure, time and capillary id. % of sample plug and corresponding % of remaining length available for separation is also indicated. Injection time x pressure, mbars

Vp, nL Ip, mm % of sample plug

% of remaining length for separation

50 1.5 0.75 0.15 99.8 200 6.0 3.00 0.62 99.4 500 15.0 7.50 1.55 98.5 1000 30.0 15.00 3.09 97.0 1500 45.0 22.50 4.64 95.4 2000 60.0 30.00 6.19 93.8 2500 75.0 37.50 7.73 92.3

Vp = plug volume L = 48.5 cm η = 1 Ip = plug lengths T = 25˚C

There was an increase in peak height for TCDF till it reached a maximum at

30 s while for TCDD it reached a maximum at 40 s before decreasing with a further

increase in injection time, s (Figure 5.5B). This may be due to the sample plug being

too long generating a strong laminar flow. This laminar flow is the result of a

mismatch of the EOF velocity in the sample and buffer zones (Wang, 2003). There

is also an increase in peak height followed by a reduction in peak efficiency. Peak

efficiency for TCDF was good (above hundred thousand) till a 40 s injection time is

used. While for TCDD, the peak efficiency deteriorated from 10 s onwards. In order

87

to maintain high separation efficiency and reasonable peak height and area, the

optimized injection time chosen is 10 s at 50 mbar.

Figure 5.4: Electropherograms showing the effect of different injection times on peak shapes of TCDD and TCDF. Separation buffer: 20 mM sodium cholate, 20 mM sodium tetraborate-decahydrate and 5% v/v MeCN-MeOH (3:1) at a final buffer pH 9.16-9.22. Separation wavelength, 225 nm; separation voltage, 25 kV; hydrodynamic injection of samples varied from 1s, 4s, 10s, 20s, 30s, 40s and 50s. Capillary total length, 48.5 cm; effective length, 40 cm.

88

Figure 5.5: Influence of injection time on (A1) peak area, (A2) enlarged for TCDF peak area, (B) peak height and (C) efficiency on the separation of TCDF and TCDD. Hydrodynamic injection pressure constant at 50 mbar.

5.3.2 Calibration Lines, Linearity (r2), LODs

The calibration curves for both TCDF and TCDD are constructed with

concentrations 10 times lower than in NM-MEKC and using the optimum injection

time of 10 s. Each run was done in triplicates. The calibration curves based on peak

heights are shown in Figure 5.6 while the calibration equations, linearity and LODs

at S/N = 3 are shown in Table 5.4. The calibration curves for TCDF and TCDD are

linear in the range of 1-4 ppm for TCDF and 0.25-1.5 ppm for TCDD. Both curves

show r2 values of greater than 0.995 and the LOD of 0.34 ppm and 0.05 ppm for

TCDF and TCDD respectively.

89

Table 5.4: Equation of calibration curves, r2, and LODs (for S/N = 3) based on the calibration curves in Figure 5.6.

ANALYTES EQUATION r2 LOD (S/N = 3), ppm TCDF y = 0.1827x + 0.1572 0.9950 0.34

TCDD y = 3.3013x + 1.6329 0.9993 0.05

Figure 5.6: Calibration curves based on peak height for (A) TCDF and (B) TCDD using NSM-MEKC. Injection time at 50 mbar for 10 s. Separation conditions as in Figure 5.4.

5.3.3 Repeatability, Reproducibility and Efficiency

The repeatability of migration time, peak height and peak area for each

analyte are given in Table 5.5. Migration times of both TCDF and TCDD peaks are

highly reproduced with RSDs below 1 % while RSDs for peak heights and peak

areas are in the acceptable range of 0.9-6%. Electropherogram of three intraday

replicated runs are shown in Figure 5.7 as carried out on 17 November 2006.

90

Table 5.5: Repeatability of migration time, peak height and peak area for TCDF and TCDD using NSM. ANALYTES % RSD, n=3


Figure 5.7: Electropherogram of three successive replicated runs for the separation TCDF and TCDD in NSM-MEKC under same conditions as in Figure 5.4 but with an injection time of 10 s.

91

Reproducibility (n=5) for migration time, peak area and peak height in the

separation of TCDF and TCDD over four consecutive days with n=5 per day are

given in Table 5.6. The migration time % RSD for both analytes are in the

acceptable range of 3.8-4.8 % while for peak area it was at 9.7 % for TCDF and for

TCDD it was reasonably low at 5.5 %. The peak height % RSD for TCDF was 10.8

% and for TCDD was acceptable at 7.0 %. Interday RSDs are higher compared to

intraday RSDs due to different surrounding temperature on different days which

might influence the capillary temperature and hence the migration time, peak height

and peak area. Furthermore, this might be due to the different ways of preparing

buffers on different days. In this experiment, buffers are freshly prepared after every

four runs in order to maintain stability. To avoid errors, the buffers were prepared in

large quantities for 4 days for 20 runs.

Table 5.6: Reproducibility of migration time (min), peak area (mAUs) and peak height (mAU) for TCDF and TCDD ANALYTES % RSD, n=5


* Note: Analysis was carried out for four consecutive days with (n=5) per day.

Peak efficiency, N, greatly increased for TCDF from 1 to 3 ppm then dropped

at 4 ppm. Generally the efficiency is lower compared to NM-MEKC with

efficiencies below 100 000. While for TCDD, the efficiency remained satisfactory

and better compared to TCDF for all concentrations with efficiencies above 110 000

with a minimum at 0.5 ppm (Figure 5.8). However the efficiency is poorer compared

to NM-MEKC due to the longer sample plug being injected which gave broader

sample zones although N is above 110 000. N can be concentration dependent as a

higher concentration is more prone to peak broadening due to longitudinal diffusion

with a higher concentration.

92

Figure 5.8: Variations in N in the concentration range for (A) TCDF and (B) TCDD used in the calibration curve in NSM-MEKC.

5.3.4 Stacking Efficiency

Table 5.7 shows the comparison of LOD obtained for TCDF and TCDD using

NM-MEKC and NSM-MEKC. Stacking effiencies in terms of peak height

(SEFheight), peak area (SEFarea), and LOD (SEFLOD) are calculated for each test

analyte to evaluate quantitatively the degree of stacking to compare between NSM

and NM. A stacking efficiency of 10 and 100 is comparable to one and two orders of

magnitude improvement respectively. Stacking efficiencies in the form of sensitivity

enhancement factors were calculated based on the ratio of the peak height, peak area

and LOD obtained by NSM-MEKC to NM-MEKC multiplied by the dilution factor

of 10. All three enhancement factors are shown in Table 5.7.

Table 5.7: Sensitivity Enhancements Factor (SEF) in NSM-MEKC over NM-MEKC in the separation of TCDF and TCDD.

ANALYTES SEFheight SEFarea SEFLOD* LODNM-

MEKC (ppm)

LODNSM-

MEKC (ppm)

TCDF 7.4 53 65 2.20 0.34

TCDD 32 24 329 1.71 0.05

SEFheight, SEFarea and SEFLOD: see text, *LODs are based on peak heights. Conditions as in Figure 5.5 and Figure 5.1.

93

SEFarea gave a much higher value for both analytes compared to SEFheight but

this implies that with stacking, peaks broadened in NSM. Similarly, LODs were

improved by almost 65 times lower for TCDF and 329 fold lower for TCDD. A

sensitivity enhancement of 7.4-32 fold was obtained with the NSM-MEKC (based on

peak height). However the LOD obtained so far is still insufficient for real sample

analysis of TCDF and TCDD. Therefore, additional on-line preconcentration

methods needs to be investigated such as reversed electrode polarity switching mode

(REPSM).

5.4 Reversed Electrode Polarity Stacking Mode (REPSM)

Reversed electrode polarity stacking mode was used as it has been able to

provide enhancement approximately up to a hundred fold improvement in detection

sensitivity (Süsse and Müller, 1996; Nevabo et al., 2002 and Carabias-Martínez et

al., 2003). As the efficiency of stacking is dependent on the analyte’s retention

factors, k (Quirino and Terabe, 1997b; and Puig et al., 2005). Higher k values imply

higher hdyrophobicity thus are more efficiently stacked. Therefore, REPSM would

stand a good chance in reducing the detection sensitivities of both analytes.

5.4.1 Influence Of Various Stacking Periods

The concentration effect depends strongly on the chosen period during the

stacking step with reversed polarity. The right moment for switching polarity is very

important to avoid back-flush of the more polar analyte causing sample loss (Núñez

et al., 2001 and Laamanen et al., 2006). This depends on the electrophoretic velocity

of the sodium cholate micelle, the electroosmotic flow and the distribution of the

analytes between the micellar and aqueous phase. Figure 5.9 shows the effect of

polarity reversal time on the peak area and peak height intensity of both analytes.

94

020406080

100120140

19 21 23 25 time,s

Pea

k ar

ea, m

AUs

TCDFTCDD

05

101520253035

19 21 23 25 time,s

Peak

hei

ght,

mA

U

TCDFTCDD

0

2

4

6

8

10

19 21 23 25 27

time, s

Pea

k ar

ea, m

AU

s

TCDF

1.451.501.551.601.651.701.751.80

19 21 23 25 27

time, s

mA

U

TCDF

Figure 5.9: Effect of polarity switching time on (A1) peak area and (B1) peak height for both analytes. (A2) enlarged line graph of peak area for TCDF. (B2) enlarged line graph of peak height for TCDF.

The stacking period for polarity reversal gave a more profound effect on the

peak area and peak height intensity for TCDD as compared to TCDF. This is

because TCDD is more retained effectively in the micelle due to its higher

hydrophobicity compared to TCDF. Based on Figure 5.9, the most intense peak

height for both analytes was obtained at 20 s which was also obtained for the most

intense peak area. Therefore, for further quantization purposes, 20 s was chosen as

the optimized polarity reversal time. Table 5.8 shows the corresponding percentage

of current with the polarity switching time. Figure 5.10 is the electropherogram of

separation of TCDF and TCDD at different stacking periods.

A1 B1

A2 B2

95

Table 5.8: Corresponding percentage of original current, % with polarity switching time,s used in REPSM.

Polarity Switching Time,s Percentage of Original Current, %

19.0 97 20.0 82 23.0 94 24.5 89 25.0 96

Higher percentages of current gave lower intensities for the peak area and

peak height intensities for both analytes. At 20 s, the percentage of current obtained

was at 82%. Increasing the stacking period from 20 s onwards increased peak

broadening for both analytes especially for TCDD. Switching the polarity later

would result in the loss of the more polar analytes as can be seen in Figure 5.10

where the intensity of TCDF decreases with later polarity switching due to increase

in the percentage of current. For a solute that is not stacked (low retention factors),

reducing the percentage of current when the polarities are switched will increase the

volume of injected sample compared to the volume injected when polarity is

switched later (eg 99%) (Quirino and Terabe, 1997b). In the case of polar analytes, if

the percentage of current is too high, focalization of these analytes would not occur

as they would have been pushed out off the capillary by the EOF. Based on literature

review, most polarity switching occurs when the percentage of current reaches 90-

99% (Smyth et al., 1997; Leung et al., 2002 and Hernández-Borges et al., 2005).

However, this percentage range was not suitable as under 99%, TCDF peak was not

detected. Therefore, the ideal percentage of current chosen was at 82%.

96

Figure 5.10: Electropherogram of REPSM with different polarity switching times. TCDF at 4 ppm (circled peak) while TCDD at 1.5 ppm. Separation buffer: 20 mM sodium cholate, 20 mM sodium tetraborate-decahydrate and 5% v/v MeCN-MeOH (3:1) at a final buffer pH 9.16-9.22. Separation wavelength, 225 nm; separation voltage, 25 kV. Hydrodynamic injection of sample at 50 mbar for 100 s followed by electrokinetic injection of buffers at -25 kV for 20 s. Total capillary length: 48.5 cm. Effective length: 40.0 cm.

97

5.4.2 Calibration lines, linearity (r2) and LODs

The calibration curves for TCDF are constructed with concentrations 20 times

lower than in NM-MEKC and each run is done in triplicates. For TCDD the

concentration was 100 times lower compared to NM-MEKC. The calibration curves

based on peak heights are shown in Figure 5.11 while the calibration equations,

linearity and LODs at S/N = 3 for each analyte are shown in Table 5.9. Both graphs

are linear in the concentration range 0.5 to 2.0 ppm for TCDF and 0.025 to 0.15 ppm

for TCDD. The correlation coefficient, r2 are 0.997 for TCDF while it is 0.9998 for

TCDD. LODs calculated based on peak heights for TCDF are 130 ppb while for

TCDD it is much lower at 3 ppb as shown in Table 5.9.

Table 5.9: Equation of calibration curves, r2, LODs (for S/N = 3) on the basis of calibration curves in Figure 5.11 using REPSM.

ANALYTES EQUATION r2 LOD (S/N = 3), ppb TCDF y = 0.3054x + 0.831 0.9970 130

TCDD y = 109.46x + 2.3197 0.9998 3

Figure 5.11: Calibration curves based on peak height for (a) TCDF and (b) TCDD using REPSM. Injection time set at 50 mbar for 100 s. Electrokinetic injection at -25 kV for 20 s. Separation conditions as in Figure 5.10.

98

5.4.3 Repeatability and Efficiency

Repeatabilities of migration time, peak height and peak area for each analyte

are given in Table 5.10. Migration times of both TCDF and TCDD peaks are

uniquely reproduced with % RSDs below 1% while %RSDs for peak heights and

peak areas are in the acceptable range of 0.11-9.4% for online concentration

techniques (Carabias-Martínez et al., 2003).

Table 5.10: Repeatability of migration time, peak area and peak height in the separation of TCDF and TCDD using REPSM.

ANALYTES %RSD, n=3 Migration time Peak Height Peak Area

TCDF 0.12 7.75 2.16 TCDD 0.32 0.11 9.37

N greatly decreased for TCDF from 0.5 to 1 ppm before increasing to 2 ppm.

Generally, N was lower compared to NM-MEKC with efficiencies below 100,000.

TCDD gave a lower efficiency compared to TCDF for all concentrations as shown in

Figure 5.12. The efficiency for TCDF and TCDD was poorer compared to NM-

MEKC. This maybe due to the longer sample plug being injected which gave

broader sample zones.

Figure 5.12: Variations in N with the concentration for (A) TCDF and (B) TCDD used in the calibration curve in REPSM-MEKC. Conditions as in Figure 5.10.

99


Stacking effiencies in terms of peak height (SEFheight), peak area (SEFarea),

and LOD (SEFLOD) are calculated for each test analyte to evaluate quantitatively the

degree of stacking to compare between REPSM and NM. Stacking efficiencies in

the form of sensitivity enhancement factors are calculated based on the ratio of peak

heights, peak areas and LODs obtained by REPSM to NM which are multiplied by

dilution factors. All three enhancement factors are shown in Table 5.11.

Table 5.11: Sensitivity enhancement factors (SEF) in REPSM over NM-MEKC in the separation of TCDF and TCDD.

ANALYTES SEFheight SEFarea SEFLOD* LODNM-

MEKC (ppb)

LODREPSM (ppb)

TCDF 42 124 338 2200 130

TCDD 666 997 57000 1710 3

SEFheight, SEFarea and SEFLOD: see text, *LODs are based on peak heights.

SEFarea gave a much higher value for both analytes compared to SEFheight but

this implies that with stacking, peaks broadened in REPSM. Similarly, LODs were

improved by almost 338 times lower for TCDF and 57 000 times lower for TCDD.

The sensitivity enhancement for TCDD was much better compared to TCDF as

TCDD was more retained in the micelle compared to TCDF. Due to this, TCDF was

easily removed from the capillary when polarity reversal was used as it is being

pushed out by the EOF more easily compared to TCDD.

5.5 High Conductivity Sample Stacking Mode (HCSSM)-MEKC

Stacking in high salt matrix is only evident when the sample matrix conductivity

is higher than the separation buffer conductivity. This caused stacking of the

micelles at the sample/separation buffer interface. The critical factors for stacking to

occur are that the sample anion has a larger charge-to-mass ratio than the run buffer

100

micelle and the ionic strength of the sample matrix is higher than the run buffer

(Palmer and Landers, 2000 and Palmer, 2004). The sample matrix must contain a co-

ion with a higher intrinsic electrophoretic mobility than the micelle to guarantee the

formation of a pseudo-steady-state boundary between the micelle and co-ion

component in the sample region (Jiang and Lucy, 2002). Thus in this study, we

would like to study the feasibility of HCSSM in improving the detection sensitivity

of TCDD and TCDF using sodium cholate as the micelle and addition of sodium

chloride to the sample matrix to initiate stacking.

5.5.1 Optimization of Sodium Chloride Concentration

In high conductivity sample stacking mode (HCSSM), samples are prepared

in a high conductivity medium in order to focalize the micelles in the sample-

separation buffer interface; consequently, neutral analytes are swept by the grouped

micelles at the interface. It is a simple procedure as it only requires the addition of a

high salt concentration to the sample. This work studied the effect of addition of

sodium chloride in the range of 0-300 mM on the stacking of TCDD and TCDF.

Samples with sodium chloride concentration in the range of 0-300 mM were

prepared and the effect it had on peak area, peak height and efficiency is shown in

Figure 5.13. Figure 5.14 shows the electropherogram of HCSSM under different

concentrations of sodium chloride.

The trend shown in Figure 5.13 was also observed by Palmer and Landers

(2000). Peak area and peak height intensity increased for both analytes from

0-200 mM followed by depreciation at 300 mM. While for separation efficiency, the

efficiency, N decreased from 50 mM to a minimum at 300 mM. At 300 mM NaCl,

peak shape deteriorated badly. There was serious peak tailing for the TCDD peak at

300 mM. A possible explanation for the decrease in stacking efficiencies at higher

sodium chloride concentrations maybe due to the high sodium ion concentrations

producing a co-ion layer around the surfactant micelle that is less diffuse than with

101

lower sodium chloride concentrations (Palmer, 2004). This hinders the interaction

between the analyte and the micelle thus reducing the stacking efficiency. The

deterioration of peak efficiency could be due to the disappearance of the pseudo-

steady-state boundary between the micelle and co-ion component. The conductivity

of the sample matrix increases, thus the electrophoretic movement of co-ion against

EOF is further slowed down causing poor stacking (destacking). Therefore, we

choose 200 mM NaCl as the optimal NaCl concentration.

Figure 5.13: Effect of different concentrations of NaCl (0-300 mM) on (A) peak area, (B) peak height and (C) efficiency for both TCDF and TCDF. Separation buffer: 20 mM sodium cholate, 20 mM sodium tetraborate-decahydrate and 5% v/v MeCN-MeOH (3:1) at a final buffer pH 9.16-9.22. Separation wavelength: 225 nm. Separation voltage: 25 kV. Hydrodynamic injection of sample at 50 mbar for 10 s. Total capillary length: 48.5 cm. Effective capillary length: 40 cm.

102

Figure 5.14: HCSSM-MEKC electropherograms with different concentration of NaCl. TCDF at 2 ppm while TCDD at 0.3 ppm. Peak for TCDF is shown by the circle drawn around the peak. Separation buffer: 20 mM sodium cholate, 20 mM sodium tetraborate-decahydrate and 5% v/v MeCN-MeOH (3:1) at a final buffer pH 9.16-9.22. Separation wavelength: 225 nm. Separation voltage: 25 kV. Hydrodynamic injection of sample at 50 mbar for 10 s. Total capillary length: 48.5 cm. Effective capillary length: 40 cm.

103

5.5.2 Calibration lines, linearity (r2) and LODs

The calibration curves for TCDF and TCDD are constructed with

concentrations 10 times lower than in NM-MEKC and each run was done in

triplicates. The calibration curves based on peak heights are shown in Figure 5.15

while the calibration equations, linearity and LODs are shown in Table 5.12.

The calibration curves are linear in the concentration range of 1-4 ppm for

TCDF and 0.25-1.5 ppm for TCDD with good correlation, r2 of 0.9999. The LOD

obtained for both analytes with a signal to noise ratio of three are within the ppb

range with the LOD for TCDF at 46.0 ppb while TCDD at 18.5 ppb. The LOD for

TCDF is better with HCSSM compared to REPSM (130 ppb), however for TCDD,

the LOD is better with REPSM (3 ppb) compared to HCSSM.

Table 5.12: Equation of calibration curves, r2, LODs (at S/N = 3) for TCDF and TCDD based on calibration curves in Figure 5.15.

ANALYTES EQUATION r2 LOD (S/N = 3), ppb TCDF y = 0.1549x + 0.3888 0.9999 46.0

TCDD y = 0.4581x + 1.5843 0.9999 18.5

Figure 5.15: Calibration curves based on peak height for (a) TCDF and (b) TCDD using HCSSM. Injection time set at 50 mbar for 10 s. NaCl concentration in sample matrix at 200 mM. Separation conditions similar to Figure 5.14.

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5.5.3 Repeatability, Reproducibility and Efficiency

Repeatability and reproducibility of migration time, peak height and peak

area for each analyte are given in Table 5.13. The reproducibility of migration time,

peak height and peak area for each analyte are also given as in Table 5.14.The RSDs

were determined by triplicate injections of 2 ppm TCDF and 0.3 ppm TCDD. While

in the determination of reproducibilities, five injections (n=5) were carried out daily

for four consecutive days. The repeatability of migration times of both TCDF and

TCDD peaks were highly reproduced with RSDs below 0.3% while RSDs for peak

heights and peak areas were in the acceptable range of 0.6-3.9%. The

reproducibilities of the migration times for both analytes did not exceed 4.28% while

for peak height it was in the range of 10.33-19.71%. As for peak area, the range was

from 7.34-10.93%.

Table 5.13: Repeatability of migration time (min), peak area (mAUs) and peak height (mAU) in the separation of TCDF and TCDD. ANALYTES %RSD, n=3


Table 5.14: Reproducibility over four consecutive days with n=5 per day of migration time (min), peak area (mAUs) and peak height (mAU) in the separation of TCDF and TCDD using HCSSM. TCDF is at 2 ppm while TCDD is at 0.3 ppm. ANALYTES %RSD, n=3


105

For TCDF, N greatly decreased from 1 to 2 ppm then increased for 4 ppm.

Generally the value of N was lower compared to NM-MEKC with efficiencies below

100,000. While for TCDD, the efficiency remained lower compared to TCDF for all

concentrations with efficiencies below 50 000 with a minimum at 0.5 ppm as in

Figure 5.16. Yet the efficiency was poorer compared to NM-MEKC due to the longer

sample plug being injected which gave broader sample zones.

Figure 5.16: Variations in efficiency, N in the concentration range for (A) TCDF and (B) TCDD used in the calibration curve in HCSSM. Separation conditions similar to Figure 5.14.



and LOD (SEFLOD) were calculated for each test analyte to evaluate quantitatively

the degree of stacking to compare between HCSSM and NM. Stacking efficiencies

in the form of sensitivity enhancement factors were calculated using the ratio of peak

heights, peak areas and LODs obtained from HCSSM and NM which were multiplied

with the dilution factors. All three enhancement factors are shown in Table 5.15.

106

Table 5.15: Sensitivity Enhancements Factor (SEF) in HCSSM-MEKC over NM-MEKC in the separation of TCDF and TCDD.

ANALYTES SEFheight SEFarea SEFLOD* LODNM, ppb

LODHCSSM, ppb

TCDF 11.6 4.0 478 2200 46.0 TCDD 22.5 18.7 924 1710 18.5


SEFheight gave a much higher value for both analytes compared to SEFarea

which implies that with high salt stacking, peak broadening did not occur as

compared to previous stacking methods such as NSM and REPSM. Similarly, LODs

were improved by almost 478 fold lower for TCDF and 924 fold lower for TCDD.

The sensitivity enhancement factor for TCDD was much better compared to TCDF

as TCDD is more retained in the micelle compared to TCDF. This is because

efficient high-salt stacking is dependent upon a strong analtye/micelle interaction.

REPSM has a sensitivity enhancement which is selective towards highly

hydrophobic analytes especially TCDD which gave the best sensitivity enhancement

factor. With HCSSM, the selectivity was not limited to TCDD but also to TCDF.

This was because the sensitivity enhancement factor was much better for TCDF with

HCSSM compared to that for REPSM which only gave an enhancement factor of 338

fold.

5.6 Sweeping

In sweeping, the sample was prepared in a matrix void of micelles at a buffer

concentration of 5 mM, 10 mM and 20 mM. The conductivity was adjusted to that of

the running buffer with drops of borate buffer till it match the BGS conductivity. It

was shown that only at 10 mM sodium cholate, were the peak for TCDF and TCDD

observed (Figure 5.17) which gave a buffering conductivity of 1.29 mS/cm. Yet the

enhancement efficiency was very poor as compared to NM-MEKC mode. This may

be due to the analytes not having a very high capacity factor that is required for

107

sweeping since it has already been concluded that the EOF velocity has no role in

enhancing the sensitivity. It may also be due to the pseudostationary phase used

which influences the capacity factor of the analytes which appears to be unsuitable

for the analyte used.

Figure 5.17: Electropherogram of sweeping with different concentration of sodium cholate in sample matrix at (A) 5 mM, (B) 10 mM and (C) 20 mM. Separation buffer: 20 mM sodium cholate, 20 mM sodium tetraborate-decahydrate and 5% v/v MeCN-MeOH (3:1) at a final buffer pH 9.16-9.22. Separation wavelength, 225 nm; separation voltage, 25 kV. Hydrodynamic injection of sample at 50 mbar for 5 s. Total capillary length: 48.5 cm. Effective length: 40.0 cm. TCDF at 2 ppm while TCDD at 0.5 ppm.

5.7 Field-Enhanced Sample Injection (FESI)

Field-enhanced sample injection, FESI was tried out on both analytes as

sweeping was not successful in improving the detection. Furthermore, the injection

of a water plug before electrokinetic injection of sample provides a higher electric

field at the tip of the capillary, which will eventually improve the sample stacking

108

procedure. In this procedure, sample preconcentration is obtained by

electrokinetically injecting a sample of lower conductivity compared with the BGS.

Here, samples were prepared in 20 mM sodium cholate, 20 mM sodium tetraborate

without the mixed modifier. Despite being under basic conditions, negative polarity

under electrokinetic injection was feasible due to the injection of a long water plug

beforehand. In this case, only neutral analytes associated with the micelle can be

concentrated. Sample was electrokinetically injected at negative polarity, -20 kV for

100 s yet no peaks were detected. Decreasing the voltage to -15 kV was carried out

as higher voltage make the analytes get through the boundary between the water plug

and the buffer and disperse into the buffer during injection procedure thus decreasing

stacking efficiency (Quirino and Terabe, 1998 and Wang et al., 2006). Yet, this too

was not successful in obtaining peaks. Increasing the length of water plug injected

from 0 to 5 s could also increase sensitivity. The reason is that the water plug

provides a higher electric field at the tip of the capillary, which will eventually

improve the sample stacking procedure. However, no peaks were detected even after

increasing the water plug injection time to 5 s (Figure 5.18).

Figure 5.18: Electropherogram of TCDF (2ppm) and TCDD (0.5 ppm) under FESI-MEKC conditions. Analytes were mixed with 20 mM sodium cholate and deionized water. Samples were electrokinetically injected at -20 kV, 100 s after hydrodynamic injection of water plug for 5 s at 50 mbar. Separation conditions as in Figure 5.18.

109

5.8 Off-line Preconcentration Technique With Solid Phase Disc Extraction

(SPDE)

In this section, we are combining off-line preconcentration with on-line

preconcentration techniques in order to reduce the existing LOD for both analytes.

The optimized on-line preconcentration technique used was HCSSM which gave

LODs in the ppb range. For environmental samples, the ability to detect within ppb

range is still not sufficient. Therefore, we decided to combine both off-line (SPDE)

and on-line preconcentration (HCSSM) techniques in order to improve on the LODs

through sample enrichment during extraction. Furthermore, off-line preconcentration

(SPDE) are also used as sample clean-up prior to CE analysis.

Both TCDF and TCDD are present at very low concentrations in water with a

high proportion in the particulate matter. Solid phase disc extraction (SPDE) has

proven to be an effective method in extracting PCDDs and PCDFs from both the

water and the particle phase as it avoids previous separation of the two phases, the

liquid-liquid extraction for the aqueous and the Soxhlet extraction for the particulate

phase thus avoiding the formation of emulsions between both phases (EPA Method

8290A, 1998 and Gawlik et al., 2000). As deionized water was used as the sample

matrix, therefore we assume that this is particulate-free samples thus PCDDs and

PCDFs tend to adsorb to the walls of the sample container or filtration (Taylor et al,

1995). Therefore, it is critical to rinse the sides of the reservoir and the sample

container to achieve the best possible recoveries. According to the EPA Method 613,

the method detection limit for industrial wastewater using gas chromatography was

0.002 µg/L (U.S. EPA Method 613, May 1984).

Our experiment during the validation step shows that no clogging of the disc

was observed for the spiked deionized water since it was free of particulate matter.

Thus filtering was not necessary in this study. 1 L deionized water was spiked with

50 ng of TCDF and TCDD both in separate 1 L glass flasks before being loaded onto

the reservoir. The total extraction time from the time of conditioning is around 45

minutes. The time of analysis is dependent on the type of disc used (Pujadas et al.,

2001). Analysis with Speedisk gave an extraction time of 15 min.

110

After spiking into water, the actual concentration of both analytes were 0.05

ppb once diluted into 1 L of deionized water This is then followed by recovery

studies of the method. Table 5.16 shows the results of the recovery studies with 50

ng of spiked analytes. Figure 5.19 shows the electropherogram of the extraction of

both concentrations used in the validation study for TCDF and TCDD.

Table 5.16: Recovery and repeatability of extraction of TCDF and TCDD from spiked water using SPDE. Congener Spiked amount Recovered

amount Recovery (%) Repeatability

(%RSD) TCDF 50 ng 54 ng 108 5.9 TCDD 50 ng 55 ng 110 6.9

Based on the recovery study, the spiking at 50 ng gave good recovery for

TCDF, 108% and for TCDD, 110%. Based on the results, this proves that SPDE is

sufficient for quantitative analysis at very low concentrations. This method has

proven to be repeatable with RSDs % falling within the acceptable range of 5.9-

18.17% (Kiguchi et al., 2007). The preconcentration factor calculated from spiking

into 1 L of water to reconstitution to 1 mL is 1000 fold. Thus this shows that this

method is able to detect up to a concentration of 0.05 ppb. Therefore, the LODs for

coupling offline concentration methods (SPDE) with online concentration technique

(HCSSM-MEKC) for TCDF is 46 ppt while it is 18.5 ppt for TCDD.

The optimized SPDE-HCSSM technique was then applied to real sample

analysis. The sample used was effluent samples from a pulp mill. The same sample

preparation and extraction procedures were carried out as in the method validation

procedures. The effluents were divided into two categories which were untreated

effluent (A and B) and treated effluent (A and B). The effluents at 475 mL each had

to be filtered twice due to the presence of suspended particles. The results of the

extraction are shown in Table 5.17 and their electropherograms in Figure 5.20.

Based on the results, it shows that treating the effluent lowers the amount of TCDF

111

and TCDD till it was not detected in sample treated B for TCDD. Overall, the study

proves that the concentration of both these analytes fall within the ppb range. This

shows that the method SPDE-HCSSM developed was sufficient to detect TCDF and

TCDD for environmental samples.

Figure 5.19: Electropherograms of SPDE-HCSSM of TCDF and TCDD spiked with 50 ng. Separation buffer: 20 mM sodium cholate, 20 mM sodium tetraborate-decahydrate and 5% v/v MeCN-MeOH (3:1) at a final buffer pH 9.16-9.22. Separation wavelength, 225 nm; separation voltage, 25 kV. Hydrodynamic injection of sample at 50 mbar for 10 s. Total capillary length: 48.5 cm. Effective capillary length: 40 cm.

Table 5.17: Concentration of TCDF and TCDD discovered in pulp-mill treated and untreated effluent. ND: Not detected. Effluent TCDF, ppb TCDD, ppb Untreated A 87 14 Untreated B 19 2 Treated A 22 10 Treated B 10 ND

112

Figure 5.20: Electropherograms of pulp mill effluent water obtained as tabulated in Table 5.17. (A) treated A, (B) treated B, (C) untreated A and (D) untreated B.

113

5.9 Conclusions

Five types of online concentration were investigated which were normal

stacking mode (NSM-MEKC), reversed electrode polarity stacking mode (REPSM),

high conductivity sample stacking mode (HCSSM), sweeping and field-enhanced

sample injection (FESI-MEKC). The optimized on-line preconcentration technique

was HCSSM as it gave good LODs for both TCDF (46 ppb) and TCDD (18.5 ppb)

within the ppb range. Yet this was still insufficient to be applied to environmental

samples such as water samples. Off-line preconcentration such as solid phase disc

extraction (SPDE) are used to improve on the existing LOD by offering a sample

enrichment step which can go as high as 1000 fold. Furthermore, SPDE is also used

for sample clean-up by reducing interference from the sample matrix. TCDF and

TCDD were successfully extracted from 1 L deionized water using SPDE and using

HCSSM-MEKC as the on-line preconcentration method. The total enrichment

factors achieved were 1000 fold and existing LOD was successfully reduced by 1000

fold. The HCSSM-MEKC-SPDE method achieved a LOD of 46 ppt for TCDF and

18.5 ppt for TCDD with reproducibilities within the acceptable range of 5.9-18.17%.

Recoveries were in the range of 76.9-110%. The developed method was able to

detect both analytes within the ppb range as shown in the pulp-mill effluent samples.

Treating the effluent before release to surface water helps to reduce the presence of

TCDF and TCDD which should be a practice to avoid environmental pollution.

114

CHAPTER VI

SEPARATION OF HYDROPHILIC ORGANOPHOSPHOROUS

PESTICIDES USING MICELLAR ELECTROKINETIC

CHROMATOGRAPHY WITH ACIDIC BUFFER

6.1 Introduction

Organophosphorous pesticides (OPPs) are divided into hydrophobic and

hydrophilic categories. The study focuses on 3 selected hydrophilic OPPs which are

phosphamidon, dicrotophos and monocrotophos whose structures are shown in

Figure 2.3 while their properties are shown in Table 2.3. In this study, optimized

separation parameters such as surfactant concentration, buffer concentration, the

presence of organic modifier, buffering pH and separation voltage have already been

determined from a previous study (Alam, 2005). The main objective of this work is

to reduce the detection limits in order to be applied to environmental samples. From

the previous study, the LOD (S/N) attained was in the sub-ppm level (Alam, 2005).

The detection used was a UV detector set at 230 nm. In the current study a diode-

array detector (DAD) was used at the optimized wavelength (195, 205, 220, 225 and

230 nm).

115

6.2 Reverse Mode Micellar Electrokinetic Chromatography

Separation for the three OPPs were conducted under acidic conditions but

with the help of diode-array detection (DAD). The DAD allowed the analysis of the

three OPPs under five different wavelengths simultaneously in order to find the best

wavelength for the respective analytes which were not possible in the previous study.

Obtaining the best wavelength for all three analytes would greatly improve on the

detection sensitivity of the three analytes.

6.2.1 Wavelength Optimization

The DAD wavelength detection was optimized at several values (Table 6.1).

Irrespective of the wavelength of detection, the migration times of all the pesticides

did not vary greatly. Dicrotophos gave the fastest migration time at 2.30 min

followed by monocrotophos at 6.90 min and phosphamidon at 7.68 min. The

analytes at 100 ppm each were analyzed in mixture form at five wavelengths

simultaneously and the results are summarized as in Table 6.1 while the

electropherograms for the respective analytes at their optimized wavelengths are

shown in Figure 6.1. Phosphamidon gave the longest migration time as it is the most

hydrophobic among the three OPPs thus it complexes strongly with the micelle.

Monocrotophos showed the highest absorbance at 225 nm while dicrotophos and

phosphamidon at 195 nm.

116

Table 6.1: Summary of wavelength optimization for dicrotophos, monocrotophos and phosphamidon. Separation conditions: separation buffer contained 20 mM phosphate (pH 2.3), 10 mM SDS and 10% v/v methanol; samples at 100 ppm each prepared in methanol; applied potential -25kV; hydrodynamic sample injection for 10 s at 50 mbar. Total capillary length, 48.5 cm; effective length, 40 cm.

Dicrotophos Monocrotophos PhosphamidonExperimental wavelength, nm

195 √ √ √ 205 √ √ √ 220 √ √ √ 225 √ √ √ 230 √ √ √

Optimized wavelength, nm

195 √ √ 205 220 225 √ 230

Figure 6.1: Comparison of intensity of (1) dicrotophos, (2) monocrotophos and (3) phosphamidon at different detection wavelengths which are 225 nm and 195 nm under the same running buffer conditions. Separation buffer: 20 mM phosphate at buffer pH 2.3, 10 mM SDS and 10% v/v methanol. Separation wavelength, 225 nm; separation voltage, -25 kV; hydrodynamic injection for 50 s at 50 mbar. Capillary total length, 48.5 cm; effective length, 40 cm.

min

min

117

Figure 6.2 shows the detector response on peak area intensity of dicrotophos,

monocrotophos and phosphamidon at different wavelengths simultaneously. Figure

6.3 shows the peak height intensity of the three analytes at different wavelengths

simultaneously. Analysis were done at different wavelengths simultaneously to find

the optimum wavelength for each analyte. Based on Figures 6.2 and 6.3, different

analytes gave different responses at different wavelengths. Dicrotophos and

phosphamidon gave the best peak area and peak height intensity at 195 nm while

monocrotophos gave the best peak area intensity at 220 nm and peak height at 225

nm. But as peak area is also influenced by peak broadening therefore, in

constructing the calibration graph, 225 nm was chosen as the optimum wavelength

for monocrotophos. Although all OPPs were best measured at different wavelengths,

a compromise in choice of wavelength needs to be made as it would be impractical if

different wavelengths were to be used on systems that do not use diode array

detection. Thus, 225 nm was chosen as the optimized wavelength.

0

10

20

30

40

50

60

dicrotophos monocrotophos phosphamidon

Peak

are

a, m

AUs 195 nm

205 nm220 nm225 nm230 nm

Figure 6.2: The influence of different wavelengths on peak area response for dicrotophos, monocrotophos and phosphamidon. Separation conditions as in Figure 6.1.

118

0

5

10

15

20

25

dicrotophos monocrotophos phosphamidonP

eak

heig

ht, m

AU 195 nm205 nm220 nm225 nm230 nm

Figure 6.3: The influence of different wavelengths on peak height intensity for dicrotophos, monocrotophos and phosphamidon. Separation conditions is similar to Figure 6.2.

6.2.2 Calibration Lines, r2 and LODs

LODs (S/N=3) were calculated based on peak area and peak height using

calibration curves obtained from three standard mixtures (100, 200, and 300 ppm).

The calibration curves based on peak areas and peak heights are shown in Figures 6.4

A and 6.4 B, respectively and their linear regression equations, r2 and LODs are

shown in Table 6.2.

Based on Table 6.2, the linearity and LOD for all analytes based on peak

areas were poorer compared to peak height. There have been some works whereby

peak height is preferred compared to peak area as peak area is more prone to

broadening (Quirino and Terabe, 1997; Quirino et al., 2000a and Carabias-Martínez

et al., 2003). This shows that all analytes are vulnerable to peak broadening. In

addition to that, high LODs may be attributed to lower regression values as compared

to those obtained using peak heights. Therefore in further studies, peak height was

used to construct calibration curves. Monocrotophos gave the lowest LOD (3.75

ppm) while dicrotophos gave the highest LOD (32.1 ppm). The LOD obtained here

during RM-MEKC is not suitable to be used for the analysis of real samples from

water matrix.

119

Table 6.2: Regression equation, r2, LODs (S/N = 3) on the basis of calibration curves in Figure 6.4. Dicrotophos Monocrotophos PhosphamidonPeak Area

Regression Equation

y = 0.0099x + 1.9787 y = 0.2216x + 5.9466 y = 0.1738x + 2.6492

R2 0.9944 0.9657 0.9577 LOD,

ppm 89.1 80.0 89.2

Peak Height

Regression Equation

y = 0.0112x + 2.7425

y = 0.0774x + 5.6633

y = 0.0328x + 2.1086

r2 = 0.9944 = 0.9999 = 0.9981 LOD,

ppm 32.1 3.75 18.6

Figure 6.4: Calibration curves based on (A) peak areas and (B) peak heights for the separation of hydrophilic OPPs in RM-MEKC. Separation conditions remain the same as in Figure 6.3.

6.2.3 Repeatabilities and Efficiency, N

Repeatability in the form of % RSDs for migration time, peak area and peak

height in the separation of dicrotophos, monocrotophos and phosphamidon are given

in Table 6.3. The %RSDs for all analytes for migration time, peak height and peak

area are all lower than % except for the peak area of phosphamidon at 7.24%. Figure

6.5 shows the electropherograms of 100 ppm of the analytes in triplicates.

120

Table 6.3: Intraday %RSD of migration time (min), peak area (mAUs) and peak height (mAU) in the separation of dicrotophos, monocrotophos and phosphamidon at three replicates each at 100 ppm. ANALYTES %RSD, n=3

Migration time (min)

Peak Height (mAU)

Peak Area (mAUs)

Dicrotophos 2.95 4.47 2.96 Monocrotophos 1.06 2.37 1.73 Phosphamidon 1.09 4.72 7.24

Figure 6.5: Electropherogram of (1) dicrotophos, (2) monocrotophos and (3) phosphamidon at 100 ppm each when injected in triplicates. Separation conditions as in Figure 6.4.

Figure 6.6 shows the effect of different concentration on the separation

efficiency of the three analytes. N increases from 100-200 ppm for dicrotophos and

monocrotophos then decreases sharply when concentration increases to 300 ppm.

For phosphamidon, N does not change significantly on increasing concentration.

Efficiency is significantly poor for dicrotophos peak as compared to other peaks. As

all three peaks are well separated, therefore there is the possibility of increasing the

injection volume in order to improve sensitivity which may also result in poorer

efficiency.

min

min

min

121

0

50000

100000

150000

200000

250000

300000

0 100 200 300 400concentration, ppm

Effic

ienc

y, N


Figure 6.6: Variations in efficiency, N in the concentration range for dicrotophos, monocrotophos and phosphamidon using RM-MEKC. Separation conditions of Figure 6.5.

6.3 Stacking With Reverse Migrating Micelles

Stacking with reverse migrating micelles (SRMM) technique employs an

acidic micellar background solution (BGS) to reduce the electroosmotic flow (EOF).

Therefore, the velocity of the micelles are higher than the EOF in SRMM and

samples are prepared in purified water or in low conductivity matrix and injected for

a longer time compared to the normal injection as shown in Figure 6.7. Figure 6.7

illustrates the basic model of sample stacking. Sample stacking occurs as the neutral

analytes complexed with the charged micelles cross a boundary that separates regions

of the high electric field (low conductivity) sample zone and the low electric (high

conductivity) BGS zone. Sample solutions are introduced at the cathodic end of the

capillary and then separation voltage is applied with negative polarity at the injection

end. As the buffer used is in acidic conditions, thus the EOF would be suppressed

greatly which moves towards the anodic end. This creates a push for the sample

matrix to move toward the cathodic end for detection.

122

Figure 6.7: Schematic diagram of the principle of stacking with reverse migrating micelles in MEKC. EOF, electroosmotic flow; SB, stacking boundary.

Sample solutions are introduced hydrodynamically in this experiment at the

cathodic end of the capillary and then separation voltage is applied with negative

polarity at the injection end. In this work, hydrodynamic injection at 50 mbar for 50

s was used instead of 100 s as it was discovered that the LOD at 100 s was higher

compared to at 50 s. In addition to that, there was peak broadening when 100 s was

used.

6.3.1 Calibration Lines, r2 and LODs

LODs (S/N=3) were calculated based on peak area and peak height using

calibration curves obtained from three standard mixtures at a concentration 10 times

lower than the concentrations used in RM-MEKC (10, 20 and 30 ppm). The

calibration curves based on peak areas and peak heights are shown in Figures 6.8 A

and 6.8 B and their respective linear equations, r2 and LODs are shown in Table 6.4.

Three electropherograms from each mixture of OPPs are shown in Figure 6.9.

123

Table 6.4: Regression equation, r2, LODs (for S/N = 3) using SRMM-MEKC based on calibration curves in Figure 6.8. Dicrotophos Monocrotophos PhosphamidonPeak Area

Regression Equation

y = 1.5331x + 21.69 y = 2.3252x - 4.3489 y = 0.8289x + 2.534

r2 0.9926 0.9987 0.9896 LOD,

ppm 3.66 1.50 4.35

Peak Height

Regression Equation

y = 1.5036x + 0.6266

y = 0.5592x + 4.1214

y = 0.2402x + 0.5414

r2 1 = 0.9999 1 LOD,

ppm 0.15 0.39 0.22

Figure 6.8: Calibration curves based on (A) peak areas and (B) peak heights for the separation of hydrophilic OPPs in SRMM-MEKC. Separation buffer: 20 mM phosphate at buffer pH 2.3, 10 mM SDS and 10% v/v methanol. Separation wavelength: 225 nm. Separation voltage: -25 kV. Hydrodynamic injection for 50 s at 50 mbar. Capillary total length: 48.5 cm. Effective length: 40 cm.

Based on Table 6.4, this shows that the linearity and LOD for all analytes

based on peak areas were much poorer as compared to peak height as reported in

Section 6.2.2. This could only be attributed to the lower regression values in

calibration curves using peak areas. Therefore in further studies, calibration curves

based on peak heights was used. Dicrotophos gave the lowest LOD (0.15 ppm)

while monocrotophos gave the highest LOD (0.39 ppm). The LODs remained in the

sub-ppm region thus off-line preconcentration was needed in order to improve on

sensitivity further. The LOD obtained with the current study is from the range of

0.18-0.37 ppm by Alam, 2005 which was insufficient for environmental analysis. In

this work the results improved except for monocrotophos which gave a higher LOD

at 0.39 ppm compared to the previous study at 0.24 ppm. The graph was linear in the

124

concentration range studied (10-30 ppm for the three OPPs). The correlation

coefficient, r2 for peak height is excellent showing good correlation between peak

height and concentration of OPPs (much better compared to peak area).

Figure 6.9: Separation of hydrophilic OPPs using SRMM-MEKC at mixture at (A) 10 ppm, (B) 20 ppm and (C) 30 ppm for (1)dicrotophos, (2) monocrotophos and (3) phosphamidon. Separation conditions as in Figure 6.8.

6.3.2 Repeatability and Efficiency, N

Table 6.5 shows the repeatability (%RSD) of migration time, peak height and

peak area for dicrotophos, monocrotophos and phosphamidon. The repeatabilities of

the three parameters for the three analytes were satisfactory with a value of less than

5%. This shows that SRMM-MEKC under acidic conditions is stable and gives good

reproducibility. A study conducted by Quirino et al. (2000b) that showed SRMM-

MEKC gave poor reproducibility in the range of 5-7% for migration time which was

due to difference in local electroosmotic flow between the sample zone and

background solution (BGS) zone. In this study, this issue did not cause a major

problem in the migration time reproducibility.

125

Table 6.5: Repeatability of migration time (min), peak area (mAUs) and peak height (mAU) in the separation of dicrotophos, monocrotophos and phosphamidon at three replicates each at 10 ppm. ANALYTES %RSD, n=3

Migration Time Peak Area Peak Height Dicrotophos 1.10 4.36 2.14 Monocrotophos 1.40 4.79 4.68 Phosphamidon 1.71 3.72 1.42

Figure 6.10 shows the effect of different concentration on the separation

efficiency of the three analytes. Efficiency worsened significantly for dicrotophos

peak with increasing concentration as compared to the other two peaks. The

efficiency for both monocrotophos and phosphamidon decreased after 20 ppm. This

shows that increasing the injection time further would be detrimental to the

efficiency of these analytes particularly dicrotophos.

0

50000

100000

150000

200000

250000

0 10 20 30 40

concentration, ppm

Effic

ienc

y, N


Figure 6.10: Variations in N in the concentration range for dicrotophos, monocrotophos and phosphamidon using SRMM-MEKC. Separation conditions similar to Figure 6.9.

6.3.3 Stacking Efficiencies (SEF)


and LOD (SEFLOD) were calculated for each test analyte to evaluate quantitatively

126

the degree of stacking to compare between RM-MEKC and SRMM-MEKC. Stacking

efficiencies in the form of sensitivity enhancement factors were calculated using the

ratio of peak heights, peak areas and LODs obtained from SRMM-MEKC and RM-

MEKC which were multiplied with the dilution factors. Here the dilution factor is

10. All three enhancement factors are shown in Table 6.6.

Table 6.6: Sensitivity Enhancement Factors in SRMM-MEKC over RM-MEKC in the separation of dicrotophos, monocrotophos and phosphamidon. Conditions as in Figure 6.10.

ANALYTES SEFheight SEFarea SEFLOD* Dicrotophos 76 132 2140 Monocrotophos 7 9 96 Phosphamidon 7 6 85


SEFarea gave a higher value for dicrotophos and monocrotophos compared to

SEFheight but this implies that with stacking, peaks broadened in SRMM-MEKC

especially for dicrotophos. LODs were calculated based on peak heights as peak

height gave a much better linearity compared to peak areas. Dicrotophos was most

efficiently stacked achieving a stacking efficiency (SEFLOD) of 2140 fold.

Monocrotophos is the least efficiently stacked. This was due to monocrotophos

being the least hydrophobic thus it is the least complexed with the micelle.

127

6.4 Off-line Preconcentration Technique With Solid Phase Disc Extraction

(SPDE)

In the determination of pesticides in water, a sample pretreatment step

includes the analyte enrichment as well as the removal of matrix components. This is

commonly carried out using solid-phase extraction (SPE) in the form of cartridges

and disc devices. SPE benefits from low intrinsic costs, shorter processing times, low

solvent consumption, simpler processing procedures and easier automation. SPE has

been commonly used in pesticide multiresidue analysis such as in fruits and

vegetables, eggs and wine with gas chromatography detection methods. There have

also been coupling of SPE with on-line stacking methods in capillary electrophoresis

in order to lower the detection sensitivity especially in environmental analysis of

pesticides using SPE catridges (Silva et al., 2003 and Süsse and Müller, 1996). In

this study, solid phase extraction in the form of discs was used in these two step

enrichment process of dicrotophos, monocrotophos and phosphamidon with the use

of SRMM-MEKC as an online stacking method.

The maximum concentration of individual pesticides in drinking water

allowed by the European Community legislation is 0.1 µg/L. Therefore, analyte

preconcentration prior to MEKC determination is required in order to reach

sensitivity below the legal limits. To provide a reference value, blank pond water

was passed through the SPE discs too and no peak signals were detected. Figure 6.11

shows the electropherograms of blank runs and spiked pond water at 1 µg and 0.5 µg

into 250 mL of pond water. The concentration of the analytes once diluted in 250

mL of pond water is 4 ppb and 2 ppb respectively. The enrichment factor used is 250

times thus the final concentration when reconstituted to 1 mL of water before CE

analysis is 1 ppm and 0.5 ppm.

Based on the recovery studies as stated in Table 6.7, the spiking at higher

concentration (1.0 µg) gave better recovery for the three analytes in the range of

(96.2-132)%. While at 0.5 µg the recovery was lower in the range of (74-117)%.

Based on the results, this proves that SPDE is sufficient for quantitative analysis at

low concentrations as after being diluted in 250 mL of water, the concentration of the

128

spiked analytes were in the ppb range (2-4 ppb). The repeatability of the extraction

was higher for lower concentrations in the range of (10-12)%RSD as compared to

higher concentration within (0.81-5.36)%RSD. The preconcentration factor

calculated from spiking into 250 mL of pond water to reconstituting to 1 mL is 250.

Thus the LOD for the three analytes has been reduced by 250 times for dicrotophos

(0.6 ppb), monocrotophos (1.56 ppb) and phosphamidon (0.88 ppb). The limit

attained with off-line SPDE in combination with SRMM-MEKC is still not sufficient

for OPPs analysis for water samples set by European Commision (0.1 µg/L).

However, this is the first study on hydrophilic OPPs using SRMM-MEKC and

further studies in reducing the LOD needs to be investigated but beyond the scope of

the current work.

Table 6.7: Recovery and repeatability of extraction Congener Spiked

amount, µg Mean, µg Recovery (%) Repeatability

(%RSD) Dicrotophos 0.50 0.37 74.0 10.0 1.0 0.96 96.2 4.7 Monocrotophos 0.5 0.56 112.0 12.0 1.0 1.04 104.4 0.81 Phosphamidon 0.5 0.59 117.0 10.9

1.0 1.32 132.0 5.36

129

Figure 6.11: Electropherograms of extracted blank pond water (A) and of dicrotophos (1), monocrotophos (2) and phosphamidon (3) at (B) 0.5 µg and (C) 1 µg. Separation buffer: 20 mM phosphate at buffer pH 2.3, 10 mM SDS and 10% v/v methanol. Separation wavelength, 225 nm; separation voltage, -25 kV; hydrodynamic injection for 50 s at 50 mbar. Capillary total length, 48.5 cm and effective length, 40 cm.

A

B

C

130

CHAPTER VII

CONCLUSIONS AND FUTURE DIRECTIONS

7.1 Conclusions

MEKC separations of the TCDF and TCDD were successfully performed with

the running buffer of 20 mM di-sodium tetraborate decahydrate buffer containing 20

mM of sodium cholate as the chiral surfactant with 5% v/v mixed modifier MeCN-

MeOH (3:1). It is noteworthy to mention that baseline separation was achieved with a

total analysis time of below 8 min with an optimum wavelength at 225 nm, separation

voltage at 25 kV with hydrodynamic injection for 10 s at 50 mbar. This is the first ever

study conducted specifically on TCDF and TCDD using MEKC with DAD. Although

literature review states that sodium dodecyl sulphate (SDS) as the surfactant with

phosphate as the running buffer is sufficient for good separation, yet it was not achieved

in this study. Instead SDS failed to provide any separation of the two analytes.

Phosphate was also investigated as the running buffer during our preliminary

investigation but in terms of baseline stability and peak intensity, borate performed

superior to phosphate. Based on the influence of different sample matrices, the analytes

were then prepared in water for further optimization as it gave a superior intensity and

baseline stability as compared to buffer matrix and organic matrix (in ethanol).

131

After obtaining the optimized conditions from optimization of parameters which

include organic modifier content and sample matrix, on-line preconcentration techniques

were then carried out. A summary of the on-line preconcentration techniques LODs

were shown in Table 7.1. While Table 7.2 shows the %RSD of migration time, peak

area and peak height for both analytes using the selected on-line preconcentration

methods used. Five on-line preconcentration techniques were used which were normal

stacking mode (NSM-MEKC), reversed electrode polarity stacking mode (REPSM),

high conductivity sample stacking mode (HCSSM), sweeping and field-enhanced

sample injection (FESI-MEKC). Overall, HCSSM gave the best performance to be

applied to water samples giving an LOD of 46 ppb for TCDF while for TCDD it was

18.5 ppb. Korytár et al. obtained a LOD of 70 fg for TCDF while for TCDD was at 90

fg using gas chromatography (Korytár et al., 2004). As stated by the EPA Method 613,

the LOD for TCDF and TCDD were 0.002 ppb thus, in order to achieve that level of

detection, off-line concentration techniques using solid phase disc extraction (SPDE) is

needed for sample enrichment. The detection limits was reduced by 1000 times after

reducing 1 L of deionized water to 1 mL, thus achieving an LOD of 46 ppt and 18.5 ppt

for TCDF and TCDD, respectively. These limits are still above the detection limits set

by the EPA using gas chromatography. As this was the first study conducted on the

usage of MEKC to the detection of these two specific congeners, the results achieved

appear to be promising if compared to the results achieved thus far for TCDD and TCDF

using MEKC which was only in the sub-ppm range (Otsuka et al., 1999). The combined

method using HCSSM-SPDE achieved 46 000 fold SEF for the LOD of both analytes.

Table 7.1: LODs (for S/N = 3) obtained using the three online preconcentration techniques as compared to normal mode (NM) for TCDF and TCDD.

ANALYTES LODNM (ppb)

LODNSM (ppb)

LODREPSM (ppb)

LODHCSSM (ppb)

LODHCSSM-

SPDE (ppt) TCDF 2200 340 130 46 46 TCDD 1710 50 3 18.5 18.5

132

Table 7.2: Repeatabilities for migration time, peak area and peak height for both analytes using the three online preconcentration techniques as compared to normal mode (NM) for TCDF and TCDD. Sweeping and FESI were not successful in enhancing the detection of both analytes.

%RSD, n=3 TCDF TCDD NM Migration

Time 0.16 0.58

Peak Area 3.70 4.36 Peak Height 10.08 3.08

NSM Migration Time

0.50 0.34


REPSM Migration Time

0.12 0.32


HCSSM Migration Time

0.29 0.12


Hydrophilic organophosphorous pesticides which consisted of dicrotophos,

monocrotophos and phosphamidon were successfully separated using acidic buffer

consisting of 20 mM sodium dihydrogenphosphate, 10 mM sodium dodecyl sulphate

and 10% v/v methanol using as reverse mode MEKC (RM-MEKC). Baseline separation

was achieved for the three analytes in less than 10 min which was much shorter

compared to the previous study which achieved an analysis time of below 30 min

(Alam, 2005). The separation voltage applied was -25 kV at detection wavelength 225

nm. This was due to the shorter capillary used in this study. The LODs using RM-

MEKC obtained were 32.1 ppm (dicrotophos), 3.75 ppm (monocrotophos) and 18.6 ppm

(phosphamidon). According to the European Community legislation for the detection

limits for OPPs is 0.1 µgL-1 in water samples.

133

In order to achieve lower detection limits, on-line preconcentration techniques

was needed. Stacking with reverse migrating micelles (SRMM) at a longer

hydrodynamic sample injection time of 50 s at 50 mbar was chosen. Higher injection

times were not chosen as there were peak broadening and reduction in peak efficiency.

In SRMM, the samples were dissolved in water to enhance stacking by providing a low

conductivity zone. The usage of stacking has been able to reduce the LODs by varying

stacking efficiencies with dicrotophos being stacked most efficiently achieving an

SEFheight of 76. The SEFheight obtained for the three OPPs were in the range of 7-76. In

order to enhance detection sensitivity, solid phase disc extraction (SPDE) was employed

to provide sample enrichment and to extract out the analytes from pond water sample

obtained from UTM Lake. Natural water sample was chosen as these pesticides are

mostly found in water due to their high hydrophilicity. With a sample enrichment factor

of 250, the detection limits were successfully improved with reasonable recoveries

ranging from (74-132)%. The LODs achieved were 0.6 ppb for dicrotophos, 1.56 ppb

for monocrotophos and 0.88 ppb for phosphamidon when applied to real water sample.

More work is required in order to achieve the stipulated detection limits in for

application in real sample analysis thus making capillary electrophoresis as a method

comparable to high performance liquid chromatography and gas chromatography.

Overall, this study has shown that with MEKC it is possible to achieve detection

limits of ppt range for TCDF and TCDD and ppb range for the three OPPs. This is an

important development as it shows with further developments, MEKC can be

established as alternative methods to gas chromatography (GC) and high performance

liquid chromatography (HPLC) for environmental analysis. Furthermore, with the

flexibility it offers in optimizing various parameters to enhance separation, MEKC is

more economical compared to GC and HPLC. Continuation of off-line SPDE reduce

the LOD 1000 fold for TCDF and TCDD and 250 fold for the three hydrophilic OPPs.

134

7.2 Future Directions

As the coupling of SPDE and HCSSM-MEKC is the first method conducted on

the analysis of TCDF and TCDD, it is advisable to devise an extraction method that is

more economical and environmentally friendly. This is because the SPDE used in this

method requires large volumes of organic solvents which would not be economical in a

commercial lab. Thus, an alternative extraction method such as single drop

microextraction (SDME) or solid phase microextraction (SPME) that does not require

large volume of organic solvent would be investigated in the future. In addition, Z-

shaped detection cells or high sensitivity capillaries could be used in improving the

detection sensitivity of polychlorinated compounds and the three hydrophilic

organophosphorous pesticides. As for both analytes, water samples were used as the

real sample. The analysis can be extended by using biological fluids such as blood and

animal fatty tissue as the real sample. This is important especially for the

polychlorinated compounds which are highly hydrophobic and thus reside more in

animal fat rather than being excreted into the urine. For the analysis of hydrophilic

OPPs, different online concentration methods such as high salt stacking and non-

aqueous capillary electrophoresis (NACE) could be investigated in their response

towards the detection of the three hydrophilic OPPs. This research can also include

various types of pesticides and organic pollutants with varying hydrophobicities.

Further research and development is required as the usage of MEKC involving

polychlorinated compounds and hydrophilic OPPs is scarce.

135

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152

APPENDICES

LIST OF PUBLICATIONS

1. Wan Aini Wan Ibrahim, Sharain Liew Yen Ling, Mohd Marsin Sanagi, “Organic

Modifier Influence on the Separation of 2,3,7,8-TCDD and 2,3,7,8-TCDF using

Micellar electrokinetic Chromatography”, Buletin Kimia, 22, 2006, 1-10 (ISSN

0127-8711).

2. Wan Aini Wan Ibrahim, Sharain Liew Yen Ling, Mohd Marsin Sanagi, “Sample

Matrix Effect on the Separation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

and 2,3,7,8-tetrachlorodibenzo-p-furan TCDF) using Micellar Electrokinetic

Chromatography”, ACGC Chemical Research Communications, 2007, 21, 30-

33.

3. Wan Aini Wan Ibrahim, Sharain Liew Yen Ling, Mohd Marsin Sanagi,

“Analysis of dioxin and furan related compounds and organophosphorous

pesticides using micellar electrokinetic choramtography (MEKC)” (under

preparation for submission to J. Chromatogr. A.).

153

LIST OF PRESENTATIONS

1. Wan Aini Wan Ibrahim, Sharain Liew Yen Ling, Mohd Marsin Sanagi, “On-line

preconcentration in micellar electrokinetic chromatography for analysis of

dioxin-related compounds.” 30th International Symposium On Capillary

Chromatography. Dalian, China. 5th-7th June 2007.

2. Wan Aini Wan Ibrahim, Sharain Liew Yen Ling, Mohd Marsin Sanagi,

“Preliminary Investigation on the Separation of 2,3,7,8-TCDF and 2,3,7,8-TCDD

using Micellar Electrokinetic Chromatography (MEKC).” Poster presentation at

the 18th Simposium Kimia Analisis Malaysia (SKAM-18). Universiti Teknologi

Malaysia, Skudai, Johor, Malaysia. September 12-14, 2005.

Documents

VOT 75149 A NOVEL CAPILLARY … · vot 75149 a novel capillary electrophoretic method for the analysis of dioxins and furans (kaedah novel elektroforesis rerambut untuk analisis dioksin