DETERMINATION OF BISPHENOL A IN ENVIRONMENTAL WATER SAMPLES BY ON-LINE NANOFIBROUS EXTRACTION WITH COLUMN-SWITCHING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY By LOH THUNG YING S45379 A research method proposal submitted in partial fulfilment of the requirements for the award of the degree of Bachelor of Science

DETERMINATION OF BISPHENOL A IN ENVIRONMENTAL WATER SAMPLES BY ON-LINE NANOFIBROUS EXTRACTION WITH COLUMN-SWITCHING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

By
LOH THUNG YING
S45379

A research method proposal submitted in partial fulfilment of
the requirements for the award of the degree of
Bachelor of Science (Analytical and Environmental Chemistry)

SCHOOL OF MARINE SCIENCE AND ENVIRONMENTAL
UNIVERSITY MALAYSIA OF TERENGGANU
2018
TABLE OF CONTENTS
PAGE
TITLE PAGE i
CONTENT ii
LIST OF TABLES iv
LIST OF FIGURES v
LIST OF ABBREVIATIONS vi

CHAPTER 1 INTRODUCTION
1.1 Research Background 1
1.2 Problem Statement 2
1.3 Research Objectives 3
CHAPTER 2 LITERATURE REVIEW
2.1 Bisphenol A 4
2.1.1 Physical Properties and Chemical 4
Properties of Bisphenol A
2.1.2 Human Exposure and Toxic 6
Effects of Bisphenol A
2.1.3 Bisphenol A Exposure to Environment 6
2.1.4 Legislation of Bisphenol A 7

2.2 Extraction Technique 7
2.2.1 Liquid-liquid Extraction (LLE) 7
2.2.2 Solid-phase Extraction (SPE) 8
2.2.3 Solid Phase Microextration (SPME) 8
2.2.4 Liquid-phase Microextraction (LPME) 8

2.3 Analysis Technique 9
2.3 Analysis Technique 9
2.3.1 Liquid Chromatography 9
2.3.1.1 High Performance Liquid 9
Chromatography with Fluorescence
Detection (HPLC-FLD)
2.3.1.2 High Performance Liquid 10
Chromatography with Mass
Spectroscopy (HPLC-MS)
2.3.2 Gas Chromatography 10

2.4 Summary of Past Reports on Bisphenol 11
A Analysis

CHAPTER 3 METHODOLOGY
3.1 Chemicals and Reagents 12
3.2 Apparatus and Instrumentation 12
3.3 Preparation of Standard Solution 13
3.4 Preparation of Sample 13
3.5 Preparation of Electrospun Nanofibers 13
3.6 Preparation of Nanofibrous Extraction 13
Pre-columns
3.7 UHPLC Column-switching 14
3.8 Optimization and Validation of 14
On-line SPE-UHPLC
Nanofibrous Extraction

CHAPTER 4 EXPECTED RESULTS
4.1 Expected Result 15
REFERENCES 16
LIST OF TABLES

Table No. Page
Table 2.1 Physical and Chemical properties of Bisphenol A 5
Table 2.2 Summary of Past repost in Bisphenol A Analysis 11

LIST OF FIGURES

Figure No. Page
Figure 2.1 Molecular structure of bisphenol A 5
Figure 2.2 Synthesis of BPA 5
Figure 2.3 Fluorescence spectra for BPA in 1 10

LIST OF ABBREVETIONS

Abbreviation
BPA Bisphenol A
DDLME-SFO liquid-liquid phase microextraction based on solidification of floating organic
DLLME Liquid-liquid Microextraction
EPA Environmental Protection Agency
FDA Food and Drug Administration
GC Gas Chromatography
HF-LPME Hollow Fibre Liquid-phase Microextraction
HPLC High Performance Liquid Chromatography
HPLC-FLD High Performance Liquid Chromatography with Fluorescence Detection
HPLC-MS High Performance Liquid Chromatography with Mass Spectroscopy
LC-MS Liquid Chromatography-Mass Spectrum
LLE Liquid-liquid extraction
LOD Limit of Detection
LOQ Limit of Quantification
LPME Liquid-phase Microextraction

MISPE-CE Molecularly Imprinted Solid Phase Extraction-Capillary
PA6 Polyamide 6
PTFE Polytetrafluoroethylene
RSD Precision Values
SD-LPME Single-drop Liquid-phase Microextraction
SPE Solid phase extraction
SPE-UHPLC Solid Phase Extraction- Ultra-High Performance Liquid Chromatography
SPME Solid-phase Microextraction
UHPLC Ultra-High Performance Liquid Chromatography

CHAPTER 1

INTRODUCTION

1.1 Research Background
Bisphenol A (BPA) is used in the production of polycarbonate plastics and epoxy resins. BPA is produced in large amount (U.S. Environmental Protection Agency (EPA), 2014). Moreover, daily items are mainly contains BPA (Rochester, 2013).
Most people are exposed to BPA through their diet and this is due to present of BPA in food and beverages (National Institute of Environmental Health Sciences, 2010). This make human easily expose to BPA. In addition, inhalation and transdermal can lead to exposure to BPA (Konieczna, Rutkowska & Racho?, 2015). Moreover, the BPA source of contamination can happen through wastewater, air, dust and soil (Valentino et al., 2016).
BPA can be leach into our food and water supplies as it is function to coat the lining inside of food and drinks cans. BPA can bring many health problems to human. BPA is an endocrine disruptor and it can influence secretion, transportation and elimination of natural hormones. It can lead to reproductive disorders which may lead to infertility (Nordqvist, 2017).

There are several common methods to extract BPA from environmental water samples. Liquid-liquid extraction (LLE) and solid-phase extraction are regularly been used to extract BPA. Both of these methods involved very complicated procedure, time-consuming and used of large quantities of organic solvents. (Sarafraz-Yazdi & Amiri, 2010).
There are some new methods introduced to compensate for the less-ideal behaviour of LLE and Solid phase extraction (SPE). Dispersive liquid-liquid microextraction (DLLME) and solid-phase microextraction (SPME) are used to meet the green chemistry requirements. These methods have some advantages include fast, inexpensive, easy to operate and consumes low volume of organic solvents (Rezaee, Yamini & Faraji, 2010).

1.2 Problem Statement
At present, people are concerned about BPA because BPA can migrate to human in various ways such as through food, beverages, environmental water and even baby bottles. Human exposure to BPA will have higher chance to get heart disease, diabetes, and reproduction defects. Newborns can have reduced birth weight and abnormal development of brain function. Besides, BPA can affect human’s endocrine function. The instability, insensitivity and low accuracy of instruments have made it difficult to screen BPA. Therefore, a new method should be developed to compensate for the inadequate of previous methods such as LLE and SPE.

1.3 Research Objectives
i To develop an on-line Nanofibrous extraction couple with column-switching high performance liquid chromatography for the extraction of bisphenol A in environmental water samples.
ii To apply on-line nanofibrous extraction for the extraction of bisphenol A in environmental water samples.
iii To determine present of bisphenol A in various environmental water samples.

CHAPTER 2

LITERATURE REVIEW

2.1 Bisphenol A
Bisphenol A (BPA) is a chemical that is used to manufacture many commercial products which include food container and hygiene products (Petre, 2016). BPA is in dental sealants, water bottles, the inside lining of canned foods and drinks, compact discs, medical devices and many other daily products. There is a high possibility for human to take in BPA through diet, air, water and dust (Micha?owicz, 2014). Moreover, BPA is best known for its application in producing polycarbonate plastics and epoxy resins which include those in beverage cans and baby bottles. The occasion of using epoxy resins is to avoid corrosion and stay inert (Rogers, n.d).

2.1.1 Physical Properties and Chemical Properties of Bisphenol A
Bisphenol A or 4, 4′-Isopropylidenediphenol is commonly known as BPA is a white powder with mild odour. BPA with molecular formula of C15H16O2 contain two phenol functional groups. Besides, it has become a difunctional building block of various important plastics and plastics additives (“Bisphenol A,” 2005). BPA is one of
the largest quantities chemical in the world (Rasheed, 2014). The figure 2.1 showed the molecular structure of bisphenol A (Vandenberg et al., 2009).

Figure 2.1: Molecular structure of bisphenol A
BPA is first synthesized from phenol and acetone through condensation process. This process requires a strong acid to act as catalyst such as hydrochloric acid (Neagu, 1998). The figure 2.2 showed the synthesis process of BPA (Negau, 1998). The physical and chemical properties of bisphenol A is listed in table 2.1 (Rasheed, 2014).
Figure 2.2: Synthesis of BPA
Parameter Value
Formula C15H16O2
Molecular Weight 228.29 g/mol
Boiling Point 220 °C(at 4mm Hg)
Melting Point 150-155°C
Specific Gravity 1.195 at 25°C
Octanol/ Water Partition Coefficient log Kow = 3.32
Water Solubility 200°C
Table 2.1: Physical and Chemical properties of Bisphenol A
2.1.2 Human Exposure and Toxic Effects of Bisphenol A
There are numerous ways that human can expose to BPA, particularly from food and beverage containers made with BPA. BPA is widely used to coat the surface of cans whereas polycarbonate is used in food and beverage containers (Rasheed, 2014). Migration of BPA from metallic cans to human is mainly affected by the storage time and temperature (MUNGUIA-LOPEZ, 2002). Moreover, trace amount of BPA may leach from the lining of the cans to the food due to heating process for shipping (Takao et al., 2002). The other possible route of exposure to BPA is through inhalation due to there is detectable amount of BPA in indoor air correlated with dust (Taylor, Welshons & Saal, 2008). According to the researcher, BPA is present in human’s urine, tissues, breast milk and even follicular fluid (Vandenberg, n.d).
BPA is an endocrine disruptor. BPA can act as antagonist which can blocks the reaction of the natural ligand. Children and babies face the greatest danger of the endocrine disruptor due to low biological resistance. Moreover, BPA can inhibit some enzyme activity which subsequently affects human health (Preethi, 2014). BPA can prohibit the pathway of thyroid hormone which may affect human embryogenesis and neonatal development. This happened when the thyroid hormone binds to BPA and function as an antagonist (Rubin, 2011). Furthermore, BPA is trusted of causing hormone-related problems such as early puberty in girls and also birth defects in human (Chouhan, 2012). A study revealed that BPA level in the urine of normal people is significantly higher than the control group of people towards direct contact with BPA products. Researcher suspected that disruptive role of BPA will contribute to early puberty and premature breast development (Leonardi et al., 2017).

2.1.3 Bisphenol A Exposure to Environment
Bisphenol A does not naturally produce in environment. The sources of environmental BPA can be categorized as preconsumer and postconsumer items. Preconsumer source contain materials that used to manufacture BPA-containing products. On the other hand, postconsumer source is corresponds to discharge from municipal wastewater, break down of plastics and leaching from the landfills (Flint et al., 2012). Rivers, lakes and ocean are the major deposition place for BPA. This brings adverse negative impacts to aquatic organisms. From a research study, the researcher claimed that the main route of exposure for the fish is not through their diet, but inhalation through the gills (Kang, Asai, ; Katayama, 2007). Hence, it is not astounding that BPA will continuously affect the fish by estrogenic effects. There are also evidence in showing BPA has remarkable effect on antagonistics activity towards endocrine disruptor (Mitsui, Tooi, ; Kawahara, 2007).

2.1.4 Legislation of Bisphenol A
Matters over BPA’s toxic effects to human health brings the government of Canada become the first throughout the world to classify BPA is hazardous and ban it in manufacturing baby’s bottles (“Regulation,” n.d). Besides, European Union legally prohibited sales of BPA-containing baby’s bottles. In 2003, France has banned BPA in children products. France has started to ban BPA on food packaging in 2015. It is stated that the U.S. Food and Drug Administration (FDA) prohibits the use of BPA in manufacturing children and baby’s items in 2012. In addition, infant formula packaging materials is banned from using BPA in 2013 (“Regulations,” 2017). China and Malaysia have joined to the team to ban BPA to protect their citizens (Bardelline, 2011).

2.2 Extraction Technique
There are many methods can be used to extract BPA from environmental water sample.

2.2.1 Liquid-liquid Extraction (LLE)
Liquid-liquid extraction (LLE) is most commonly used extraction method to extract analyte from water sample. LLE use the principal of different solubility in two different liquid solvents usually water and organic solvent to separate analytes in interested sample (Peake, 2016). The sample is commonly dissolves in ethyl acetate, chloroform or dichloromethane. This may due to adequate sensitivity of result provided from this method. However, LLE can cause a change in actual concentration of the analyte in the sample (Liao ; Kannan, 2012).

2.2.2 Solid-phase Extraction (SPE)
Solid phase extraction (SPE) is commonly used to replace liquid-liquid extraction (LLE). This is due to SPE has numerous advantages including low consumption of organic solvents, easy to operate and high recovery (Zhao et al., 2009). The principle of SPE is to increase the concentration of targeted analytes in aqueous phase which having very low concentration. Solid phase is used to absorb the target analytes then a small volume of solvent such as methanol and acetone is used to desorb the analytes (Cunningham, 2014).

2.2.3 Solid Phase Microextration (SPME)
Solid phase microextraction (SPME) can extract the target analyte onto a solid support (Razaee, Yamini ; Faraji, 2009). This method is simple, rapid and portable. This is follow by introduction of different geometries of SPME such as fiber, stir bar, membrane, magnetic nanoparticles and thin film microextraction (Piri-Moghadam, Ahmadi ; Pawliszyn, 2016). Stir bar sorptive extracation (SBSE) is one of the green microextration. SBSE is used due to its simplicity, selectivity and sensitive especially in detecting BPA. In addition, SBSE is mainly coupled with high performance liquid chromatography (HPLC) (Lin et al., 2010). On the other hand, a fiber-packed needle extraction is developed and it has higher efficiency than classical sample preparation methods such as LLE and SPE (Ogawa et al., 2009).

2.2.4 Liquid-phase Microextraction (LPME)
Liquid-phase microextraction (LPME) is an organic-free extraction and it is known as alternative green microextraction. LPME is mainly used to extract analytes from volatile and semivolatile ionisable sample. Moreover, LPME is very efficient method and affordable technique (Zhang, Su ; Lee, 2005). There are mainly three categories of LPME such as single-drop liquid-phase microextraction (SD-LPME), hollow fibre liquid-phase microextraction (HF-LPME) and dispersive liquid-liquid microextraction (DLLME). SD-LPME is an unstable method and sample may be lost because this extraction method is based on a hanging drop (Liu ; Dasgupta, 1996). Next, HF-LPME is introduced by Pedersen-Bjergaard and Rasmussen by using the principle of using polypropylene hollow fibers as membrane (Pedersen-Bjergaard ; Rasmussen, 1999).Dispersive liquid-liquid extraction (DLLE) is a microextraction technique introduced to compensate for the high usage of organic solvent in classical extraction technique (Hong et al., 2017). DDLE has several advantages include environmental-friendly sample preparation, fast, inexpensive, easy to operate and high recoveries (Rezaee, Ya mini ; Faraji, 2010). After that, dispersive liquid-liquid phase microextraction based on solidification of floating organic (DDLME-SFO) is developed to replace DLLE by using less toxic solvents which can solidify at low temperature for easy sample extract (Hong et al., 2017). In addition, DDLME-SFO is a time dependent method due to its non-exhaustive properties.

2.3 Analysis Technique
Trace amount of bisphenol A (BPA) can be determine by high sensitivity, selectivity instrument.

2.3.1 Liquid Chromatography
Reversed-phase C18 column is commonly used to detect trace amount of BPA. Reversed phase is when a non-polar stationary phase is used simultaneously with a polar mobile phase (Driskell, 2003).

2.3.1.1 High Performance Liquid Chromatography with Fluorescence Detection (HPLC-FLD)
Emission and excitation wavelength are important factors to increase the sensitivity of instrument. BPA shows optimal excitation in 230nm. In addition, 305nm is the highest peak for emission wavelength (Xiong et al., 2017). The fluorescence spectra are show in figure 2.3 (Ballesteros-Gómez, Rubio ; Pérez-Bendito, 2009).

Figure 2.3: Fluorescence spectra for BPA in 1

2.3.1.2 High Performance Liquid Chromatography with Mass Spectroscopy (HPLC-MS)
HPLC-MS need injection of large amount of sample. Hence, this will subsequently affect the cartridges and decrease the sensitivity of mass spectroscopy (Li ; Franke, 2015). Mass spectrometry is always used due to it can provide fast and accurate result in a short of time. Furthermore, MS can detect even the sample is at low concentration (Battal et al., 2014). Therefore, it is frequently as a coupled method to determine BPA in sample.

2.3.2 Gas Chromatography (GC)
GC needs derivatization step compared to LC, but this make it more selective and sensitive (Jurek ; Leitner, 2017). Derivatization makes the target analytes more selective and sensitive by improving its volatility and thermal stability (Jeannot et al., 2002). Separation and detection for BPA uses GC in order to be improved.

2.4 Summary of Past Reports on Bisphenol A Analysis
The table shows analysed data from several researchers regarding bisphenol A (BPA).
Sample Matrix Method Linearity Range LOD Reference
Milk HPLC-FLD 10-100µg/kg 3.1µg/kg Xiong et al., 2017
Waste water SPME 0.34-195ng/mL – Reza et al., 2017
Water GC-MS – 99% purity) will be purchased from Sigma-Aldrich, Chromasolv methanol and Chromasolv acetonitrile. Ultra-pure water will be purified by Mili-Q (Millipaore, Bedford, MA, USA). Nylon 6 will be purchased from BASF (Prague, Czech Republic).

3.2 Apparatus and Instrumentation
A Nexera X2 UHPLC system (Shimadzu Corporation, Kyoto, Japan) will be provided with LC-30AD solvent delivery system, a DGU-20 A5R degassing tube, a SIL-30AS autosampler, a CBM-20A module and will be attached to an SPD-M30A DAD and RF-10AXL detector. In addition, a CTO-20AC column oven and a FCV-12AH high-pressure six-port switching valve. The evaluation of data will be accomplished by Shimadzu LC Lab Solution software version 5.57 (Shimadzu Corporation, Kyoto, Japan). A Nanospider NS1WS500U (Elmarco, Czech Republic) laboratory machine and patented technology will be used to developed the nanofibers.
3.3 Preparation of Standard Solution
A standard solution will be prepared by dissolving bisphenol A in acetonitrile which have concentration of 1000mg/L. The standard solution will be stored at 40C in the dark.

3.4 Preparation of Sample
Water samples will be stored in the glass bottles at 40C. Samples will be filtered by 0.45µm PTFE syringe filters before analysis.

3.5 Preparation of Electrospun Nanofibers
Polyamide 6 (PA6) will be dissolved in a mixture solution of formic acid and acetic acid (1:2 v/v) at 12 wt% concentration of PA6. Electrospinning is caused by a nanospider. The temperature and humidity level during electrospinning are 32% and 22.10C respectively. Nanofibers will be collected on nonwowen which caused a motion at the collecting electrode.

3.6 Preparation of Nanofibrous Extraction Pre-columns
About 40g of PA6 nanofibers will be packed into a column cartridge (5 x 4.6 mm) and then will be fixed to the guard pre-column holder. UHPLC fittings will be used to connect the extraction pre-column to the system. 100% acetonitrile will be used to activate the sorbent for 15 minutes with an increasing flow rate. Then, wash with water for 5 minutes at flow rate of 1 mL min-1.

3.7 UHPLC Column-switching
Pre-column with nanofibers will be used for extraction of samples. Supelco Ascentis® Express C18 (10 cm x 4.6 mm) column with 5µm of particle size will be used for separation. Water and methanol 95:05 (v/v) will be used as washing mobile phase. Water (solvent A) and acetonitrile (solvent B) will be used as gradient elution mobile phase.
50µL sample solution will be injected into the pre-column. The washing mobile phase will be used as a clean-up for the column. Pre-column will be washed for 1 minute at a flow rate of 1 mL min-1. The analytical column will be equilibrated to the initial conditions. BPA will be preconcentrated on the pre-column. Then, BPA will be eluted onto analytical column from the pre-column. The analysis will begin at 50% B. The concentration will be changed to 60% when 2 minutes is reached. Thereafter, concentration will be changed at 0.5 minute to 100% B. At 3.0rd minutes, the equilibration of the analytical column will be switched back to its initial conditions. The analytical and extraction column’s temperature will be set at 350C. The fluorescence detector will set for an excitation wavelength at 225 nm and an emission wavelength at 320 nm to detect BPA. 4.30 minutes will be used to run all the steps include extraction.

3.8 Optimization and Validation of On-line SPE-UHPLC Nanofibrous Extraction
Several extraction parameters will be thoroughly investigated to enhance the extraction efficiency of the proposed extraction technique, an on-line SPE-UHPLC nanofibrous extraction. The parameters include composition and the flow rate of the washing mobile phase, the time taken of the extraction step, stability of nanofibers and column packing. This method is then assessed for linearity, relative recovery and reproducibility, limit of detection (LOD), limit of quantification (LOQ) and precision values (% RSD).

CHAPTER 4

EXPECTED RESULTS

4.1 Expected Results
A new method which is to use polyamide 6 nanofibers as solid phase will be developed to be coupled with column switching method in order to determine bisphenol A in environmental water samples. The parameters that will affect the extraction efficiency include column packing process and the amount of nanofiber polymer. This research will purpose a multiple reuse nanofibrous polymer in high pressure system. BPA level for water samples will be detected. The results will have high recovery, good precision and low limit of detection.