Extended Essay Chemistry by Andrew Brown

EXTENDED ESSAY: Measuring the Effects of Temperature, Microwaves and Acidity on Bisphenol A Leaching

RESEARCH QUESTION: How much Bisphenol A (BPA) leaches into water from a polycarbonate Rubbermaid Bouncer速 Pitcher when treated with different temperatures or microwaves; does the acidity of the liquid affect the amount BPA leached?

NAME: IB CANDIDATE NUMBER: RESEARCH SUPERVISORS: Mauricio Albrizzio John Teare, Ph. D. Principal Process Development Scientist

DISCIPLINE OF ESSAY: CHEMISTRY WORD COUNT: 3972

Abstract Bisphenol A is a phenolic chemical used in the manufacturing of polycarbonate plastics, which are present in everyday household items such as plastic bottles and food containers. However, the production process of polycarbonate plastics usually results in traces of non-reacted BPA, which have been shown to leach from the plastic into its contents and be ingested by humans. Recent experiments link the ingestion to many health concerns. Studies suggest that the amount of BPA leached depends on different conditions; this essay investigates how much BPA leaches into water from a polycarbonate Rubbermaid BouncerÂŽ Pitcher when treated with different temperatures or microwaves; does the acidity of the liquid affect the amount BPA leached? To determine the presence of leached BPA, an experiment was designed in which both control and experimental liquid samples were produced and then tested using high performance liquid chromatography. Standard solutions were mixed and test samples were exposed to temperature, microwave radiation, and acidity in the pitcher. The standard solutions were used to determine the retention time for BPA and produce a standardization curve correlating peak area to BPA concentration, which was then used to determine the concentration of leached BPA in the test samples. The results indicate that traces of BPA (11.2ng/mL) leach from the polycarbonate at room temperature into water as well as vinegar. A higher temperature gave (up to) a 157% increase of the concentration leached BPA. Microwave radiation also promoted the dissolution into water, giving an increase of 580%. This suggests that polycarbonates become unstable when exposed to heat and, especially, microwaves; this causes the chain of polycarbonate monomers to separate partly, releasing traces of BPA. The acidic vinegar significantly encouraged leaching only at high temperatures. However, even the highest concentration found, 76.2ng/mL, would be deemed harmless compared to the EPA's reference dose (50Âľg/kg/day).

(Word Count: 300) ii

Table of Contents Research Question:.............................................................................................................1 Introduction:.......................................................................................................................1 Bisphenol A:....................................................................................................1 Polycarbonate:................................................................................................2 Leaching/Health Concerns:...........................................................................3 High Performance Liquid Chromatography (HPLC):..................................6 Experimental Details:.........................................................................................................9 Standards:.......................................................................................................10 Samples:..........................................................................................................11 Stationary Phase:............................................................................................12 Mobile Phase:.................................................................................................12 Chromatographic Parameters:......................................................................13 Gradient Scheme:...........................................................................................14 Results:...............................................................................................................................15 Standards:.......................................................................................................15 BPA Concentration Standard Curve:............................................................16 Test Samples:..................................................................................................17 Conclusion:........................................................................................................................18 Evaluation:........................................................................................................................21 Bibliography:.....................................................................................................................24 Appendices:.......................................................................................................................28 Appendix 1: Preparation Methods:...............................................................28 Appendix 2: HPLC Methods:........................................................................30 Appendix 3: HPLC Results:...........................................................................32 Appendix 4: Calculations:..............................................................................37

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Measuring the Effects of Temperature, Microwaves and Acidity on Bisphenol A Leaching

Research Question: How much Bisphenol A (BPA) leaches into water from a polycarbonate Rubbermaid Bouncer® Pitcher when treated with different temperatures or microwaves; does the acidity of the liquid affect the amount BPA leached?

Introduction: Bisphenol A: Bisphenol A, with a formula of (CH3)2C(C6H4OH)2, is an important ingredient in the production of polycarbonate plastics and epoxy resins. The small white crystals have a relatively heavy molecular mass of 228.3gmol-1. It was first synthesized in Germany in the early 20th century, but only in the 1950s did it become a useful material in the production of polycarbonate plastics and epoxy resins (which are then used to manufacture kitchenware, bottles, food cans, etc.). As the popularity of plastic containers and kitchenware grew, so did the use of BPA for its "optical clarity, shatter-resistance and high heat-resistance"1; its high melting point (152 - 153˚C) and boiling point (250 - 252˚C) contributed to its suitability. It is poorly soluble, if at all, in water, but dissolves in organic solvents, and reacts violently with acid anhydrides, acid chlorides, strong bases and strong oxidants.2 Thus, it is a chemical that may be reacted in a laboratory but should be stable when exposed to the average household. As shown in Figure 1, BPA is an organic compound made up of 15 carbon, 16 hydrogen, and two oxygen atoms.

1 2

http://www.bisphenol-a.org/about/bpa-info/bpa-synthesis.html http://www.cdc.gov/niosh/ipcsneng/neng0634.html iv

Figure 1: Bisphenol A Structure3

Polycarbonate: Polycarbonate, with the abbreviation PC, is a thermoplastic polymer that is used to make a wide variety of objects: kitchenware items such as bottles and food storage containers, shatterproof windows, and eyeglass lenses to name a few.4 Figure 2 presents a unit of the polymer, which is made up mainly of Bisphenol A and phosgene (COCl2):

Figure 2: Polycarbonate Structure Unit5 To produce polycarbonate, the two molecules6 undergo a condensation reaction7; both of the hydroxyl groups of BPA are treated with sodium hydroxide (NaOH), which produces two water molecules and two sodium ions from the heterolytic cleavage. The BPA molecule is now a

http://en.wikipedia.org/wiki/File:Bisphenol_A.svg http://www.pslc.ws/macrog/pc.htm 5 http://en.wikipedia.org/wiki/File:Lexan.png 6 Bisphenol A and phosgene 7 A small molecule is lost to create new bonds between the monomers BPA and phosgene. 4

salt, and both oxygen ions are negatively charged. A phosgene molecule then binds to one of the charged oxygen ions on the BPA molecule, displacing a chlorine ion. The chlorine ion reacts with one of the sodium ions produced to give sodium salt (NaCl).

Figure 3: Equation for the Synthesis of Polycarbonate8

Another Bisphenol A molecule treated with NaOH may attach to the chloroformate9 formed, thus creating the long chain which makes up the polymer polycarbonate.10 The polycarbonate may then be heated and molded to its desired shape.

Leaching/Health Concerns: The polymerization of polycarbonate will occur only as long as there are enough of the reactants (BPA, phosgene) in their molar ratio. As a result, some non-reacted BPA (as well as other reactants) may stay in the plastic. This trace BPA may migrate from the plastic into contents. Also, if the polycarbonate is exposed to conditions (heated water, etc.) that cause it to hydrolyze, it may release BPA. 11

Synthesis by Solvent Method. Digital image. Polycarbonate Resin and Bisphenol A. Polycarbonate Resin Manufacturing Group, June 2003. Web. 28 Aug. 2012. 9 chloroformate: a salt or ester of chloroformic acid 10 http://www.pslc.ws/macrog/pcsyn.htm 11 Polycarbonate Resin & Bisphenol A. Rep. Polycarbonate Resin Manufacturing Group, June 2003. Web. 26 Aug. 2012. vi

In the past decade, BPA leaching from polycarbonate products has become an increasingly controversial topic. Scientific studies have caused doubts of the apparent safety of BPA when it leaches from polycarbonate containing food/liquids and is then ingested by humans.12 These studies link the exposure to BPA (both long- and short-term) to a wide array of health problems: breast/prostate cancer, diabetes, impaired development and learning, obesity, and various genetic diseases.13 To determine the possible health effects, scientists injected several species (mice, rats, monkeys) with BPA and monitored the side effects. In some cases, BPA will mimic the body's hormones such as estrogen, resulting in "reduced daily sperm production and increased prostate gland weight in males and alteration in the development and tissue organization of the mammary gland, disruption of sexual differentiation in the brain, long-term deleterious effects in the vagina, and accelerated growth and puberty in females."14 The possibility of these detrimental effects in humans is proof enough for most consumers to remove Bisphenol A from their household; others argue that the ingestion of the chemical will not have the same effects as injection.15 Instead of immediately entering the blood stream, like in the laboratory animals, it tends to pass through the digestive tract in humans. The majority is then expelled in the urine16. However, another more recent research experiment involved feeding pregnant monkeys fruit containing BPA; the female offspring then displayed "changes in the [mammary] glands that give rise to dense tissue - something that in humans is a risk factor for 12

BPA may also be transmitted by inhalation and dermal absorption: http://www.fertilitycoach.com/wp/featured/bpa-lowers-male-fertility-minimize-exposure/ 13 http://www.baumhedlundlaw.com/bpa/bpa_side_effects.php 14 Kuklenyik, Zsuzsanna, John Ekong, Caroline D. Cutchins, Larry L. Needham, and Antonia M. Calafat. "Simultaneous Measurement of Urinary Bisphenol A and Alkylphenols by Automated Solid-Phase Extractive Derivatization Gas Chromatography/Mass Spectrometry." 15 Reddy, Sanhita. "BPA, Health, and Nuance." Columbia Journalism Review 16 Pottenger, L. H., J. Y. Domoradzki, D. A. Markham, S. C. Hansen, S. Z. Cagen, and J. M. Waechter. "The Relative Bioavailability and Metabolism of Bisphenol A in Rats Is Dependent upon the Route of Administration." National Center for Biotechnology Information. U.S. National Library of Medicine vii

breast cancer".17 The responses to these findings has caused a conflict between scientists and the government: while hundreds of lab animal experiments have produced distressing results about the potential health effects of BPA, the U.S. Food and Drug Administration does not consider this sufficient evidence to support a ban on the material18. Therefore, the phenolic chemical can be found in almost every household in some form or other, considering the variety of uses of polycarbonate plastics and epoxy resins: baby bottles, plastic dinnerware, plastic bottles, and the linings of food cans. The average American is exposed to the chemical on a daily basis: the "2003-2004 National Health and Nutrition Examination Survey (NHANES III) conducted by the Centers for Disease Control and Prevention (CDC) found detectable levels of BPA in 93% of 2517 urine samples from people six years and older."19 So, as BPA is currently not banned from the production of polycarbonates and epoxy resins, the "U.S. Environmental Protection Agency (US EPA) has established 50 Âľg/kg body weight/day as the reference dose (RfD) for BPA."20 Given this guideline, I want to determine the concentration of BPA that leaches into liquids contained in a popular polycarbonate pitcher when the liquids are heated, exposed to microwaves, or have a lower pH. Previous studies have shown that boiling water promotes the separation of BPA into the contents of the container21, releasing the chemical "15 to 55 times faster" (than at room temperature). Radiation from microwaves also makes BPA more unstable and likely to migrate.22 Furthermore, acidic contents are said to increase the leaching of BPA.23 17

"Study Hints at BPA Tie to Breast Cancer." San Francisco Chronicle 8 May 2012, Nation sec.: A7. Print. 18 http://www.sfgate.com/cgi-bin/article.cgi?f=/c/a/2012/03/30/MNJK1NSN8T.DTL 19 http://www.niehs.nih.gov/news/sya/sya-bpa/ 20 Lateef, Syed S. Analysis of Bisphenol A Leaching from Baby Feeding Bottles. Agilent Application Solution. Agilent Technologies 21 Cooper, James E., Eric L. Kendig, and Scott M. Belcher. Assessment of Bisphenol A Released from Reusable Plastic, Aluminium and Stainless Steel Water Bottles. ScienceDirect. 22 http://ehp03.niehs.nih.gov/article/info:doi%2F10.1289%2Fehp.1003220 viii

In order to measure the concentration of BPA that leaches out of the pitcher under the different conditions, samples were manipulated in the pitcher and then tested for traces of BPA using high performance liquid chromatography (HPLC). Liquid chromatography was selected because it can separate the different components of a mixture and measure the abundance of the components, allowing precise quantitation of leached Bisphenol A in the solution. This not only allows me to quantify the concentration of BPA ingested by a consumer, but also whether the average human using such a container consumes a hazardous concentration of BPA.

High Performance Liquid Chromatography (HPLC): The basic principle of HPLC is that a sample of a tested mixture is injected into a silica column (called the stationary phase), which contains spherical silica particles, maximizing the surface area to allow interaction with the liquid sample. Silica particles are used for HPLC columns as they are "stable under high operating pressures for longer periods", "exhibit lower backpressures and longer lifetimes", and "stable during use with various solvent types" under a pH of 8.24 However, since the silica pores are polar, reversed-phase chromatography (RPC)25 was used for this experiment, which uses a modified column featuring long hydrocarbon chains (the number of chains ranges from 8 to 18) attached to the surface of the silica pores, so that the surface is non-polar. This method is more effective to analyze non-polar compounds such as Bisphenol A.26 Non-polar compounds from the mixture interact with the hydrocarbon surface,

http://www.plasticalbatross.org/get-informed/bpa/ "5.2 Characteristics of Columns and Column Packings." Practical HPLC Method Development. 2nd ed. 25 Reversed-phase chromatography is one of the two major sub-categories of HPLC. The other is known as normal phase chromatography, and is used to separate polar compounds. 26 For polar compounds, normal phase chromatography is usually employed. 24

and will be attracted to the pores according to their polarity due to intermolecular forces like van der Waals forces. Then, the mobile phases (A and B) are run through the column. The mobile phases make up a non-polar organic solvent that travels through the column, flushing out compounds of the mixture depending on whether they are attracted more to the solvent or the hydrocarbon chains. The polarity of the solvent can be modified by changing the percentages of the mobile phases (which have different polarities): as the concentration of the mobile phase B is increased over time, the solvent becomes more non-polar, eventually flushing out even the most non-polar compounds.27 The time (in min) a certain component of the sample spends in the column before being carried out with the mobile phases is called the retention time, and this is then used to identify the different compounds28. The fluorescence detector (FLD) then measures the abundance of the different compounds, displaying them as peaks on a chromatograph. The FLD is made up of a flow cell to receive excitation light axially and a photocell to receive light radially. The purpose is to detect only substances that fluoresce, so a Pyrex glass shields the photocell from the excitation light. When a substance then passes through the FLD and is hit by the excitation light, it will fluoresce and be sensed by the photocell, which will process the output electronically and produce the peaks on the chromatograph.29 The area under each peak represents the relative amount of that specific compound. To identify a specific substance on a chromatograph, control samples with a known concentration of the substance are made and tested in the HPLC. This allows determination of the retention time, which can then be used to pinpoint the same substance in a test sample. The peak 27

http://www.chemguide.co.uk/analysis/chromatography/hplc.html Because each compound has a different polarity, each has a specific retention time. 29 http://www.chromatography-online.org/topics/fluorescence/detector.html 28

areas of the control samples represent the abundance and can be used to correlate area to concentration of a compound. The following diagram presents a simplified overview of process of HPLC:

Figure 4: Block Diagram of HPLC Method30

Image 1: Example of an Agilent Technologies HPLC Apparatus31

http://www.protein.iastate.edu/images/hplc.jpg http://upload.wikimedia.org/wikipedia/commons/thumb/4/4b/Agilent1200HPLC.jpg/220pxAgilent1200HPLC.jpg

Experimental Details: To investigate how much BPA leaches from an average polycarbonate pitcher, liquid samples of water and distilled vinegar were manipulated in the pitcher and then collected. The vinegar, which has a pH of 2.4, may mimic the behavior of drinks with a similar acidity, such as Coca-Cola or some juices32. The polycarbonate used in the experiment is a four-year-old Bouncer® Pitcher33 from Rubbermaid Commercial Products, used abundantly in U.S. restaurants and homes to distribute drinks. It is made of "durable polycarbonate", and should be "dishwasher safe"34. Therefore, it should be able to resist high temperature liquids. This experiment evaluates how durable it really is, and whether the average person is consuming hazardous amounts of BPA, unknowingly encouraging detrimental effects which may change their life.

Most of the work of the experiment lies in the preparation of the standard and test samples. For this, the following materials were used: •

Bisphenol A

•

~6L Distilled Water (Arrowhead, pH 5.6 - 7.035)

•

~2L Distilled White Vinegar (Safeway, 5% Acidity, pH 2.436)

•

Rubbermaid Bouncer® Pitcher, 60oz.

•

~1.6L 95% Ethanol

http://www.21stcenturydental.com/smith/pH_drinks.htm Model number: 3338 34 http://www.rubbermaidcommercial.com/rcp/products/detail.jsp?rcpNum=3338 35 http://www.ehow.com/about_4623914_ph-distilled-water.html 36 http://www.apple-cider-vinegar-benefits.com/properties-of-vinegar.html 33

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The aim of the preparatory work is to create 1) five standards (solutions of a known concentration), to produce a standard curve, and 2) eight test samples. It is crucial to make accurate measurements when making the standards, as the test samples on the chromatograph will then be measured in relation to the standards. Standards: Standards were made from a 0.0100g/L BPA STOCK solution by dilution (in distilled water and ethanol in a 1:1 volumetric ratio) so that there are five standard solutions with the following concentrations: a. 10ng/mL BPA. b. 20ng/mL BPA. c. 100ng/mL BPA. d. 500ng/mL BPA. e. 1000ng/mL BPA. See appendix entry 1.1 for a detailed method of the standard preparation.

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Samples: Using the polycarbonate pitcher, prepare eight test samples with the following conditions: Table 1.1: Test Sample Descriptions Sample #

Solvent (500mL)

Temperature (in Ë&#x161;C)

Time (in mins)

Water37

~2538

Vinegar39

~25

Water

~82

Vinegar

~82

Watts (W) 5

Water

1250

Vinegar

1250

Water

1250

Vinegar

1250

Test samples were collected and diluted with ethanol for analysis using HPLC. For a detailed method, see appendix entry 1.2. Measurement of the concentration of BPA in the samples was then carried out at the Bayer (HealthCare) Corporation in Berkeley, CA, under the supervision of John Teare, Ph.D. Principal Process Development Scientist (for Kogenate Analytical Development in Hematology/Cardiology), using an HPLC apparatus from Agilent Technologies.40

All mentions of 'water' in this experiment refer to distilled water unless otherwise noted. This is done to avoid contamination. 38 Room temperature, ~ 25Ë&#x161;C 39 All mentions of 'vinegar' in this experiment refer to distilled white vinegar, 5% acidity. 40 The HPLC apparatus consisted of parts from Agilent Technologies, from both the 1100 Series and 1200 Series. xiv

Stationary Phase: In this case, a ZORBAX Eclipse Plus C18 column from Agilent Technologies41 with 18 carbon atoms are attached to the silica surface was used. This gives it a non-polar, hydrophobic internal surface. The dimensions of the column are 4.6x100mm, with a silica particle size of 5Âľm.

Image 2: A HPLC Column from Agilent Technologies42 Note: The image does not present the column used for this particular experiment; it is an example HPLC column.

Mobile Phase: For reversed-phase chromatography, the mobile phases are usually made up of an aqueous solvent and an organic solvent.43 A study of BPA leaching from baby bottles from Agilent Technologies44 was used as an example for the parameters. The two mobile phase gradient was prepared to elute the samples: mobile phase A was 10mM monobasic sodium phosphate

The column was newly purchased for this experiment to avoid any contamination. Furthermore, it was washed several times before conducting the experiment to optimize the stationary phase. 42 http://www.vgdusa.com/images/agilent-zorbax-extend-columns.jpg 43 http://www.ionsource.com/tutorial/chromatography/rphplc.htm 44 Op. cit. Lateef, Analysis of Bisphenol A Leaching from Baby Feeding Bottles. xv

(NaH2PO445) in HPLC-grade water46, and mobile phase B was 100% Acetonitrile47(CH3CN), a suitable organic solvent. The HPLC system was then washed to remove any impurities. Appendix entry 2.2 describes the cleaning method. Finally, the Experiment Method was set up on the HPLC software. The following data table gives the parameters of the experiment:48

Table 1.2: Chromatographic Parameters Parameters

Agilent 1100/1200 Series HPLC System

Column

Agilent ZORBAX Eclipse Plus C18 4.6x100mm 5 µm

TCC49 Temperature

40˚C

FLD50

Excitation: 230.nm ;Emission: 316nm

Mobile phase A

10mM monobasic sodium phosphate in water

Mobile phase B

100% Acetonitrile

Flow

0.9mL/min

Injection Volume

100µL

Pressure

400bar

NaH2PO4: monobasic sodium phosphate The Agilent Technologies Study originally uses 10mM monobasic potassium phosphate in water; here, 10mM monobasic sodium phosphate is used instead due to availibility. 47 See appendix entry 2.1 for the preparatory method of the mobile phases. 48 The choice of parameters is based partly on the aforementioned Agilent Technologies Study: Lateef, Syed S. Analysis of Bisphenol A Leaching from Baby Feeding Bottles. 49 TCC: Thermostatted Column Compartment 50 FLD: Fluorescence Detector 46

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Table 1.3 gives the gradient of the mobile phases at the different time intervals for each run. The polarity of the mobile phase is decreased as the percentage of Acetonitrile is increased: Table 1.3: Gradient Scheme Time51 (mins)

Percentage of mobile phase B: 100% Acetonitrile

2.1

12.5

12.6

18.1

10mL of each standard and each test sample were injected into separate glass vials using an ergonomic pipette52. The glass vials were inserted into the wells in the HPLC before starting the runs.53

The time signifies the minutes that have gone by in a run. In total, one run will take 23mins. The pipette is from Rainin. 53 See appendix entry 2.3 for the method of cleaning the HPLC apparatus after use. 52

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Results: Standards: The following chromatograph and table display the results of the five standards: Chromatograph 1.1: Standards54

The chromatographs produced by the HPLC software were studied for the possible presence of BPA. The aforementioned Agilent Technologies Study suggests that BPA has a retention time of around 11 minutes. The chromatographs of the standards revealed a peak 54

For all chromatographs in this study, the x-axis (independent variable) is the (retention) time (min). The y-axis (dependent variable) is the relative fluorescence detector (FLD) signal response. xviii

between 10-11 minutes. With the increasing concentration of BPA, the peak area increased, which suggests that the substance identified at ~11 minutes is BPA. Table 1.4: Standards Chromatograph Analysis Concentration BPA (ng/mL)

Ret. Time (min)

Area

10.905

10.946

100

10.937

500

10.941

152

1000

10.938

682

The peak area was used to create the standard curve shown below. The areas do not produce a linear relationship (as they theoretically should), which suggests an error in the preparation of the standard. The 500ng/mL BPA standard skewed the results significantly more than the others, so it was omitted for the standardization curve55:

Figure 5: BPA Concentration Standard Curve56

55 56

See appendix entry 3.1 for the justification of omitting the 500ng/mL BPA standard. For further data about the standard curve (i.e. slope), see appendix entry 3.2. xix

Test Samples: The chromatographs of the test samples were analyzed for peaks that could indicate the presence of BPA. Furthermore, fresh vinegar and water samples were run through the HPLC as a control to discern that there are no disturbances that could give a false positive. Based on the appearances of peaks at a retention time of 10-11mins, the presence was confirmed in all test samples. The peak areas of the different samples varied greatly, indicating different concentrations of BPA. Using the HPLC software, the peak areas of the test samples were integrated into the standardization curve (above). The relative comparison of the peak areas determine the concentrations of BPA in each test sample: Table 1.5: Test Sample Chromatograph57 Analysis

Sample

Ret. Time (min)

Water, 25˚C Vinegar, 25˚C Water, 82˚C Vinegar, 82˚C Water, 1250W, 2mins Vinegar, 1250W, 2mins Water, 1250W, 5mins Vinegar, 1250W, 5mins

10.879 10.873 10.888 10.906 10.890 10.920 10.881 10.930

Peak Area

BPA Concentration (ng/mL) (after dilution)

BPA Concentration (ng/mL) (original)*

10 10 12 16 32 9 30 13

5.6 5.6 8.5 14.4 38.1 4.1 35.2 10.0

11.2 11.2 17.0 28.8 76.2 8.2 70.4 20.0

*Since ethanol was used to dilute the test samples to 50% of the original concentrations, the concentrations derived through chromatography half the actual concentrations in the test samples.58

57 58

See appendix entry 3.3 for the chromatograph(s) of the test samples. Using the law of dilution: M1V1 = M2V2 xx

Conclusion: The standard solutions are the groundwork of the study, as they allow for quantitative analysis of the test samples. Appropriately, those with higher concentrations of BPA produced larger peak areas at the expected retention time. Giving consideration to human error, the standards proved fitting to produce a standardization wave: the r2 value was very close to 1 at 0.999859. This shows that if measurements are performed correctly, the preparatory method is effective in creating BPA solutions for chromatographic analysis. On the chromatographs of all the test samples, a peak was identified at the retention time (10.873 - 10.930mins) according to that of BPA. Therefore, it was deduced that all of the samples were contaminated with BPA to various degrees. The samples at 25˚C both resulted in the same concentration of leached BPA. At 11.2ng/mL, there is very little leaching, but the value represents the amount of BPA leached in an hour, so more may have leached with time. Since both samples gave the same concentration, it seems that at 25˚C the increased acidity did not promote the dissolution of BPA from the polycarbonate. When the liquids were exposed to a higher temperature (82˚C), the concentration of BPA increased significantly. The heat may cause the plastic to decompose from a polymer into its monomers, breaking the chain.60 The heated water caused a 51.7% increase of BPA leached (than at 25˚C), demonstrating that heat promotes leaching. The heated vinegar sample showed a 157% increase from the sample at 25˚C with 28.8ng/mL BPA. Like with the water, the heat promoted the leaching of BPA, but the heated vinegar gave a significantly higher concentration than its water counterpart.

59 60

Derived from Table 2.1 in appendix entry 3.2. http://en.wikipedia.org/wiki/Plastic#Properties_of_plastics xxi

Both of the microwaved water samples gave a high increase in BPA concentration. The radiation increased the leaching by 580%61 within 2mins. When microwaved for 5mins, the water contained a slightly lower concentration BPA, which does not seem logical, but the difference in concentrations is small enough (<10% change) that there is no statistical significance, and that an error may account for the anomaly. The microwaved vinegar samples gave surprisingly low concentrations of BPA. In 2mins, the microwaved vinegar sample contained a mere 8.2ng/mL BPA, which is a 26.8% decrease from the sample at 25Ë&#x161;C. After 5mins, the microwaved vinegar produced more than twice the concentration than at 2mins, but with 20.0ng/mL it is still less than the concentration in the heated vinegar. The water samples show an increase of BPA concentration with heat, and even more so with microwave radiation, which gave the highest value of 76.2ng/mL. The fact that the microwaved vinegar sample produced a lower concentration than the water suggests that microwaves (relatively low frequency) may not affect the acetic acid as much. Interestingly, the vinegar samples do present a 145% increase in BPA concentration when treated with radiation longer. This shows that prolonged exposure is necessary to increase the leaching of BPA to acids. Also, although acidic contents were thought to encourage leaching, this is not apparent from the one hour interval; at room temperature the acidity did not increase the amount leached, and caused much lower concentrations of BPA than water when microwaved. Only when left in a hot water bath did the vinegar sample give a higher BPA concentration than the water sample. This may suggest that acidity increases the leaching of BPA only when enhanced by heat. Also, perhaps the acidity of the vinegar did not have a strong effect because vinegar is polar so there will be little interactions with the non-polar BPA.

compared to the water sample at room temperature. xxii

The range of results from 8.2-76.2ng/mL BPA suggests that the factors pH (of the contents), heat, and microwaves may affect the dissolution of BPA in different ways. The fact that even at room temperature BPA leached from the polycarbonate presents how unstable the plastic is, and that the leaching does not occur only under extreme conditions. Whether these concentrations can be harmful to a daily user of these polycarbonate products is questionable; these levels of BPA may possibly pass through the digestive tract and be egested. The EPA has called 50Âľg/kg/day BPA to be the safe limit for the average human. The biggest concentration of BPA found in the test samples was 76.2ng/mL; if an average male adult weighs approx. 80kg62, and drinks 2L daily of water (or another liquid) with 76.2ng/mL BPA, then he would consume 1.90Âľg/kg/day63, which is significantly below the reference dose. Considering that this is the highest concentration detected by the HPLC apparatus, all of the other samples are yet less harmful. In conclusion, BPA is a substance that leaches from polycarbonates under standard conditions and even more so under extreme conditions; a higher temperature has been shown to increase the amount of leached BPA (up to a 157% increase), especially if the liquid is acidic. Microwaves increase the concentration (of BPA) greatly in water samples (up to almost 7x as much), while in the acidic vinegar sample there is no significant effect. The greatest concentration found (76.2ng/mL) was well below the reference dose, and would be deemed harmless, but the results exemplify how readily BPA will leach under extreme conditions, wearing off the polycarbonate. So, while BPA in low doses is deemed safe by the EPA, consumers should be aware of it nevertheless. Fortunately, Rubbermaid Commercial Products has

62 63

http://www.theaveragebody.com/average_weight.php For calculations, see appendix entry 4.1 xxiii

removed Bisphenol A from the product two years ago, so the results of this experiment are not applicable to new Bouncer速 Pitchers64.

Evaluation: There are some limitations in the preparatory part of the experiment, as the HPLC software and equipment is highly developed and exact. Therefore, the measurements and dilution made have to be made very carefully and accurately, and random human errors are easily made. For example, the standard that was meant to have 500ng/mL of BPA did not adhere to the relative proportionality to which the others did, and the microwaved vinegar test samples did not give the expected increase in leaching; this could be due to random errors in dilution, such as adding too much ethanol, which would change the relative concentration. To minimize these limitations, suitable measurement tools should be used and the individual experiments should be repeated, giving more trials and decreasing uncertainties. The samples should also be run through the HPLC several times, which time did not allow in this case. This experiment introduces questions for many more studies; there are several variables and details relating to BPA leaching into food/drinks that could be explored. While the dose found in the results should not be enough to harm an average adult male, it may pose a risk to infants, or even fetuses, if passed through the umbilical cord. Even if infants are fed with bottles that do not contain BPA, it may be passed to the child through breast milk65. Also, if BPA may mimic estrogen, it could affect females more than males. A long-term study monitoring pregnant women exposed to 'safe' doses of BPA could be conducted by measuring BPA concentrations in 64

Rubbermaid Commercial Products, Michelle D. "Customer Service Request." Message to the author. 6 Mar. 2012. E-mail. 65 http://www.health.state.mn.us/divs/eh/risk/chemhazards/bisphenola.html xxiv

bodily fluids and examining the mammary glands routinely. Another aspect to consider is whether the average adult may be absorbing/ingesting BPA from any other sources, like food cans or even eyeglass frames66, which could increase the dose. Also an important question is how much BPA is egested in the urine in comparison to how much is absorbed by the body, which could be determined by measuring BPA levels in urine compared to blood (and breast milk). These would be elaborate studies using human trials and taking considerations such as age, weight, ethnicity, etc. into consideration. There are other more technical and chemical aspects to consider; whether the surface area to volume ratio of the polycarbonate product (to contents) increases the migration rate. Controlled sample sizes could be manipulated in differently shaped containers with varying surface areas, and then tested for BPA levels. While an acidic sample was tested in this experiment, it may be useful to test a more basic substance to further explore the effects of pH. Also, since BPA is considered a non-polar compound, it could be tested with a fatty liquid, such as milk, to see whether that will affect the leaching due to interactions between the non-polar molecules67. This could be an important point when considering increased health risks for infants ingesting BPA.68 Another question would be if the amount of leached BPA increases with each use of the polycarbonate product, as the stability of the polycarbonate chain decreases as BPA separates. The same product could be treated under the same conditions for repeated time intervals, taking a sample of the contents after each interval, and comparing BPA levels. It may be beneficial to perform longer time trials than 60mins to receive more detectable results. For these past three research proposals, HPLC could effectively be used to detect BPA traces. The behavior of BPA in different conditions has 66

http://www.washingtonpost.com/wp-dyn/content/article/2008/04/26/AR2008042602038.html The alkaline and/or milk could be treated like the vinegar/water samples in this experiment, and equally tested for BPA contamination using HPLC. 68 http://www.environmentalhealthnews.org/ehs/newscience/BPA-11-times-higher-in-babies/ 67

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not yet been explored to its full extent, and will certainly give rise to intriguing discoveries in the future; in the meantime, it could be advantageous to limit exposure until its danger has been definitely evaluated.

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Bibliography: "117 Years of BPA." Washington Post. The Washington Post, 27 Apr. 2008. Web. 01 Aug. 2012. <http://www.washingtonpost.com/wpdyn/content/article/2008/04/26/AR2008042602038.html>. 3338 Bouncer Pitcher. Advertisement. Rubbermaid Commercial Products. Rubbermaid Commercial Products, 2012. Web. 25 July 2012. <http://www.rubbermaidcommercial.com/rcp/products/detail.jsp?rcpNum=3338>. "5.2 Characteristics of Columns and Column Packings." Practical HPLC Method Development. 2nd ed. N.p.: John Wiley & Sons, 1997. 178-79. Print. Agilent Technologies HPLC Zorbax Extend Columns. Digital image. Agilent Zorbax Extend Columns. Agilent Zorbax Extend Columns, n.d. Web. 27 July 2012. <http://www.vgdusa.com/agilent-zorbax-extend-columns.htm>. Allard, Patrick, and Monica P. Colaiacovo. Bisphenol A Impairs the Double-strand Break Repair Machinery in the Germline and Causes Chromosome Abnormalities. Rep. Ed. Joan Ruderman. PNAS, 6 Oct. 2010. Web. 27 July 2012. <http://www.pnas.org/content/107/47/20405.full>. "Average Weight." TheAverageBody.com. TheAverageBody.com, 2012. Web. 01 Aug. 2012. <http://www.theaveragebody.com/average_weight.php>. "Bisphenol A (BPA) Synthesis and Use." Bisphenol A. Bisphenol A, n.d. Web. 23 July 2012. <http://www.bisphenol-a.org/about/bpa-info/bpa-synthesis.html>. "Bisphenol A." Minnesota Department of Health. N.p., 25 Aug. 2011. Web. 01 Aug. 2012. <http://www.health.state.mn.us/divs/eh/risk/chemhazards/bisphenola.html>. "Bisphenol A Won't Be Banned by FDA." San Francisco Chronicle 30 Mar. 2012, NATION sec.: n. pag. San Francisco Chronicle. Hearst Communications, 30 Mar. 2012. Web. 23 July 2012. <http://www.sfgate.com/nation/article/Bisphenol-A-won-t-be-banned-byFDA-3448971.php>. "BPA." Plastic Albatross. Plastic Albatross, 2010. Web. 28 Aug. 2012. <http://www.plasticalbatross.org/get-informed/bpa/>. Calvero. File:Bisphenol A.svg. Digital image. Wikipedia. Wikimedia Foundation, Aug. 2006. Web. 24 July 2012. <http://en.wikipedia.org/wiki/File:Bisphenol_A.svg>. Chadwick, Janet. "BPA Lowers Male Fertility - How to Minimize BPA Exposure." Fertilitycoach.com. Wordpress, 10 June 2011. Web. 26 Aug. 2012. xxvii

<http://www.fertilitycoach.com/wp/featured/bpa-lowers-male-fertility-minimizeexposure/>. Clark, Jim. "High Performance Liquid Chromatography - HPLC." High Performance Liquid Chromatography - HPLC. Jim Clark, 2007. Web. 26 July 2012. <http://www.chemguide.co.uk/analysis/chromatography/hplc.html>. Cooper, James E., Eric L. Kendig, and Scott M. Belcher. Assessment of Bisphenol A Released from Reusable Plastic, Aluminium and Stainless Steel Water Bottles. ScienceDirect. Elsevier Ltd., 8 July 2011. Web. 26 July 2012. <http://www.sciencedirect.com/science/article/pii/S004565351100717X>. File:Agilent1200HPLC.jpg. Digital image. Wikipedia.org. N.p., 11 Aug. 2008. Web. 26 July 2012. <http://en.wikipedia.org/wiki/File:Agilent1200HPLC.jpg>. "Fluorescence Detector." Chromatography-Online.org. Library4Science.com, 2008. Web. 31 Aug. 2012. <http://www.chromatographyonline.org/topics/fluorescence/detector.html>. "FULL LIST OF HEALTH PROBLEMS LINKED WITH BPA." Baum, Hedlund, Aristei & Goldman, PC. Baum, Hedlund, Aristei & Goldman, PC, n.d. Web. 29 Aug. 2012. <http://www.baumhedlundlaw.com/bpa/bpa_side_effects.php>. Guzzetta, Andrew. "Reverse Phase HPLC Basics for LC/MS." IonSource. IonSource, 22 July 2001. Web. 27 July 2012. <http://www.ionsource.com/tutorial/chromatography/rphplc.htm>. "International Chemical Safety Cards - BISPHENOL A." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 20 Apr. 1994. Web. 24 July 2012. <http://www.cdc.gov/niosh/ipcsneng/neng0634.html>. Kuklenyik, Zsuzsanna, John Ekong, Caroline D. Cutchins, Larry L. Needham, and Antonia M. Calafat. "Simultaneous Measurement of Urinary Bisphenol A and Alkylphenols by Automated Solid-Phase Extractive Derivatization Gas Chromatography/Mass Spectrometry." Laiosa, Michael D., and Wendy Hessler. "Study Predicts BPA in Babies 11 times Higher than Adults." Environmental Health News. Environmental Health Sciences, 12 Jan. 2009. Web. 01 Sept. 2012. <http://www.environmentalhealthnews.org/ehs/newscience/BPA-11-timeshigher-in-babies/>. Lateef, Syed S. Analysis of Bisphenol A Leaching from Baby Feeding Bottles. Rep. no. 5990xxviii

9398EN. N.p.: Agilent Technologies, 2011. Analysis of Bisphenol A Leaching from Baby Feeding Bottles. Agilent Technologies, Inc., 1 Dec. 2011. Web. 23 July 2012. Lawrence, Joel D. Typical Liquid Chromatography System. Digital image. Example Paper. N.p., 2006. Web. 26 July 2012. <http://xnet.rrc.mb.ca/davidb/new_page_27.htm>. "Making Polycarbonates." Making Polycarbonates. Polymer Science Learning Center, 2005. Web. 25 July 2012. <http://www.pslc.ws/macrog/pcsyn.htm>. Moore, Shelley. "What Is the PH of Distilled Water?" EHow. Demand Media, 01 Dec. 2008. Web. 01 Aug. 2012. <http://www.ehow.com/about_4623914_ph-distilled-water.html>. "Plastic." Wikipedia. Wikimedia Foundation, 31 Aug. 2012. Web. 01 Sept. 2012. <http://en.wikipedia.org/wiki/Plastic>. "Plastics That May Be Harmful to Children and Reproductive Health." Environment and Human Health, Inc. EHHI, n.d. Web. 30 July 2012. <http://www.ehhi.org/reports/plastics/bpa_health_effects.shtml>. "Polycarbonates." Polycarbonates. Polymer Science Learning Center, 2005. Web. 25 July 2012. <http://www.pslc.ws/macrog/pc.htm>. Polycarbonate Resin & Bisphenol A. Rep. Polycarbonate Resin Manufacturing Group, June 2003. Web. 26 Aug. 2012. Pottenger, L. H., J. Y. Domoradzki, D. A. Markham, S. C. Hansen, S. Z. Cagen, and J. M. Waechter. "The Relative Bioavailability and Metabolism of Bisphenol A in Rats Is Dependent upon the Route of Administration." National Center for Biotechnology Information. U.S. National Library of Medicine "Properties of Vinegar." Apple-cider-vinegar-benefits.com. Apple-cider-vinegar-benefits.com, 2012. Web. 26 July 2012. <http://www.apple-cider-vinegar-benefits.com/properties-ofvinegar.html>. Reddy, Sanhita. "BPA, Health, and Nuance." Columbia Journalism Review Rubbermaid Commercial Products, Michelle D. "Customer Service Request." Message to the author. 6 Mar. 2012. E-mail. Shoyrudude 555. File:Lexan.png. Digital image. Wikipedia.org. Shoyrudude 555, 30 June 2007. Web. 24 July 2012. <http://en.wikipedia.org/wiki/File:Lexan.png>. "Since You Asked - Bisphenol A (BPA)." National Institute of Environmental Health Sciences. NIEHS, 30 Mar. 2012. Web. 23 July 2012. <http://www.niehs.nih.gov/news/sya/syabpa/>. xxix

Smith, Kent. "Drinks That Eat Teeth." 21st Century Dental. 21st Century Dental, 2000. Web. 25 July 2012. <http://www.21stcenturydental.com/smith/pH_drinks.htm>. "Study Hints at BPA Tie to Breast Cancer." San Francisco Chronicle 8 May 2012, Nation sec.: A7. Print. Synthesis by Solvent Method. Digital image. Polycarbonate Resin and Bisphenol A. Polycarbonate Resin Manufacturing Group, June 2003. Web. 28 Aug. 2012. Yang, Chun Z., Stuart I. Yaniger, Craig Jordan, Daniel J. Klein, and George D. Bittner. "Most Plastic Products Release Estrogenic Chemicals: A Potential Health Problem That Can Be Solved." Environmental Health Perspectives (2011): n. pag. EHP. Web. 28 Aug. 2012. <http://ehp03.niehs.nih.gov/article/info:doi%2F10.1289%2Fehp.1003220>.

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Appendices: Appendix 1: Preparation Methods 1.1 The method for the standard solutions was as follows: 1. 0.0100g (+ 0.0001g) of the Bisphenol A were measured out on a digital scale. The scale was zeroed before weighing. 2. A STOCK solution was made with a concentration of 0.0100g/L BPA by diluting the BPA (in distilled water and ethanol in a 1:1 volumetric ratio) to 1.00 liter. A 1L flask was used for this step. 3.

A standard was made with 10.0ng/mL BPA by diluting 1.00mL of the STOCK solution to 1.00 liter. A 1mL (+ 0.012mL) pipette was used.

A 20.0ng/mL BPA standard was made by diluting 1.00mL of the STOCK solution to 500.mL. A 500mL flask was used.

A 100.ng/mL BPA standard was made by diluting 1.00mL of the STOCK solution to 100.mL. A 100mL flask was used.

6. A 500.ng/mL BPA standard was made by diluting 1.00mL of the STOCK solution to 50.0mL. A 50mL flask was used. 7. A standard with 1000ng/mL BPA was made by diluting 5.00mL of the STOCK solution to 100.mL. A 5mL (+ 0.010mL) pipette was used. Note: All dilutions were carried out with water and ethanol in a 1:1 volumetric ratio. This was done because BPA is very poorly soluble in water but dissolves readily in alcohol69, which allows the solution to be more uniform. Furthermore, rubber stoppers were used to close all of the flasks. Pipettes that were used repeatedly were rinsed after each use. 69

Allard, Patrick, and Monica P. Colaiacovo. Bisphenol A Impairs the Double-strand Break Repair Machinery in the Germline and Causes Chromosome Abnormalities. Rep. Ed. Joan Ruderman. PNAS, 6 Oct. 2010. Web. 27 July 2012. <http://www.pnas.org/content/107/47/20405.full>. xxxi

1.2 Method for preparing the test samples: 1. For the first test sample, 500mL of distilled water were measured. The liquid was poured into the test pitcher, and let stand at room temperature for 60mins. 2. Meanwhile, 20mL of ethanol were measured in a 50mL flask (using a glass funnel70) with a rubber stopper. 3. With a volumetric pipette (20mL), 20mL of the sample were collected and added to the ethanol. The rubber stopper was inserted and the flask stirred slightly. This is the sample water at RT. 4. For the second sample, steps 1., 2., and 3. were repeated with 500mL vinegar. 5. For the third sample, 500mL distilled water were measured and poured into the test pitcher. A hot water bath was prepared by filling it with distilled water and setting it to ~ 82Ë&#x161;C. The pitcher was inserted into the hot water bath as much as possible without allowing any of the water from the bath to pour into the pitcher, and covered. The pitcher stood for 60mins before being removed carefully, and let cool until it was no longer hot. Steps 2. and 3. were repeated for this sample. 6. For the fourth sample, steps 5., 2., and 3. were repeated with 500mL vinegar. 7. For the fifth sample, 500mL water were measured and poured into the pitcher. The pitcher was inserted into a microwave operating at 1250W71, and let run for 2.00mins. The pitcher was then removed and let cool. Steps 2. and 3. were repeated for this sample. 8. For the sixth sample, steps 7., 2., and 3. were repeated with 500mL vinegar.

70 71

It is significant to use a glass funnel instead of a plastic funnel to avoid contamination. The microwave used was a Panasonic 'INVERTER', 1250W. xxxii

9. For the seventh sample, 500mL water were measured and poured into the pitcher. The pitcher was inserted into the microwave, and let run for 5.00mins. The pitcher was removed and let cool. Steps 2. and 3. were then repeated for this sample. 10. For the eighth sample, steps 9., 2., and 3. were repeated with 500mL vinegar. Note: Between samples, the pitcher, funnel, and pipette were rinsed.

Appendix 2: HPLC Methods 2.1 Mobile phase A consisted of 10mM monobasic sodium phosphate. For this, 0.01mol of the monobasic sodium phosphate were measured. As the molar mass is 119.98g/mol, the required mass was calculated: m = 0.01mol x 119.98g/mol = 1.20g NaH2PO4 After zeroing a scale, 1.20g of the monobasic sodium phosphate were measured, then diluted to 1L in a 1L flask with HPLC-grade (distilled and filtered) water. This was then mixed/stirred and run through a filtration device. For this experiment, a MILLIPORE Stericup Filter Unit with a 0.22Âľm pore size and 1000mL volume was used. A vacuum was applied and the liquid poured through. This sterilized and filtered the particles in the solution, and finally gave 1L with 10mM monobasic sodium phosphate. Mobile phase B consisted of 100% Acetonitrile. Measure out 1L in a suitable glass vial. Both mobile phases were attached to the HPLC apparatus in separate glass bottles, into which a tube from the HPLC was attached.

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2.2. To wash out the HPLC apparatus, the following method was used: a) The apparatus was run with all pumps switched on with E-grade water for 5mins at a flow of 0.9mL/min. b) The flow was increased to 5mL/min and run for 5mins. c) The flow was decreased to 0.9mL/min again for 5mins., then all pumps were closed. d) The column was attached to the HPLC. e) All pumps were turned on, and the column was washed by running the HPLC apparatus for 10mins with 5% Acetonitrile. f) The concentration of Acetonitrile was increased to 70% and let run for 10mins. g) The surroundings of the HPLC were checked for any leaks. It was discerned that the waste container was available and empty, collecting the waste without spillage. Note: The HPLC was deemed as thoroughly washed when there were no significant disturbances on the chromatographs.

2.3. After using the HPLC apparatus and column, it is important to clean both, which will remove any stuck impurities and prolong the lifetimes of the equipment. The following method was employed: a) The mobile phases were switched so that mobile phase A was Q-grade water and mobile phase B was methanol. b) The column was flushed with 5% methanol for 5mins at a flow of 0.9mL/min. c) The gradient was increased to 70% methanol and the column was flushed until the pressure stabilized and no more significant disturbances were seen on the chromatograph. d) The column was then removed from the HPLC.

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e) The mobile phase B was changed to ISOPROPANOL (IPA), and the system was flushed with 100% Isopropanol for 10mins. f) All waste was disposed of safely.

Appendix 3: HPLC Results 3.1 The BPA standard curve shown on page 15 was made using the HPLC software after omitting the values for the 500ng/mL BPA standard. This was done because the standard seems to have been erroneous. The standard curve was also done including the 500ng/mL BPA standard, and is shown below. It can clearly be seen that the other standards follow a relative trend, while the 500ng/mL BPA standard does not:

BPA Standard Curve (w/ 500ng/mL BPA)

700 y = 0.6273x -‐ 15.1 R² = 0.91017

600

Peak Area

500 400 300 200 100 0 -‐100

100

200

300

400

500

600

700

800

900

1000

BPA Concentration (ng/mL)

Also, excluding the 500ng/mL BPA standard from the standard curve increased the r2 value from 0.9102 to 0.9998, which is a significant improvement and indicates a very good fit of the data points to the regression line.

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3.2. The HPLC software calculated several statistics about the BPA standard curve, shown in the table on the following page: Table 2.1 Best-fit values Slope

0.6760 ± 0.004898 6.233 ± 2.202 -9.221 1.479

Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope 0.6604 to 0.6915 Y-intercept when X=0.0 -0.7738 to 13.24 X-intercept when Y=0.0 -19.82 to 1.132 Goodness of Fit r² 0.9998 Sy.x 4.257 Is slope significantly non-zero? F 19040 DFn, DFd 1.000, 3.000 P value < 0.0001 Deviation from zero? Significant Data Number of X values 5 Maximum number of Y replicates 1 Total number of values 5 Number of missing values 0 Particularly significant here is the r2 value, which is very close to 1; this shows that the standards have a good fit to the regression line.

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3.3 On the following pages are the identified BPA peaks of the test samples. They were cut from the separate chromatographs and overlapped to allow comparison:

Chromatograph 2.1: Test Sample Peaks

In this chromatograph, the FLD response of a sample of distilled water has also been included to see whether there may be any impurity in the water that could have a similar retention time as BPA, hence causing the test samples (with water) to give a false positive. However, the water sample gives no peak at a retention time of approx. 10.9min, while the two first samples do, suggesting that the substance is indeed BPA.

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Chromatograph 2.2: Further Test Sample Peaks

Also, to give an example of the data obtained directly from the HPLC software, the FLD response and 'Area Percent Report' for the sample 'Water, 1250W, 5mins' are given on the following page(s).

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Original Chromatograph Report (includes FLD1 Response and Area Percent Report)

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Area Percent Report (continued)

Appendix 4: Calculations 4.1 Following are the calculations made to conclude approximately how much BPA an average adult male would consume daily if the maximal concentration of BPA found in this experiment were in the consumed liquid. Assuming the average adult male weighs 80kg and drinks 2 liters daily: Concentration BPA = 76.2ng/mL

1ng/mL = 0.001µg/mL

76.2ng/mL x 0.00100 = 0.0762µg/mL 0.0762µg/mL x 2000mL = 152µg in 2L 152µg/80kg/day = 1.90µg/kg/day 1.90µg/kg/day < 50µg/kg/day xl