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DETECTION OF AND STRONTIUM IN WATER

UTILIZING FUNCTIONALIZED SILVER NANOPARTICLES

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A Thesis

Presented to the Honors Tutorial College

Ohio University

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In Partial Fulfillment of the Requirements for Graduation from the Honors Tutorial College with the Degree of

Bachelor of Science in Chemistry

______by

Ashley Cobbs

May 2020 2

This thesis has been approved by

The Honors Tutorial College and the Department of Chemistry and Biochemistry

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Dr. Anthony S. Stender

Assistant Professor, Chemistry & Biochemistry

Thesis Advisor

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Dr. Lauren E. H. McMills

Associate Professor, Chemistry & Biochemistry

Director of Studies

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Dr. Donal C. Skinner

Dean, Honors Tutorial College

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Abstract

Throughout the world, pollutants in water have become commonplace, largely as the result of human activities. Environmental organizations test waterways all over the

United States for a wide array of polluting agents, from organic materials found in pesticides, to harmful bacteria, to heavy . The standard instrument used to measure the quantity of metals in water samples is an inductively coupled plasma spectroscopy.

This specialized instrument requires extensive training and is expensive to purchase, operate, and maintain. As a result, it places a burden on the testing of water samples and can prohibit water from being tested in a timely manner. Therefore, there is a current need for a faster process to evaluate water samples. The overall objective of this study was to assess the viability of utilizing functionalized silver nanoparticles to economically determine the concentrations of cations, specifically barium and strontium ions, in contaminated water samples. The ligands on the nanoparticles selectively bind the ions, which induce a color change in the nanoparticles that is detectable by a UV-Vis spectrophotometer, a relatively cheap analytical instrument. Significant progress towards the practical application of functionalized nanoparticles with environmental samples was made. After the addition of high concentrations of barium and strontium ions, nanoparticle samples shifted color from yellow to pink, signifying the feasibility of bare- eyed detection. Creating samples with a range of ions from 1 mM to 100 mM, a rough detection range for barium and strontium ions were determined. Regression analysis indicated a strong trend between plasmonic resonance absorbance wavelengths and the concentrations of ions in water. With these findings, there are many avenues for future work. To name a few, different nanoparticles could be used, the possibility of interfering factors common in water samples should be investigated, and further data at different concentrations of ions could be collected to produce more precise trend lines. 4

Table of Contents

Abstract ...... 3 List of Figures ...... 6 List of Tables ...... 8 List of Abbreviations ...... 9 I. Introduction ...... 10 1.1 Impact on Health ...... 10 1.2 Barium and Strontium ...... 11 1.3 Instrumentation...... 12 1.3.1 Instrumentation Comparison ...... 12 1.3.2 Instrumentation Theory ...... 13 1.4 Research Objectives ...... 19 1.4.1 Research Purpose ...... 19 1.4.2 Research Benchmarks ...... 20 1.5 Nanoparticles ...... 20 1.5.1 General Background ...... 20 1.5.2 Plasmonics ...... 21 1.5.3 Binding to Barium and Strontium ...... 23 II. Experimental Methods ...... 25 2.1 Water Quality Comparison...... 25 2.1.1 Background ...... 25 2.1.2 Instrumentation & Sample Preparation ...... 26 2.2 Nanoparticle Experiments ...... 28 2.2.1 Background ...... 28 2.2.2 Instrumentation & Sample Preparation ...... 30 2.2.3 Methods Development ...... 32 2.2.4 Final Method...... 34 III. Results & Discussion ...... 36 3.1 ICP Experiment, Water Quality Test...... 36 3.2 Experiment #4, Tris-HCl Study ...... 42 3.3 Experiment #5, 10 µM versus 10 mM ...... 43 3.4 Experiment #6, Barium Concentration Range ...... 45 5

3.5 Experiment #7, Red-Shift Trends ...... 47 3.5.1 Spectra ...... 47 3.5.2 Trend Analysis ...... 51 3.6 Experiment #10, Pink Color Change ...... 54 3.7 Experiment #14, Citrate versus PVP ...... 58 IV. Conclusion ...... 62 5.1 Concluding Remarks ...... 62 5.2 Future Work ...... 63 V. References ...... 66

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List of Figures

Figure 1: Diagram of ICP plasma torch, adapted from Harris’ textbook.14 ...... 15 Figure 2: listing the theoretical detection limits of samples suspended in solution for ICP-OES (in blue text) and ICP-MS (red text). Using instruments with a MS detector are extremely sensitive. Figure adapted from Evans Analytical LLC.15 16 Figure 3: Double-beam UV-Vis spectrophotometer adapted from Harris’ textbook.14 ...... 17 Figure 4: A nanoparticle with a localized surface plasmonic resonance induced by an electric field. Figure adapted from Kelly et al.20 ...... 21 Figure 5: The variable a is the radius of a gold nanosphere and is equivalent to 60 nm while the variable d is the distance between two gold nanospheres and is the independent variable in this figure. (a) illustrates the red-shifting and formation of multiple peaks as the distance between two nanospheres get closer together. Figure adapted from Romero et al.23 ...... 22 Figure 6: Diagram of how silver nanoparticles bind to a barium via thioglycolic acid ligands. In this case, the nanoparticle is silver and is represented by “Ag”...... 24 Figure 7: Experiment #4. First time using 10 nm silver nanoparticles in citrate instead of 50 nm and adding 10 mM barium and strontium solutions instead of 10 µM. Experiment performed on April 9th and 12th, 2019...... 43 Figure 8: Experiment #5. Determining the ability for the UV-vis to differentiate between 10 µM and 10 mM metal samples. Experiment performed on June 10th and 11th, 2019. ... 45 Figure 9: Experiment # 6. To observe the effect of varying barium concentrations on the absorption spectra. Experiment was performed on June 19th and 20th, 2019...... 47 Figure 10: Experiment # 7. trial of previous experiment. 5 mM metal concentration was added. Strontium metal concentrations were also measured (bottom graph). Main deviation from the previous experiment was the addition of 50 µL 100 mM tris-HCl. Experiment was performed on June 26th and 27th, 2019...... 50 Figure 11: Trend analyses for multiple spectra characteristics of the UV-Vis barium nanoparticle experiment #7. These spectra characteristics are for the shifted LSPR absorbance peak around 395 nm and include: peak location, peak height, peak area, and 7

peak width. The peak area was calculated by the UV-Vis spectrophotometer program and accounts for all the area under the merging peaks...... 53 Figure 12: Trend analyses for multiple spectra characteristics of the UV-Vis strontium nanoparticle experiment #7. These spectra characteristics are for the shifted LSPR absorbance peak around 395 nm and include: peak location, peak height, peak area, and peak width. The peak area was calculated by the UV-Vis spectrophotometer program and accounts for all the area under the merging peaks...... 54 Figure 13: Experiment # 10. Observing the effect of waiting until the day of data collection to add MSA and carousel for 2 hours instead of performing these steps on an earlier day. The top spectrum is testing with barium ions and the bottom spectrum is testing with strontium ions. Experiment was performed on July 31st and August 7th, 2019...... 57 Figure 14: Experiment #14. Comparing 10 nm nanoparticles that are suspended in PVP versus nanoparticles that are suspended in citrate. The experiment was performed on October 30th, 2019...... 61

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List of Tables

Table 1: Concentrations of standards used for the ICP water quality comparison experiment and volume of ICP multielement standard solution 6 used to make the standards...... 28 Table 2: Concentration of strontium, barium, and dissolved in various water sources. Concentrations determined by ICP-OES. Data collected on July 9th, 2019...... 41 Table 3: The final concentrations of strontium and barium ions in the nanoparticle solutions. The asterisk denotes the first color change detected by the UV-Vis on August 7th, and the carrot indicates the first noticeable dip in intensity detected by the UV-Vis in experiment #10...... 58

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List of Abbreviations

AU Arbitrary Units

DI Deionized

EPA Environmental Protection Agency

HCl Hydrochloric acid

HOMO Highest Occupied Molecular Orbital

ICP Inductively Coupled Plasma

LSPR Localized Surface Plasmon Resonance

LUMO Lowest Unoccupied Molecular Orbital

MCL Maximum Contaminant Level

MS Mass Spectrometry

MSA 2-mercaptosuccinic acid

NPDWR National Primary Drinking Water Regulations

NPL National Priorities List

OES/AES Optical Emission Spectroscopy/Atomic Emission Spectroscopy ppb Parts per billion ppm Parts per million

PVP Polyvinylpyrrolidone

UP Ultrapure

UV-Vis Ultraviolet to Visible Range

WHO World Health Organization

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I. Introduction

1.1 Impact on Health

The presence of trace metals in waterways has become an increasing problem around the world due to industrial contamination. When a metal comprises less than

0.1% of a sample of water, or less than 1,000 parts per million (ppm), it is considered a trace metal.1 The necessity for water testing and remediation has increased, especially in areas where manufacturing plants and fossil fuel industries have played an integral aspect of the economy. Due to the boom in their usage by many industrial sectors, elements such as antimony, barium, beryllium, cadmium, chromium, fluorine, mercury, and selenium are on the Environmental Protection Agency’s (EPA) National Primary

Drinking Water Regulations (NPDWR) list.2 The NPDWRs detail the legal concentration limits of commonly found contaminants found in United States public water systems that negatively impact consumers’ health.3

To illustrate the prevalence of contaminated water, at least 150,000 Americans are chronically exposed to a greater concentration of barium than the EPA’s stated maximum contaminant level (MCL) of 2 ppm.4 Of the 1,684 hazardous sites around the country that are monitored by the EPA on their National Priorities List (NPL), 798 sites have detectable amounts of barium ions and compounds.5 The fact that just under half of the

NPL sites contain barium is also very likely an underestimate, as not all NPL sites are tested for barium, and the number of sites that didn’t test for barium are unknown.5 In order to properly protect the health of citizens and enforce contamination limits to 11 polluters, water quality testing needs to be performed with more regularity for all compounds on the NPDWR list.

1.2 Barium and Strontium

Barium and strontium are the two metal cations being focused on in this study.

Barium was chosen for closer scrutiny in this work, because it increases blood pressure in humans2 and can result in ‘cardiovascular and kidney diseases, metabolic, neurological, and mental disorders’4 when in high amounts. The EPA lists the MCL for barium as 2 ppm.2 Strontium has no known health effects, and therefore no MCL is listed by the

EPA,2 but the strontium-90 is radioactive, carcinogenic, and mutagenic.6 A normal concentration of strontium in river water is 50 parts per billion (ppb).6 Therefore, strontium concentrations over 500 ppb, or 0.5 ppm would be considered an excess.

Barium and strontium ions occur naturally in waterways since soluble compounds of both are common in the Earth’s crust.7,8 Barium takes up 0.04% of the Earth’s crust and is commonly found in barite as barium and in as barium .7

Places from around the world including Morocco, China, India, and the United Kingdom mine for barite to extract barium, and barium comprises up to 3,000 mg/kg of coal.7

Strontium takes up 0.02% of the Earth’s crust and is commonly found in celestite as and in as .8 Strontium compounds can also be deposited in sediments such as gypsum, , rock , limestone and dolomite and can comprise up to 4,000 mg/kg of coal.8 12

The presence of barium and strontium in waterways only becomes concerning when their concentrations are high (2 ppm for barium, and 0.5 ppm for strontium). High concentrations of these metals strongly suggest the presence of other contaminants such as carbonate, phosphate, iron, and ,4 usually due to industry that is affecting the water nearby. These industries include hydraulic fracturing, mining, barium refineries,2

CRT TV production, , and strontium refineries.6 Nuclear reactors and nuclear bomb tests result in the production of radioactive strontium-90.6

1.3 Instrumentation

1.3.1 Instrumentation Comparison

Water testing for metals present at trace levels (barium, strontium, , antimony, etc.) is conventionally accomplished using an expensive instrument that requires extensive training, such as an inductively coupled plasma spectrometer (ICP). A new

ICP instrument costs $250,000. Additionally, the cost of gas and replacement parts for

1,000 hours of instrument use ranges from $5,800-$14,500 (depending on the instrument model and parts that need to be replaced). This means the minimum cost of running an

ICP is between 34 ¢ to 78 ¢ per sample, exclusive of labor costs.9 If a lab does not have direct access to an ICP, samples must be sent to a private company or to a government lab. It can take up to 6 months to receive results from the Ohio EPA, resulting in a lengthy delay before the start of remediation efforts.10 In that time, the chemistry of the water at the sampling site may change, altering the biome. The ramifications could be ineffective remediation or even the loss of a species with a low tolerance for 13 environmental changes. The current cost of a comprehensive trace metals ICP test locally is $15.75/sample (at Ohio State University), not including shipping or traveling.11

This option is much more expensive than the price listed above, due to the inclusion of labor.

A proposed and less costly alternative approach is the development of portable sensing technology that could detect high levels of barium or other metals within moments at the sampling site itself. For example, one could use functionalized nanoparticles that change color instantly when they bind to specific metal ions in water.

This is much easier and cheaper than the ICP method, as the color change can be detected by human eyes if the metal ions are in high enough concentration, or readout by an ultraviolet to visible range (UV-Vis) spectrophotometer at lower concentrations. A new benchtop UV-Vis spectrophotometer with a large wavelength range costs $65,000 and requires cuvettes ($145 for 500 disposable cuvettes or $128 for a pair of reusable quartz cuvettes).12,13 It is possible this method could become even cheaper by utilizing a handheld spectrophotometer. Granted, most UV-Vis instruments cannot be transported into the field, but it is still a faster and cheaper method than ICP.

1.3.2 Instrumentation Theory

An ICP is typically used for water quality assessment as it can process and detect most elemental contaminants found in the environment if the sample is in an aqueous state. These instruments are also considered the gold standard as they perform reliable and repeatable multi-elemental analysis. A single ICP run can determine the 14 concentrations of many elements at once, even at trace amounts.9 The downside to using

ICP to perform elemental analysis is that it only detects the element and “cannot reveal anything about the metal’s oxidation sate, alkylated form, or how it is bound to a biomolecule.”9

A single ICP run typically requires 1mL of sample, but sample size can be much smaller for specialty nebulizers (5uL through direct injection nebulizers or 500uL for an electrothermal vaporization nebulizers).9 Even though only 1mL of sample is needed for a run, normally each standard and sample is tested three or more times in order to obtain statistically significant data. Therefore, a minimum of 10mL of sample should be collected for analysis.

After sample uptake, the sample is transported through tubing to a nebulizer. The nebulizer will agitate the liquid to evaporate and condense off the solvent to form a fine aerosol that will mix with inert argon gas in the cyclone chamber.14 The subsequent aerosol mixture flows into the ICP plasma torch, as seen in the bottom of Figure 1. In order to break compounds down into their elemental composition, a massive amount of energy must be applied to the bonds keeping the elements together in the compound. To obtain this energy, the Tesla coil in the torch produces sparks which ionize the argon gas to Ar +, releasing electrons. The free electrons then travel to a 27 or 41-MHz radio- frequency load coil near the end of the torch where they are accelerated and collide with the incoming aerosol to atomize all the molecules in the plasma with a temperature ranging from 6,000 - 10,000 K.14

Though all ICP instruments quantify elements suspended in an aqueous solution, optical emission spectrometry (OES) and atomic emission spectrometry (AES) detectors 15 collect data differently than the mass spectrometry (MS) detector. OES and AES detectors function by promoting electrons in the highest occupied molecular orbital

(HOMO) of atoms to the lowest unoccupied molecular orbital (LUMO) using energy from the plasma. The electron falls back down to the HOMO, releasing energy as light.9

Each element has a different energy gap between the atom’s HOMO and LUMO, allowing the element to be identified by the light’s energy, detected via color.9

Figure 1: Diagram of ICP plasma torch, adapted from Harris’ textbook.14 16

Figure 2: Periodic table listing the theoretical detection limits of samples suspended in solution for ICP-OES (in blue text) and ICP-MS (red text). Using instruments with a MS detector are extremely sensitive. Figure adapted from Evans Analytical Group LLC.15 17

An MS detector functions by ionizing incoming atoms with energy from the ICP and recording the atomic mass.9 Since atomic mass is measured with an MS detector, can be differentiated unlike with an OES or AES detector, as the number of neutrons changes an element’s atomic mass but not the HOMO-LUMO energy gap.9,16

As seen in Figure 2, ICP-MS has better detection limits than ICP-OES ranging from detection limits that are 15 ppb better (silicon) to eight orders of magnitude better

(cesium).15 It is worth noting that barium and strontium are ideal candidates for testing with a method less sensitive than ICP, such as UV-Vis, as the MCL for barium is 2 ppm and an ICP-OES reliably detects barium at 0.5 ppm levels.

14 Figure 3: Double-beam UV-Vis spectrophotometer adapted from Harris’ textbook.

The important parts of a UV-Vis spectrophotometer are illustrated in Figure 3 above. A UV-Vis spectrophotometer may have one, two, or three light sources. In the example below, the spectrophotometer has two: a tungsten lamp and deuterium lamp.

Alternating between the two lamps allows a full light scanning range of 110 - 2,500 nm.14

Other versions rely on a single lamp, but have a narrower working range as a result. Next, the incoming light will pass through a scanning monochromator which allows one wavelength at a time to go through the sample.14 The monochromator repeats this 18 process with all following wavelengths until the desired spectrum goes through the sample.

The next component, a beam chopper, is specifically for double-beam spectrophotometers. A beam chopper is a rotating component that is partially covered in a mirror and has a part that is hollow, alternates between allowing light to pass through and reflecting it. The light that passes through the chopper reaches the sample cuvette with the analyte, and the light that is reflected goes through a reference cuvette that gives a constant background measurement, usually of the solvent the analyte is in.14

Lastly, the light transmitted through the sample and reference cuvettes are detected by passing through and reflecting off of a semitransparent mirror, respectively.14

The detector and amplifier can be the same device, and in the case of many UV-Vis spectrophotometers is a photo-multiplier tube. A photo-multiplier tube detects incoming signals when the photons of light impact a photosensitive surface that releases electrons with each photon impact. These electrons crash into a series of slightly more positive photosensitive surfaces in the photo-multiplier tube, which releases further electrons and amplifies the signal until all the released electrons collect on an anode.14 In this way, a single photon can cause over 106 electrons to be detected by the anode after a series of nine photosensitive surfaces.14

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1.4 Research Objectives

1.4.1 Research Purpose

This research project studied the feasibility of utilizing functionalized silver nanoparticles to selectively bind alkaline earth metals, specifically barium2+ and strontium2+, in water samples and quantify samples with a UV-Vis spectrophotometer as an alternative to ICP. Barium and strontium were chosen, because they are common ions that are found in every water source, but their presence in higher than expected levels suggests that a waterway contains additional pollutants (including but not limited to carbonate, phosphate, iron, and radium).4 This test was considered presumptive as it does not quantify the exact amount of these ions present, but shows if they are present in excess. Detecting these ions in excess will help prioritize which waterways should receive further testing with more sophisticated techniques capable of accurate quantification.

While this research is primarily focused on water quality and the impact these ions have on the environment, strontium and barium are major components of gunpowder and some low explosives, and therefore a presumptive test for these ions can be used in forensic chemistry applications as well.

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1.4.2 Research Benchmarks

i. Use a benchtop UV-Vis spectrophotometer to detect and quantify the amount of

barium and strontium in water samples utilizing functionalized nanoparticles

ii. Determine the lower and upper limits of detection for both ions using this

method

iii. Accurately quantify the ions in a field-collected water sample (tap water, then

filtered stream water)

iv. Determine if pH interferes with the detection of the ions

1.5 Nanoparticles

1.5.1 General Background

Nanoparticles are becoming popular as colorimetric assays because they are compatible with water, considered non-hazardous, have considerably higher visible light- region extinction coefficients than conventional organic dyes, and do not bleach over time like organic fluorescence-based dyes.17,18 These traits are important as barium and strontium are colorless when suspended in pure water.

Nanoparticles are considered particles that have a diameter much less than 500nm.

In some cases, materials of this scale have properties different than that of their bulk materials.19 The main focus of this project is on the optical properties of plasmonic nanoparticles, which are described below. 21

1.5.2 Plasmonics

Figure 4: A nanoparticle with a localized surface plasmonic resonance induced by an electric field. Figure adapted from Kelly et al.20

Plasmonic nanoparticles have different optical properties than their bulk material counterparts, because they exhibit a property called localized surface plasmon resonance

(LSPR). LSPR is the interaction between light and the outer electrons of the nanoparticles.21 Plasmonic nanoparticles can only exhibit their optical properties under light when their radii are much smaller (5 – 100 nm) than the wavelengths of light being used (300 – 1000 nm).19 However, the LSPR effect is only observed over a narrow range of wavelengths, not across the entire spectrum. Gold and silver are two of the metals that exhibit this effect; the majority of metals do not have this property.

The LSPR effect is permitted because the nanoparticles are small enough to sustain an induced electric dipole. An electric dipole occurs when a pair of equal yet opposite charges are minimally separated in space, like the bond between and atoms in a water molecule.22 While many systems naturally exhibit a dipole, some need to be in the vicinity of an electric field for a dipole to be induced. In either dipole system, there will be an overall net neutral charge.22 What makes metal 22 nanoparticles special is that the electrons will only collectively shift and produce a strong dipole if induced by a specific and narrow band of wavelengths.20,21 When incoming light of these wavelengths hit the nanoparticle, the dipole it induces oscillates at the same frequency as the light due to the restorative force between the negative charge of the electrons and the positive charge of the collective nuclei.19,20 This process is illustrated in

Figure 4 and is the basis of LSPR. When nanoparticles undergo the LSPR effect, they scatter and absorb light with high intensity, which makes them amenable to being observed with UV-Vis spectroscopy.

Figure 5: The variable a is the radius of a gold nanosphere and is equivalent to 60 nm while the variable d is the distance between two gold nanospheres and is the independent variable in this figure. (a) illustrates the red-shifting and formation of multiple peaks as the distance between two nanospheres get closer together. Figure adapted from Romero et al.23 23

Every nanoparticle of the same shape, size, and composition will display the same

LSPR frequency, but as particles increase in size, the LSPR band will begin to red-shift and grow wider. The red-shifting and widening of LSPR peaks are the properties that are exploited when using nanoparticles as colorimetric assays, including this study. When ions are added to nanoparticle solutions, the nanoparticles will interact with the metal and begin to cluster around it, effectively causing the nanoparticles to have a larger volume and therefore a red-shifted LSPR peak.21 Additionally, particles will start to aggregate together due to electrostatic attraction between multiple particles and the ions, increasing their size even further. When particles come into sufficiently close proximity or direct contact, it will alter the spectral profile observed from the nanoparticles, potentially into very complex patterns compared to that displayed by single, isolated nanoparticles, as shown in Figure 5.13 Finally, it should be noted that not all particles will necessarily cluster together, therefore in a UV-Vis spectrum, it would be common to see both the original LSPR peak, indicative of isolated nanoparticles, along with the shifted peak that represents aggregates, especially when the ions are present in low concentrations.

1.5.3 Binding to Barium and Strontium

The process of barium or strontium ions binding to a silver nanoparticle occurs via covalent bonding through a ligand that is bound to both the ion and the nanoparticle, illustrated in Figure 6. The ligand usually has a thiol group that binds to the nanoparticle surface via its sulfur atom.18,24,25 The other end of the ligand, in these experiments, has a carboxylic acid that interacts with the barium and strontium ions via electrostatic 24 attraction.18,24,25 Because barium and strontium ions have a 2+ charge, they can bind with two silver nanoparticles at once, and because each nanoparticle has multiple ligands, the particles end up aggregating in solution. Other ligand options include thioglycolic acid,

11-mercaptoundecanoic acid, 2-mercaptosuccinic acid, or crown ethers just to name a few.18,24,25 For these experiments, 2-mercaptosuccinic acid (MSA) was used. The lowest detection of barium ions detected via MSA in the literature is 2 µM, while it is 8 µM for strontium ions.25

Figure 6: Diagram of how silver nanoparticles bind to a barium ion via thioglycolic acid ligands. In this case, the nanoparticle is silver and is represented by “Ag”.

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II. Experimental Methods

2.1 Water Quality Comparison

2.1.1 Background

The purpose of this experiment was to determine the background levels of barium,

strontium, and calcium in different sources of faucet and filtered water. The faucet water

samples came from a cold water tap in Clippinger Laboratories room 173 (Ohio

University, Athens, OH) and a cold water tap in a residential building within Athens’ city

limits. The filtered water samples came from an ultra-pure (UP) water filtration system

within Clippinger Laboratories and the residential water going through a standard Brita

filter.

UP water is water that had its ions filtered out until the resistivity is increased to

18 MΩ*cm and the maximum ion concentration is 1 µg/L, which is approximately 1

ppb.26 On Brita’s webpage, they claim their standard filter has activated carbon to reduce

the level of chlorine and an ion-exchange resin that reduces the level of mercury,

cadmium, copper, and zinc.27 The company does not state the amount of ions that make

it through the filter nor does it mention any other ions the ion-exchange resin captures.

There are a few objectives for this ICP test. The first is to determine the amount

of barium, strontium, and calcium ions in Ohio University and Athens city faucet water.

The next is to verify that the UP water used in the lab is truly ultra-pure. Lastly, the

amount of barium, strontium, and calcium ions a standard Brita filter filters out of faucet

water, if at all. This testing provides a sense of local levels of these ions, and these data 26 are important in establishing baseline levels of ions prior to performing the colorimetric tests.

2.1.2 Instrumentation & Sample Preparation

Instrumentation

1. ICP-OES

The ICP-OES used for this experiment was a Thermo Scientific iCAP 600 series instrument. This set-up requires both argon and gas cylinders. The instrument, along with the gas cylinders and auto-sampler, are housed at Ohio University’s Institute for Sustainable Energy and the Environment.

The hardware parameters were set to have a pump speed of 50 rotations per minute (rpm) and nebulizer and auxiliary gas flow rates of 0.5 L/min. The detector simultaneously measured the barium at emission wavelengths of 455.403 nm and

233.527 nm, strontium emission wavelengths of 407.771 nm and 216.596 nm, and calcium emission wavelengths of 422.673 nm and 393.366 nm. Both barium and strontium detection wavelengths were scanned axially while both calcium detection wavelengths were scanned radially. Axial scans go through the longer dimension of the plasma effectively having a longer pathlength and are more sensitive than radial scans in which the detector goes through the narrow dimension effectively having a shorter pathlength.28 As high amounts of calcium are not uncommon in drinking water, the less sensitive radial scan was used. 27

2. Auto-Sampler

The Thermo Scientific CETAC™ ASX-520 AutoSampler was used for sample handling. This auto-sampler has a resting solution of 1% aqueous nitric acid where it rinses itself between every standard and sample. The sample uptake time was one minute and the autosampler rinse time was 30 . The software for the ICP and auto- sampler is Thermo Scientific’s Qtegra™.

Sample Preparation

1. Samples

The laboratory faucet water sample was obtained by letting the main water line in

Clippinger Laboratories room 173 run for 30 seconds before letting it directly fill a new

20 mL glass vial. The laboratory distilled water sample was obtained by pouring 20 mL of UP water from a 1 L glass reservoir in which the water originated from an UP filtration system in Clippinger Laboratories. The municipal faucet water was obtained by running a tap for 30 seconds in a residential building within the Athens city limits before filling a new 20 mL glass vial directly. The municipal faucet water through a Brita filter sample was obtained by running the same residential faucet water through an empty Brita pitcher and pouring the contents into a clean 20 mL glass vial. All vials were sealed with

Parafilm immediately after filling and capping and then stored in a refrigerator until 30 minutes before the sampling time.

2. Standards

Sigma Aldrich’s Multielement Standard Solution 6 for ICP was used to create the five standards in this experiment. This stock solution contains 23 elements including 28 barium, strontium, and calcium, all with a concentration of 100 ppm. The standards were made by diluting the volume of the stock solution displayed in Table 1 in a 25 mL volumetric flask with deionized (DI) water. DI water has a resistivity less than 18.2

MΩ*cm, which means it had more ions than the UP water sample from Clippinger

Laboratories. A study by the World Health Organization (WHO) in the Netherlands found the average concentration of barium in the groundwater to be 0.23 ppm with a maximum of 2.5 ppm.29 Based on those findings, the standard concentrations were made to have concentrations slightly below and above these values, ranging from 0.1 ppm to

5.0 ppm.

Standard # 1 2 3 4 5 Standard Concentrations 0.1 0.5 1.0 2.0 5.0 (ppm) Volume of ICP 6 used 25 125 250 500 1250 (µL)

Table 1: Concentrations of standards used for the ICP water quality comparison experiment and volume of ICP multielement standard solution 6 used to make the standards.

2.2 Nanoparticle Experiments

2.2.1 Background

Nanoparticles in colloid form normally are suspended in solution with surfactant molecules and have stabilizing ligands on them to prevent aggregation.30 When the colloidal suspension is no longer stable, nanoparticles begin to aggregate and/or dissolve, effectively expiring the nanoparticles. In order to utilize LSPR wavelength shifts due to 29 silver nanoparticles aggregating in the presence of alkaline Earth metals, the stabilizing ligands, like PVP, citrate, CTAB, etc. must be replaced. The replacement of ligands to tailor nanoparticles for specific functions is called functionalization.

Although the same functionalization ligand, MSA, is used in this project as Zhang et. al., there are a few key differences between the experiments conducted here and those by Zhang, et al.25 One is that the use of 50 nm silver nanoparticles capped with citrate,

10 nm silver nanoparticles capped with citrate, and 10 nm silver nanoparticles capped with polyvinylpyrrolidone (PVP) were investigated here instead of 13 nm gold nanoparticles capped with citrate, as (1) silver nanoparticles are cheaper than gold and (2) more ligands can bind to a larger particle, which increases the amount of cations that can be captured. Secondly, the main goal of this project is to find a mathematical model for

UV-Vis absorbance trends. In other words, the goal was to make a calibration plot from which it will be possible to determine the unknown concentration of ions in water samples. Next, said model was used to attempt to quantify barium and strontium in water samples, and the results were compared against those gathered on an ICP-OES. Lastly, an investigation on the possible interference effects of pH was done.

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2.2.2 Instrumentation & Sample Preparation

Instrumentation

1. UV-Vis Spectrophotometer

The instrument used for the nanoparticle experiments was the Shimadzu UV-VIS-

NIR Spectrophotometer UV-3600Plus. This instrument relies on deuterium and lamps to scan samples across the range of 190 – 3600 nm to determine their absorbance properties. This is a double-beam spectrophotometer which allows a sample and the blank to run simultaneously. For all the UV-Vis readouts except the one shown in Figure

14, the scan speed was set to fast and the scanning interval set to every 0.5 nm. For the

UV-Vis readout shown in Figure 14, the scan speed was set to slow and the scanning interval set to every 1 nm. Absorbances were recorded from 300 – 700 nm, as this is the expected range the nanoparticles exhibit maximum absorbance.

2. Centrifuge

An Eppendorf Centrifuge 5425 with a radius of 8.40 cm and the capacity to hold up to 24 2.0 mL centrifuge tubes was used to remove nanoparticle surfactant in these experiments. All samples were centrifuged for different lengths of time at 8,500 rpm which is equivalent to 6,785 g-forces. For centrifuges, g-force is dependent on rotation speed and rotation radius. For example, a wider centrifuge at the same rotation speed with have more g-forces. The samples in the experiment that produced Figure 7 centrifuged for 10 minutes, the samples used to produce Figure 8 centrifuged for 30 minutes, Figure 9 to Figure 12 centrifuged for 25 minutes, and Figure 13 and Figure 14 centrifuged for 20 minutes. 31

Sample Preparation

1. Nanoparticles

All experiments reported in this thesis used 10 nm silver nanospheres suspended in citrate. In the last experiment, 10 nm silver nanospheres suspended in PVP were used in conjunction with those suspended in citrate to determine if there was a significant optical response difference in how they responded to aqueous metal ions.

The first step in the preparation of the nanoparticles was to vortex the native 10 nm silver nanoparticle stock solution on high for 30 seconds. Next, 100 µL of nanoparticles were pipetted into each centrifuge sample tube and centrifuged at 8500 rpm for the amounts of time specified in the centrifuge instrumentation section. After the nanoparticles were removed from solution due to centrifugal forces, the suspension fluid

(75 – 90 µL) was removed via pipette and replaced with an equivalent volume of UP- water. All the sample tubes were sonicated to re-suspend the nanoparticles.

MSA was added to functionalize the nanoparticles. The MSA solution was first vortexed for 30 seconds. Then, 10 µL of MSA were pipetted into each sample tube.

Lastly, the sample tubes were spun on a carousel at 40% speed for 2 hours so the MSA could properly bind to the nanoparticles.

2. Standards

The barium solution was prepared by dissolving 0.49 g barium chloride dihydrate with a reported purity of 99+% in 20 mL UP water to make a 100 mM solution. The strontium solution was prepared by dissolving 0.53 g hexahydrate with a reported purity of 99-103% in 20 mL UP water to make a 100 mM solution. Then, 32 separate 1 mM, 4 mM, 7 mM, 10 mM, 15 mM, and 20 mM solutions for barium and strontium were prepared by diluting the 100 mM solutions to a total volume of 20 mL with UP water. This was completed with 0.2 mL, 0.8 mL, 1.4 mL, 2.0 mL, 3.0 mL, and

3.0 mL aliquots of 100 mL barium and strontium solutions.

The MSA solution used in the experiments was prepared by dissolving the mercaptosuccinic acid powder with a reported purity of 98%. For the experiments up until June 10th, 20 mM MSA was used. From June 10th on, 40 mM MSA was used by dissolving 0.12 g MSA into 20 mL of UP water because the intensity change was on a much smaller scale than reported in the literature and we suspected the nanoparticles were not well functionalized.

The tris hydrochloride (HCl) solution was prepared by diluting the 1 M tris-HCl buffer stock solution with a pH of 7.5. An aliquot of 0.5 mL of the 1 M stock solution was diluted to a total of 20 mL with UP water.

The sodium chloride solution used in some experiments was prepared by dissolving 0.017 g sodium chloride salt into 15 mL UP water.

2.2.3 Methods Development

In total, fifteen nanoparticle experiments were performed in order to perfect the ligand binding methodology; not all of the results are provided in this thesis. In the first three experiments, 50 nm silver nanoparticles were used, but they did not exhibit major intensity changes between metal concentrations. The 50 nm silver nanoparticles were 33 switched out for 10 nm silver nanoparticles after comparing the spectra of 50 nm conditions with the results with the 10 nm particles in experiment 4.

Next, it was suspected that the nanoparticles were not well functionalized as the intensity change was on a much smaller scale than reported in the literature. Therefore, the MSA acid stock solution was increased from 20mM to 40mM from experiment 5 onwards. Intensities greatly increased after this, but still not as expected. In order to improve the amount of metal bound to cause more aggregation, the pH was checked to see if it was still neutral when ions were added to the nanoparticle solution. The pH of all stock solutions and the pH of the nanoparticle at each step were checked in experiment 6.

The step during nanoparticle functionalization and binding with MSA had a pH between

2-3. A tris-HCl stock solution was made and added to the nanoparticle solutions in experiment 7 to compensate, and the next experiment had a pH of 8 upon the addition of metal cations.

New metal ion solutions were made for experiment 8 with concentrations of 1mM,

4mM, 7mM, 10mM, and 15mM instead of the original 1mM, 5mM, 10mM, 50mM, and

100mM solutions. However, we realized the stock metal ion solutions were not all diluted from the same original metal stock solution, and the pipette did not perform as expected. For experiment 9, all the metal stock solutions were re-made diluting solely from the original 100mM stock and using a different set of pipettes.

The order of adding chemical species and which steps required sonication was determined due to an observation made by Rachel Bracker, a graduate student in Dr.

Stender’s lab.31 In experiment 10, the nanoparticle solutions turned pink post-ion addition to varying degrees for the first time, as had been expected. Lastly, it was 34 determined through experiments conducted by Rachel that nanoparticles with citrate quickly lose color because they crash out of solution, but those in PVP don’t.32 However, upon further investigation in experiments 11 through 14, it was determined that nanoparticles coated in PVP had significantly lower absorbance intensities than nanoparticles coated in citrate, so the citrate suspension was used in further trials.

2.2.4 Final Method

The final sample preparation method is as follows. The first step is to vortex the native 10 nm silver nanoparticle stock solution on high for 30 seconds. Then, 100 µL of nanoparticles are pipetted into each centrifuge sample tube and centrifuged at 8500 rpm for 20 minutes. Next, 85 µL suspension fluid is removed via pipette and replaced with 85

µL of UP-water. Finally, all the sample tubes are sonicated for 5 minutes to re-suspend the nanoparticles.

The next step requires the addition of MSA solution. The MSA solution is vortexed on high for 30 seconds. Then, 10 µL of 40 mM MSA are pipetted into each sample tube. The sample tubes then spin on a carousel at 40% speed for 2 hours to ensure the MSA properly binds to the nanoparticles. After vortexing for 30 seconds, 37

µL of 100 µM tris-HCl solution is added to each tube.

Barium and strontium were then added to the sample tubes. The 1 mM, 4 mM, 7 mM, 10 mM, 15 mM and 20 mM barium and strontium solutions were sonicated for 30 seconds. A volume of 10 µL of each metal solution was added to a separate sample tube, along with 363 µL of UP water. Two blank samples were prepared using 373 µL of UP 35 water without the addition of metal solutions. All sample tubes were sonicated for 25 minutes, to ensure the nanoparticles and metal ions were dispersed in solution. Lastly, the solution in each sample tube was poured into disposable cuvettes and measured with the double-beam UV-Vis spectrophotometer with UP water as the background sample. 36

III. Results & Discussion

3.1 ICP Experiment, Water Quality Test

For the purpose of this discussion, the water sample that originated from the water tap in Clippinger Laboratories room 173 (Ohio University, Athens, OH) will be referenced to as ‘Lab sample’, the water sample that originated from the water tap in a residential building within Athens’ city limits will be referred to as ‘House sample’, the water sample that originated from an UP water filtration system within Clippinger

Laboratories will be referenced to as ‘UP sample’, and the water sample of the residential water going through a standard Brita filter will be referred to as ‘Brita sample’.

The EPA lists the MCL for barium as 2 ppm.2 A study in the Netherlands by the

WHO found the average concentration of barium in the groundwater to be 0.23 ppm with a maximum of 2.5 ppm.29 The barium concentrations for the Lab sample and House sample were determined to be 0.024 ppm and 0.023 ppm respectively, as seen in Table 2.

The barium concentrations of these water samples are a tenth of the Netherlands’ average groundwater barium concentration, and about one-hundredth of the EPA’s MCL for barium. As the concentration of barium in these samples are so low, water treated by

Athens municipality would not give a noticeable response via the nanoparticle test. The lowest perceptible concentration via the nanoparticle test for barium is around 3 ppm, as seen in Table 3. It is important to note, however, that these values may not be exact as

0.02 ppm is an order of magnitude lower than the lowest barium standard concentration of 0.1 ppm run on the ICP. 37

The barium concentration in the UP sample was determined to be 0.000 ppm, as seen in Table 2. This is not surprising as UP water is water that had its ions filtered out until the resistivity is increased to 18 MΩ*cm and the maximum ion concentration is 1

µg/L, which is approximately 1 ppb.26 The barium concentration in the Brita sample was determined to be 0.006 ppm, as seen in Table 2. On Brita’s webpage, they claim the standard filter only reduces the levels of chlorine, mercury, cadmium, copper, and zinc.27

The webpage does not state the degree its filter reduces these ions, nor does it mention any other ions the ion-exchange resin captures. Comparing the concentration of barium in the House sample of 0.023 ppm and the concentration of barium in the Brita sample of

0.006 ppm, the ion-exchange resin in the standard Brita filter removed nearly 75% of barium ions. It is important to note, however, that these values may not be exact as 0.02 ppm and 0.006 ppm are an order and two orders of magnitude lower than the lowest barium standard concentration of 0.1 ppm run on the ICP, respectively.

A normal concentration of strontium in river water is 50 ppb.6 Therefore, strontium concentrations over 500 ppb, or 0.5 ppm would be considered an excess. The average concentration of strontium in the Lab sample was determined to be 0.141 ppm with a range of 0.030 ppm between the two emission wavelengths that data were collected from, as seen in Table 2. The average concentration of strontium in the Home sample was determined to be 0.125 ppm with a range of 0.024 ppm between the two emission wavelengths that data were collected from, as seen in Table 2. Both water sources have higher than average strontium concentrations, but neither has a concerning excess of strontium. As the concentration of strontium in these samples are relatively low, water treated by Athens municipality would not give a noticeable response via the 38 nanoparticle test. The lowest perceptible concentration via the nanoparticle test for strontium is around 7 ppm, as seen in Table 3.

The Lab sample has an average strontium concentration 0.016 ppm greater than the House sample. This was unexpected, as both buildings receive their water from a collection of wells from the same aquifer in Athens city.33 The well water is treated at the same facility which aerates, filters, removes magnesium, calcium, and iron, and adds chlorine and fluoride before pumping the water back into resevoirs.33 The only clear difference between the Lab sample and House sample is they most likely draw their water from separate reservoirs, which may have varying conditions.

The average concentration of strontium in the UP water sample was determined to be -0.009 ppm with good agreement as seen in Table 2. The instrument may have detected a negative concentration for this ion as the reference standards were made with

DI water. DI water has a lower resistivity than UP water, and therefore the standards could have more strontium ions than the UP water sample. The average concentration of strontium in the Brita sample was determined to be 0.064 ppm with a range of 0.012 ppm as seen in Table 2. The Brita webpage did not mention that their standard filter could remove strontium ions. However, comparing the concentration of strontium in the House sample and Brita sample, the Brita filter removed a little over 50% of the strontium present in the House sample.

It is important to note that the concentration of strontium in the Brita sample may not be exact as 0.064 ppm is lower in concentration than the lowest barium standard concentration of 0.1 ppm run on the ICP. The variability in measurements between 39 emission wavelengths could be due to an interfering element emitting near the primary wavelength for strontium that the instrument recommends (407.771 nm).

‘Soft’ water is considered to be any water source with less than 60 ppm of calcium carbonate.34 As an ICP can only detect the gross amount of calcium in an aqueous solution, for the purpose of this experiment the definition will be amended to simply 60 ppm of calcium. The average concentration of calcium in the Lab sample was determined to be 36.2 ppm with a range of 2.36 ppm as seen in Table 2. The average concentration of calcium in the Home sample was determined to be 35.6 ppm with a range of 1.79 ppm as seen in Table 2. Both sources are considered to have soft water as the calcium concentration is considerably less than 60 ppm. This is a reasonable result given the treatment plant targets calcium, magnesium, and iron for removal to soften the water.33

The Lab sample has an average strontium concentration 0.589 ppm greater than the House sample. The disparity between the Lab sample and House sample may be due to the post-treatment water being held in two different reservoirs as stated earlier, since the Lab sample has a higher concentration of both strontium and calcium. It is important to note that the concentration of calcium in the Lab sample and House sample may not be exact as 35 ppm is six times higher than the highest calcium standard concentration of 5.0 ppm run on the ICP.

The average concentration of calcium in the UP sample was determined to be

0.031 ppm with a range of 0.043 ppm as seen in Table 2. This value is greater than the 1 ppb ion maximum stated for UP water. It is unclear how calcium exceeded the maximum ion concentration for UP water. Further testing would be required to determine if this 40 calcium is due to the water source or another source. The average concentration of calcium in the Brita sample was determined to be 18.9 ppm with a range of 1.01 ppm as seen in Table 2. The Brita webpage did not mention that their standard filter could remove calcium ions. However, comparing the concentration of calcium in the House sample and Brita sample, the Brita filter removed a little over 50% of the calcium present in the House sample.

It is important to note that the concentration of calcium in the UP sample and

Brita sample may not be exact as 0.03 ppm is lower than the lowest calcium standard concentration of 0.1 ppm and 18 ppm is higher than the highest calcium standard concentration of 5.0 ppm run on the ICP. The variability in measurements between emission wavelengths could be due to an interfering element emitting near the primary wavelength for calcium that the instrument recommends (422.673 nm).

41

Laboratory Municipal Water Laboratory Faucet Municipal Faucet (nm) Distilled Water Through Brita Water (ppm) Water (ppm) (ppm) Filter (ppm)

Sr 0.156 0.137 -0.009 0.070 (407.771)

Sr 0.126 0.113 -0.008 0.058 (216.596)

Sr Ave. 0.141 0.125 -0.009 0.064

Ba 0.024 0.023 0.000 0.006 (455.403)

Ba 0.024 0.023 0.000 0.006 (233.527)

Ba Ave. 0.024 0.023 0.000 0.006

Ca 37.396 36.533 0.009 19.408 (422.673)

Ca 35.041 34.739 0.052 18.309 (393.366)

Ca Ave. 36.219 35.636 0.031 18.859

Table 2: Concentration of strontium, barium, and calcium dissolved in various water sources. Concentrations determined by ICP-OES. Data collected on July 9th, 2019.

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3.2 Experiment #4, Tris-HCl Study

The experimental conditions specific to this experiment are: 10 nm silver nanoparticles suspended in citrate were used for the first time instead of 50 nm nanoparticles suspended in citrate; bare nanoparticles, nanoparticles with barium, and nanoparticles with strontium were compared with samples with those same conditions with the addition of 10 µL of 100 mM tris-HCl; and barium and strontium solutions with concentrations of 10 mM were used instead of 10 µM solutions to observe if intensities increased. The bare nanoparticle sample is considered the blank for this experiment and was prepared using the same method as the other samples, except the volume of metals and/or tris-HCl added to the other samples was substituted with the same volume of UP water.

Looking at the peak locations in the UV-Vis readout shown in Figure 7, the blank’s plasmonic absorbance wavelength is approximately 395 nm. With the addition of only barium or strontium to the solution, the peak locations did not shift. This implies that the nanoparticles did not aggregate around either metal ion when the ions are simply added into solution with them. However, with the addition of barium or strontium with tris-HCl, the peaks red-shifted by about 10-15 nm and the peaks widened unsymmetrically towards higher wavelengths. This detectable color change is indicative of an LSPR shift from an increased functional diameter of the nanoparticles due to the nanoparticles clustering around barium and strontium ions.

This experiment is significant because it was the first to exhibit a detectable color change in its UV-Vis readout. Therefore, it was determined that tris-HCl needed to be 43 added to the nanoparticle samples in order to get the nanoparticles to cluster around the metal ions and induce a detectable LSPR red-shift. The purpose of the tris-HCl is to serve as a buffer and neutralize the acidity due to MSA. The dependence of the color change on tris-HCl indicates this reaction is pH dependent. To verify this, the pH will be recorded and studied in subsequent experiments.

Figure 7: Experiment #4. First time using 10 nm silver nanoparticles in citrate instead of 50 nm and adding 10 mM barium and strontium solutions instead of 10 µM. Experiment performed on April 9th and 12th, 2019.

3.3 Experiment #5, 10 µM versus 10 mM

Experiment #5 used both 10 µM and 10 mM concentrations of barium and strontium ion solutions in the nanoparticle samples in order to determine the concentrations of barium and strontium that are required for the particles to aggregate. 44

The two blanks in this experiment were prepared identically, by substituting the volume of metal ion solution added to the other samples with UP water.

Looking at the peak locations in the UV-Vis readout in Figure 8, the blank’s plasmonic absorbance wavelength was approximately 395 nm, as expected. The addition of 10 µM barium or strontium solutions resulted in attenuation of the intensity while the peak positions did not change. This indicates the nanoparticles were not aggregating sufficiently to cause an LSPR shift. The addition of 10 mM barium or strontium ions resulted in the peak around 395 nm to nearly disappear. The 10 mM barium sample showed a new absorbance peak around 560 nm and the 10 mM strontium sample had a low intensity, stretched peak in the range of 380 nm to 530 nm. This peak shift and widening are indicative of nanoparticle aggregation and LSPR shifting. The final pH of all the samples were determined using pH paper to be in the range of 5-6. This result is not ideal, as the final solution should be nearly neutral, not moderately acidic.

The significance of this experiment was that the order of magnitude for metal ion concentrations to use for further experimentation was determined and the pH was noted as a potential issue. Going forward, only metal ions in the mM range were used to perfect the experimental methods and determine the trend between metal ion concentration and absorbance peak location. 45

Figure 8: Experiment #5. Determining the ability for the UV-vis to differentiate between 10 µM and 10 mM metal samples. Experiment performed on June 10th and 11th, 2019.

3.4 Experiment #6, Barium Concentration Range

The experimental conditions specific to this experiment were: a wide range of barium ion solutions from 1 mM to 100 mM were added to different nanoparticle samples and the pH of samples at multiple steps was determined with pH paper. The blank was prepared as previously described.

The nanoparticle solutions had pH values of 5-6 after the replacement of most of the citrate suspension fluid with UP water. The addition of MSA to the nanoparticle solutions resulted in pH values between 2-3. After the samples were diluted with UP water and right before they were scanned via the UV-Vis spectrophotometer, the samples had a pH around 6, similar to the previous experiment and confirming the addition of tris- 46

HCl buffer to the nanoparticle solutions is necessary to balance the pH drop due to the addition of MSA.

Looking at the peak locations in the UV-Vis readout shown in Figure 9, the blank’s plasmonic absorbance wavelength is approximately 395 nm. The peak for the 1 mM barium sample has a slight peak around 460 nm. This implies the formation of a second peak due to minor nanoparticle aggregation. With the addition of 10 mM of barium, two merging peaks are noticeable around the original 395 nm LSPR peak as well as a new peak around 535 nm. The new peak is the development of a new LSPR absorbance wavelength and is a clear sign of the nanoparticles aggregating. The spectra of the 50 mM barium sample and 100 mM barium sample are very similar to the spectra of the 10 mM barium sample, except with lower absorbance intensities. The nanoparticle samples with 10 mM to 100 mM barium concentrations have positive UV-Vis readout responses to the presence of barium.

The significance of this experiment is absorbance peak intensities noticeably decrease with increased barium ion concentration until a concentration of 50 mM. This potentially means that the addition of 50 mM barium ions is either the upper detection limit or above the upper detection limit, and therefore no barium concentrations past 50 mM will provide a different response. This hypothesis was investigated for both barium and strontium ions in the next experiment. Additionally, due to the low pH of the final samples it was determined the tris-HCl buffer should be added to the nanoparticle solutions in future experiments. 47

Figure 9: Experiment # 6. To observe the effect of varying barium concentrations on the absorption spectra. Experiment was performed on June 19th and 20th, 2019.

3.5 Experiment #7, Red-Shift Trends

3.5.1 Spectra

The experimental conditions for this Experiment #7 were the same as the previous experiment except 50 µL of 100 mM tris-HCl buffer was added to the nanoparticle solutions right before the addition of the metal ion solutions. Samples with an aliquot of

5 mM metal ions solutions were prepared and strontium ions were used in five extra nanoparticle samples. The blanks for this experiment were both prepared as previously described, including the addition of tris-HCl, except 10 µL of the metal solution was substituted for 10 µL of UP water. 48

The pH was measured at three different stages of the experiment. The pH of 40 mM MSA stock was between 1-2. The pH of the nanoparticle solutions after the addition of MSA and spinning on the carousel for 2 hours ranged between 4-5. Lastly, the pH of the nanoparticle solutions after the addition of tris-HCl was about 8 and higher than the pH of 6 for previous experiments. Therefore, the addition of tris-HCl worked to counter- act the low pH of MSA. However, this pH is just as basic as the previous experiments were acidic, therefore less tris-HCl should be used in the future to obtain a pH closer to 7.

As the UP-water used in these experiments has a pH of 7, it is important to note the average pH of freshwater is between 6 – 8.5 and the amount of tris-HCl buffer needed to neutralize the samples would vary.35

Looking at the peak locations in the UV-Vis readouts shown in Figure 10, the blank’s plasmonic absorbance wavelength was approximately 395 nm. No trend between concentration and absorbance peak intensities was observed. The peaks decrease in intensity from 1 mM, 100 mM, 10 mM, 50 mM, and then 5 mM for barium and from 1 mM, 100 mM, 5 mM, 50 mM, and 10 mM for strontium.

The 1 mM metal samples showed a 10 nm red-shift for barium and a 5 nm red- shift for strontium. Both spectra had a shoulder forming at 450 nm. This implies the formation of a second peak due to minor nanoparticle aggregation. Neither 5 mM samples showed a red-shifted original LSPR absorbance peak. The 10 mM strontium sample also did not shift due to the fact that barium has a concentration of around 14 ppm at 5 mM and strontium would have a concentration around 17 ppm at 10 mM, as seen in

Table 3. Even though those samples did not have a shifted peak around 395 nm, they did exhibit the formation of a second LSPR absorbance peak starting around 475 nm, which 49 is indicative of a new LSPR absorbance wavelength and a clear sign of nanoparticle aggregation. With the addition of higher concentrations of metal ions, the two peaks become less distinct and begin to merge. This is likely due to the population of nanoparticles that did not aggregate dwindling at higher metal ion concentrations, and therefore the associated LSPR absorbance peak around 395 nm is dwindling and merging with the second absorbance peak.

The significance of this experiment is that a color change is detectable by the UV-

Vis spectrophotometer for all concentrations from 1 mM to 100 mM but is especially evident from 5 mM and beyond. With the data from Figure 10, trend analyses were performed on variables such as peak location, height, area, and width to determine if any are concentration dependent using various curve-fitting options in Microsoft Excel. The eight trend analyses are presented in Figure 11 and Figure 12 and discussed in the section below. It was also determined that a lower quantity of tris-HCl buffer should be added to the nanoparticle samples than 50 µL with a concentration of 100 mM to acquire a neutral pH prior to the addition of metal ions.

50

Figure 10: Experiment # 7. Second trial of previous experiment. 5 mM metal concentration was added. Strontium metal concentrations were also measured (bottom graph). Main deviation from the previous experiment was the addition of 50 µL 100 mM tris-HCl. Experiment was performed on June 26th and 27th, 2019.

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3.5.2 Trend Analysis

The eight trend analyses that were performed are depicted in Figure 11 and Figure

12 from data sourced from the UV-Vis readouts presented in Figure 10. The trend analyses were accomplished with various curve-fitting options in Microsoft Excel and were designed to determine which variables were dependent on barium and strontium ion concentrations present in the nanoparticle solutions. The four graphical variables investigated were peak location, peak height, peak area, and peak width. Each trend represents the curve-fitting option, between linear, polynomial up to the fourth order, exponential, logarithmic, and power function models, which had the greatest coefficient of determination (R2) value.

Evaluating the peak location trend analyses for both barium and strontium ions, the peak locations red-shifted from 1 mM to 50 mM metal concentrations, then leveled off between 50 mM and 100 mM concentrations. For the barium ions, the peak locations had shifts from 402.5-427.0 nm and for the strontium ions, the peak locations had shifts from 400.5-412.5 nm. Considering the average blank peak location was at 396 nm, even the 1 mM strontium ion concentration, the shortest peak shift, had red-shifted by 4 nm.

Although the trendline for peak locations with the best R2-value was a negative parabolic function with R2-values of 0.96 and 0.99, this trend may be logarithmic. If peak location shifting was modeled via a negative parabolic function, then the peak location would eventually have to begin blue shifting, which is possible but has not been shown to happen in the studied concentration range. If peak location shifting was modeled via a logarithmic function, there would be a cut-off concentration where all metal concentrations above said value would have the same instrumental response, which 52 has been shown to be true of concentrations from 50 – 100 mM. Having a logarithmic model would also imply that the LSPR peak red shifts more at lower concentrations and therefore this testing would be more sensitive at lower concentrations than higher ones.

The other graphical variables of peak height, area, and width did not have any trends which produced an R2-value greater than or equal to 0.70 except for the curve- fitting for strontium peak areas, which had an R2-value of 0.96 with a parabolic function.

It is unclear if this trend is meaningful or if the data randomly managed to fit one of the applied curves, as barium only had an R2-value of 0.22 for its peak area trend.

Performing the trend analyses revealed that the peak characteristic most dependent on ion concentration is the peak position. Using the curve-fitting equations for peak position, an unknown’s barium and strontium ion content can be determined by solving the equation for the unknown’s peak position. However, this will not be done using Athens, Ohio faucet water, as it was shown in the ICP results in Table 2 that the barium and strontium concentrations in those samples are considerably below the detectable range shown in Table 3. Furthermore, a calibration plot will need to be made anew each time unknown samples run as the absorbance intensities of the same standard concentrations are different each time the experiment is run. 53

Figure 11: Trend analyses for multiple spectra characteristics of the UV-Vis barium nanoparticle experiment #7. These spectra characteristics are for the shifted LSPR absorbance peak around 395 nm and include: peak location, peak height, peak area, and peak width. The peak area was calculated by the UV-Vis spectrophotometer program and accounts for all the area under the merging peaks. 54

Figure 12: Trend analyses for multiple spectra characteristics of the UV-Vis strontium nanoparticle experiment #7. These spectra characteristics are for the shifted LSPR absorbance peak around 395 nm and include: peak location, peak height, peak area, and peak width. The peak area was calculated by the UV-Vis spectrophotometer program and accounts for all the area under the merging peaks.

3.6 Experiment #10, Pink Color Change

The experimental conditions of this experiment were similar to those described in the experiment above, except for these changes: the addition of MSA and subsequent spinning on the carousel which functionalizes the nanoparticles were performed on the same day as the samples would be scanned by the UV-Vis; half of the nanoparticle 55 samples were delayed before functionalization to ensure these samples were not settling while the other half were scanned in the UV-Vis; the barium and strontium ion concentration ranges were changed from 1 mM, 5 mM, 10 mM, 50 mM, and 100 mM to

1 mM, 4 mM, 7 mM, 10 mM, 15 mM, and 20 mM as it was verified samples from 50 mM onwards did not provide a different instrumental response; and 37 µL of 100 mM tris-HCl buffer instead of 50 µL was added to each sample.

Looking at the peak locations in the UV-Vis readouts shown in Figure 13, the blank’s plasmonic absorbance wavelength was approximately 395 nm and retained the same yellow color as the initial stock nanoparticle solution as this is the typical plasmonic resonance wavelength of these silver particles. This experiment was the first time some of the nanoparticle samples’ color change could be observed by the naked eye as a shift from yellow to pink.

For the samples with metal ions, the UV-Vis readout illustrates the pink color change by the second peak that starts to appear around 520 nm, which is evidence of plasmonic coupling between aggregating nanoparticles. In barium, this change starts to arise when using 4 mM, and in strontium by 10 mM. In the top of Figure 13, the 4 mM,

7 mM, and 10 mM barium spectra were very similar in shape and intensity, but for the 15 mM and 20 mM barium spectra, the two peaks began to merge into a single peak around

430 nm. In the bottom of Figure 13, there is a noticeable decrease in intensity from 1 mM to 15 mM of strontium. The 20 mM barium and strontium peaks were both merged more than the 15 mM peaks.

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The significance of this experiment is that this was the first time the nanoparticle samples visibly changed color and turned pink, and an estimate for the lower limit of detection was determined. Looking at Table 3, the UV-Vis detected a color change via the formation of a second LSPR absorbance peak by 11 ppm for barium and 17ppm for strontium. There was also a noticeable dip in intensity and the appearance of a slight peak on the original LSPR absorbance peak in comparison of the blank without a distinct color change, or appearance of another peak, by 3 ppm for barium and 7 ppm for strontium.

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Figure 13: Experiment # 10. Observing the effect of waiting until the day of data collection to add MSA and carousel for 2 hours instead of performing these steps on an earlier day. The top spectrum is testing with barium ions and the bottom spectrum is testing with strontium ions. Experiment was performed on July 31st and August 7th, 2019.

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Metal Stock Metal Final Ba (ppm) Sr (ppm) Concentration (mM) Concentration (µM)

1 20 3^ 2

4 80 11* 7^

7 140 19 12

10 200 27 17*

15 290 40 26

20 390 54 34

Table 3: The final concentrations of strontium and barium ions in the nanoparticle solutions. The asterisk denotes the first color change detected by the UV-Vis on August 7th, and the carrot indicates the first noticeable dip in intensity detected by the UV-Vis in experiment #10.

3.7 Experiment #14, Citrate versus PVP

The experimental conditions of this experiment were: using nanoparticles that were suspended in citrate for half of the samples and using nanoparticles that were suspended in PVP for the other half of samples to assess the viability of using nanoparticles suspended in PVP, and the UV-Vis scan rate was changed from fast with

0.5 nm increments to slow with 1 nm increments.

Looking at the peak locations in the UV-Vis readouts shown in Figure 14, the blank for the nanoparticles that were suspended in citrate had a plasmonic absorbance wavelength around 395 nm and the blank for the nanoparticles that were suspended in

PVP had a plasmonic absorbance wavelength around 400 nm. The LSPR absorbance peak for the citrate blank is just below 0.15 arbitrary units (AU) and the lowest citrate 59 peak is around 0.04 AU. The LSPR absorbance peak for the PVP blank is just below

0.025 AU and the lowest PVP peak is around 0.005 AU. Both ends of the PVP peak range are an order of magnitude less intense than the citrate peak range. This means the nanoparticles suspended in PVP are considerably less sensitive than those suspended in citrate to detect barium and strontium ions and is also likely the reason the PVP spectra appear rough compared to the citrate spectra.

Looking at the UV-Vis readout for nanoparticles that were suspended in citrate, every metal concentration has two merging peaks. The second peak has a shifting location depending on the metal concentration. For the 7 mM barium sample, the second peak is simply a shoulder on the original LSPR peak with a maximum around 525 nm.

For the 15 mM and 20 mM barium samples, the second peaks are around 560 nm and 550 nm respectively and have higher intensities than the original LSPR absorbance peaks.

For strontium, the second peak is simply a large peak with a maximum around 460 nm off the original 7 mM peak, then both the 15 mM and 20 mM samples have merging peaks that decrease around 550 nm and 525 nm, respectively. Barium has more sensitive responses with nanoparticles than strontium because barium has a higher ppm concentration at the same mM concentrations as strontium as seen in Table 3, and nanoparticles detect metals based on mass and size, not the raw number of particles.

Looking at the UV-Vis readout for nanoparticles that were suspended in PVP, none of the concentrations for either metal has a second peak. This means there were no color changes and therefore no nanoparticle aggregation occurring at any of the metal concentrations. All measurements, including the blank, also had tailing occurring. The 60 ideal peak shape for all spectra, especially the blank, is a Gaussian distribution, not tailing.

The significance of this experiment is 10 nm silver nanoparticles suspended in

PVP are not a viable alternative to those suspended in citrate to serve as a detector for barium or strontium ions in water.

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Figure 14: Experiment #14. Comparing 10 nm nanoparticles that are suspended in PVP versus nanoparticles that are suspended in citrate. The experiment was performed on October 30th, 2019.

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IV. Conclusion

5.1 Concluding Remarks

The overall objective of this study was to assess the viability of utilizing

functionalized silver nanoparticles to economically determine the concentrations of

cations, specifically barium and strontium ions, in contaminated water samples

employing a UV-Vis spectrophotometer. There were many challenges to reach this goal.

There were vital gaps in the literature documenting the experimental conditions related to

using nanoparticles in this way. Multiple trials were performed to determine which

experimental conditions were conducive to aggregating the nanoparticles around barium

and strontium ions. Another difficulty was that samples made with nanoparticles

suspended in citrate were stable for less than two hours. Therefore, samples had to be

prepared in a staggered time frame to prevent deterioration while awaiting to be scanned.

Lastly, an excessive amount of strontium in a waterway is greater than 0.5 ppm and the

lower detection limit for strontium with this test was estimated to be around 7 ppm, as

seen in Table 3. A detection limit closer to 0.5 ppm would be desired for practical

application.

After the addition of barium and strontium ions to the nanoparticle solutions in

Experiment #10, a few samples visibly changed color from yellow to pink opening the

door for bare-eye detection of barium and strontium ions in water, even though these ions

are colorimetrically silent by themselves. From the absorbance data acquired in

Experiment #7, the trend analyses in Figure 11 and Figure 12 were performed. These

analyses illustrate the LSPR absorbance peak locations are dependent on the 63 concentration of barium and strontium ions, and the relationship can be modeled mathematically with minimal error. The upper detection limit was determined to be slightly less than 50 mM, which is around 80 ppm for strontium. These levels will likely never be reached in the field. As shown in Table 3, the lower detection limit for barium ions was around 3 ppm and around 7 ppm for strontium ions. These values are significantly higher than the concentrations of barium and strontium found in Athens,

Ohio treated water as determined by ICP in Table 2, which means treated water samples should not procure a positive result with this test.

Although the overall objective to test contaminated water samples with the nanoparticles was not fully accomplished, significant progress was made towards this practical application. The successes of this project include:

• after the addition of high concentrations of barium and strontium ions,

nanoparticle samples shifted color from yellow to pink, signifying the feasibility

of bare-eyed detection,

• creating samples with a range of ions from 1 mM to 100 mM, a rough detection

range for barium and strontium ions were determined, and

• regression analysis indicating a strong trend between plasmonic resonance

absorbance wavelengths and the concentrations of metal ions in water.

5.2 Future Work

With the information gathered through this study, there are many avenues for future research. The first avenue would be to continue investigating possible interfering 64 experimental conditions on the optical response of the nanoparticles. The effects of moderately acidic and basic pH’s, of sonicating the nanoparticles at different points of the experiment, the spectral difference of using 10 nm nanoparticles versus 50 nm nanoparticles, and the effect of using nanoparticles suspended in PVP instead of citrate were all investigated.

Other possible experimental factors that could be investigated include investigating nanoparticles suspended in a variety of solutions, investigating nanoparticles of sizes below 10 nm or between 10 and 50 nm, investigating using less nanoparticles or a higher nanoparticle-to-water ratio, and many more. The effects of common compounds found in water sources on LSPR absorbance peaks can also be investigated. For example, the spectral effects due to the presence of other group II elements like beryllium, calcium, and magnesium or the presence of other common metal water contaminants like copper, iron, and zinc or the presence of common organic contaminants like nitrogen and phosphorus containing compounds and pesticides could be investigated.

Another avenue to explore would be to use the same experimental design in section 2.2.4 Final Method) to obtain further statistical data. For example:

• Experiment #10 could be performed again but with smaller jumps in metal

concentrations in order to construct a stronger curve-fitting function,

• Experiment #10 could be performed numerous times to determine if the

instrumental response varies too much from day-to-day to create a library

calibration plot (instead of creating a wide range of standards every time an

unknown sample is tested), and 65

• a water sample known to be contaminated and is filtered of all sediment and

lifeforms could be run as an unknown against a wide range of standards in the

UV-Vis and then compared to the true concentrations reported with an ICP. 66

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