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1 Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2013 Accurate and Precise Determination of Low Concentration Iron, Arsenic, Selenium, Cadmium, and Other Trace Elements in Natural Samples by Octopole Collision/ Reaction Cell (CRC) Equipped Quadrupole- ICP-Ms Angela Dial Follow this and additional works at the FSU Digital Library. For more information, please contact

2 THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES ACCURATE AND PRECISE DETERMINATION OF LOW CONCENTRATION IRON, ARSENIC, SELENIUM, CADMIUM, AND OTHER TRACE ELEMENTS IN NATURAL SAMPLES BY OCTOPOLE COLLISION/REACTION CELL (CRC) EQUIPPED QUADRUPOLE-ICP-MS By ANGELA DIAL A Thesis submitted to the Department of Earth, Ocean, and Atmospheric Science in partial fulfillment of the requirements for the degree of Master of Science Degree Awarded: Spring Semester, 2013

3 Angela Dial defended this thesis on March 28, The members of the supervisory committee were: William M. Landing Professor Directing Thesis Vincent J. M. Salters Committee Member Munir Humayun Committee Member The Graduate School has verified and approved the above-named committee members, and certifies that the thesis has been approved in accordance with university requirements. ii

4 TABLE OF CONTENTS List of Tables... iv List of Figures...v Abstract... vi 1. INTRODUCTION EXPERIMENTAL METHODS Reagents, Standards, and Sample Matrix Mass Spectrometry Quadrupole-ICP-MS High Resolution-ICP-MS RESULTS AND DISCUSSION Analytical Figures of Merit Plasma-based Interferences Matrix-based Interferences Long-term Reproducibility CONCLUSIONS...26 REFERENCES...27 BIOGRAPHICAL SKETCH...30 iii

5 LIST OF TABLES 1. Plasma and matrix based polyatomic interferences on analytes of interest in their respective matrix and sample type Instrumental settings of Q-ICP-MS (Agilent 7500cs) for hot plasma and cool plasma operations with collision reaction cell Instrumental settings of HR-ICP-MS (Thermo Finnigan ElementXR) for low and medium resolution Fe analyses Comparison of 56 Fe matrix blanks (in HNO 3 matrix), sensitivity ([Fe] analyte = 1 µg/l; High Purity Standard), and signal-to-noise (sensitivity/matrix blank) ratios of HR-ICP-MS (Thermo Finnigan ElementXR; University of Cambridge) and Q-ICP-MS (Agilent 7500cs; Florida State University) Analytical figures of merit: sensitivity (cps/µg L -1 per isotope), limit of detection (LoD, 3σ; ng/l), and signal-to-noise ratios (S/N) of analytes of interest ( 51 V, 52 Cr, 55 Mn, 56 Fe, 57 Fe, 58 Ni, 59 Co, 63 Cu, 66 Zn, 75 As, 78 Se, 80 Se, 111 Cd, and 208 Pb) in hot and cool plasma conditions with reaction mode or collision-reaction mode of CRC operation SRM NIST 1643e (Standard Reference Material - trace elements in freshwater) Fe and Cd concentration data collected over a period of 7 months (Figure 11)...25 iv

6 LIST OF FIGURES 1. HR-ICP-MS (Thermo Finnigan ElementXR) mass spectra of a 500 µg/l Fe standard solution in 0.1 M HNO 3 in low-resolution (m/ m = 300) (Fig. 1.A) and medium-resolution (m/ m = 4000) (Fig. 1.B) mode Working principle of the Octopole Collision/Reaction Cell in collision and reaction mode, adapted from the Agilent 7500cs Operator s Manual Collision mode (CM) gas flow (He) optimization for 56 Fe (3.A), 78 Se (3.B), and 75 As (3.C) in hot plasma Reaction mode (RM) gas flow (H 2 ) optimization for 56 Fe (4.A), 78 Se (4.B), and 75 As (4.C) in hot plasma Comparison of air blank, matrix blank (0.44 M HNO 3 ) and limit of detection (LoD: 3σ) of Fe in hot and cool plasma mode (red and blue bars, respectively), with and without CRC operation in RM Comparison of sensitivities (cps/1 µg L -1 ) and matrix blanks (cps) for 56 Fe (6.A), 75 As (6.C), 78 Se (6.E), and 111 Cd (6.G) in all operating conditions: hot and cool plasma, Reaction Mode (RM, H 2 = 4.7 ml/min) and Collision-and-Reaction Mode (CRM, He = 2.8 ml/min and H 2 = 2.0 ml/min) Standard calibration in 0.44 M HNO 3 of 56 Fe (circles with solid lines, y 1 -axis) and 57 Fe (squares with dashed lines, y 2 -axis) operated under hot plasma (red) and cool plasma (blue) conditions with the CRC in RM (H 2 = 4.7 ml/min) As standard calibration in mixed acid (0.048 M HNO M HCl) operated under hot plasma (red) and cool plasma (blue) conditions, with RM (H 2 = 4.7 ml/min, circles solid lines) and CRM (He = 2.8 ml/min and H 2 = 2.0 ml/min, squares with dashed lines) employed Mo standard calibration (circles) in 0.44 M HNO 3 executed in hot plasma with CRM (He = 3.0 ml/min and H 2 = 2.0 ml/min), hot plasma with RM (H 2 = 4.7 ml/min), cool plasma with CRM (He = 2.8 ml/min and H 2 = 2.0 ml/min), and cool plasma with RM (H 2 =4.8 ml/min) Comparison of sensitivity calibrations (slopes, cps/µg L -1 ) from a 111 Cd standard in 0.44 M HNO 3, operated in hot and cool plasma (red and blue bars, respectively) with the CRC in CRM and RM (white and gray regions, respectively) SRM NIST 1643e (Standard Reference Material - trace elements in freshwater) Fe and Cd concentration data collected over a period of 7 months...24 v

7 ABSTRACT An improved method for accurate and precise determination of trace quantity dissolved metals and metalloids in natural samples by Octopole Collision/Reaction Cell (CRC) equipped Quadrupole-Inductively Coupled Plasma-Mass Spectrometry (Agilent 7500cs) is reported. Our method is optimized for rapid analyses of small volume samples (~250 µl) in a variety of matrices containing HNO 3 and/or HCl. The present study focuses on elements with ICP-MS plasma- and/or matrix based interferences, in particular 56 Fe ( 40 Ar 16 O + ), 75 As ( 40 Ar 35 Cl + ), 78 Se ( 40 Ar 38 Ar + ), and 111 Cd ( 95 Mo 16 O + ). We demonstrate efficient elimination of these polyatomic interferences via the use of CRC in Reaction Mode (RM; H 2 gas) and in Collision-Reaction Mode (CRM; H 2 and He gas). In addition, the efficiency of the instrument was evaluated under both hot plasma (RF power 1500 Watts) and cool plasma (600 W) conditions. The present method is optimized to analyze elements with large mass spectrometric interferences at sub parts per billion level concentrations in a variety of natural samples and matrix compositions. We report an average external precision of ~10% for minor ( 10 µg L -1 ) elements measured in a 1:100 dilution of NIST 1643e under two different plasma conditions and CRC operational modes. Our measured concentration values for elements like Fe (99.6 µg/l), Mg (8020 µg/l), Co (26.99 µg/l), Ni (62.54 µg/l), Cd (7.68 µg/l), Sb (59.6 µg/l), and Pb (19.82 µg/l) with a large dynamic spread in concentrations in NIST 1643e are within ±12% to ±2% of the accepted / published values. vi

8 CHAPTER 1 INTRODUCTION Accurate and precise determination of trace metals in natural samples is essential to discern their source, distribution, and role in the environment. Furthermore, this knowledge is necessary to comprehend the natural biogeochemical cycles of these metals and potential changes in their natural distribution caused by anthropogenic impacts. The role of transition metals, such as Mn, Fe, Co, Cu, Ni, Zn, and Cd, as micronutrients in aqueous environments generates a significant interest in understanding the biogeochemical cycle of these metals in seawater (Brand et al, 1983; Boyd et al, 2000; Saito et al, 2005; Peers et al, 2005; Coale and Bruland, 1988; Jones and Murray, 1984; Shaked et al, 2006; Cullen et al, 1999). Iron is of special interest since it is a limiting factor in biological productivity throughout the world s oceans, particularly in high-nutrient low-chlorophyll regions (Johnson et al, 1997). Another element of interest is Cr, as Cr has variable effects on biological cycles depending on its oxidation state: chromium (III) is an essential nutrient for many organisms, however chromium (VI) is highly toxic (Mertz, 1993; Kotaś and Stasicka, 1999). Another application for trace element studies is the use of metals and metalloids like V, Zn, As, Se, Sb, and Pb as tracers of anthropogenic pollution (Bruland et al, 1974; Plant et al, 2006; Callender, 2006). Accurate documentation of these trace elements in the environment is imperative to aid in understanding how biogeochemical cycles in natural systems operate and how they have been perturbed by human activity. Advances in inductively coupled plasma mass spectrometry (ICP-MS) over the past decade have made it a popular analytical tool for rapid and simultaneous analyses of multiple elements. However, accurate measurements of trace quantity dissolved metals and metalloids in natural samples using ICP-MS is limited by the low concentration of elements in samples of interests; contamination during sample collection, handling, and storage; and sample matrixbased and ICP-MS plasma-based (mass spectrometer) isobaric and polyatomic interferences. Moreover, a high first ionization potential (IP-1), volatility in analyte matrix, and surface adsorption properties of an analyte can further compound the problems by diminishing sensitivity and increasing the carry over effect between samples (Gaboardi & Humayun, 2009). 1

9 These analytical challenges are exacerbated with certain trace elements, such as Fe, As, Se, and Cd, which have plasma- and/or matrix-based polyatomic and/or multiple charged mass interferences on the major isotopes of the analyte. A representative list of elements of interest and their major mass interferences from plasma and matrix sources are presented in Table 1. Isobaric interferences are generally due to Ar, O 2, N 2, and C based polyatomic molecules (plasma-based) or metal oxides (matrix-based), which have similar mass-to-charge (m/z) ratios as the analyte. Additionally, the nature of the sample matrix (complex or simple) can play important roles in formation of polyatomic interferences. For example, elements like V, Cr, and As all have Cl-based interferences in the presence of HCl in the matrix (Table 1). In the present study, rain water samples are analyzed in dilute aqua regia (0.048 M HNO M HCl) for stabilization of dissolved trace metals as well as volatile mercury (Landing et al, 1998), a strategy that increases the range of possible interferences due to chlorine based polyatomic ion formation. Table 1. Plasma and matrix based polyatomic interferences on analytes of interest in their respective matrix and sample type. Analyte Plasma Matrix Interference Interference Sample Application 51 V 35 Cl 16 O + Rainwater 52 Cr 40 Ar 12 C + 35 Cl 16 O 1 H + Rainwater 55 Mn Seawater/Rainwater 56 Fe 40 Ar 16 O + Seawater/Rainwater 57 Fe 40 Ar 16 O 1 H + Seawater/Rainwater 58 Ni Seawater/Rainwater 59 Co Seawater/Rainwater 63 Cu Seawater/Rainwater 66 Zn Seawater/Rainwater 75 As 40 Ar 35 Cl + Rainwater 78 Se 40 Ar 38 Ar + Rainwater 80 Se 40 Ar 40 Ar + Rainwater 111 Cd 95 Mo 16 O + Seawater/Rainwater 208 Pb Seawater/Rainwater 2

10 The success of ICP-MS analyses depends on the ability to overcome matrix- and plasmabased isobaric, polyatomic, and multiple charged interferences on an analyte. High Resolution- ICP-MS (HR-ICP-MS) is able to resolve many elements from their mass interferences using a higher mass resolution defined as the resolving power:!!!"#$% =! where m is the analyte s mass (at 100% peak height) and Δm is the mass difference between the analyte and interferent peaks (Weyer and Schweiters, 2003). However, HR-ICP-MS methods are unsuitable for measurements of mass limited samples due to significant decreases in sensitivity in mass resolution modes (m/δm) of 2000 or higher. Figure 1 exemplifies this decrease in 56 Fe sensitivity with increase in mass resolution. For analyses in medium resolution (m/δm = 4000), sensitivity is reduced by a factor of 25 from that of low resolution (m/δm = 300). A Quadrupole- ICP-MS (Q-ICP-MS) is incapable of resolving analyte peaks from any interference s using higher mass resolutions, however a Q-ICP-MS equipped with Collision/Reaction Cell (CRC) can effectively eliminate the interfering polyatomic molecules by capitalizing on their bigger collision radius and using energy discrimination (Tanner et al, 2002; Iglesias et al, 2002; Leonhard et al, 2002; McCurdy & Woods, 2004). Figure 1. HR-ICP-MS (Thermo Finnigan ElementXR) mass spectra of a 500 µg/l Fe standard solution in 0.1 M HNO 3 in low-resolution (m/ m = 300) (Fig. 1.A) and medium-resolution (m/ m = 4000) (Fig. 1.B) mode. In low-resolution (Faraday ion detection) the 56 Fe ( amu) peak is dominated by 40 Ar 16 O + ( amu) interference. In medium-resolution (Analog ion detection) a complete peak separation between 56 Fe + (left) from 40 Ar 16 O + (right) is achieved. However, there is an order of magnitude decrease in overall sensitivity from low resolution ( 56 Fe Ar 16 O + = 1.8 x 10 9 ) to medium resolution ( 56 Fe + = 1.0 x 10 8, 40 Ar 16 O + = 1.0 x 10 8 ). 3

11 In this study, we present an improved method of accurate and precise concentration determination of elements with plasma- and/or matrix-based polyatomic mass spectrometric interferences via utilization of an Agilent 7500cs Q-ICP-MS equipped with an Octopole CRC. The CRC is an off-axis chamber, 2 ml in volume, with a positive potential bias along the ion flow path. The CRC can be flooded with low molecular weight gases, such as H 2 (reaction mode) and/or He (collision mode), which can collide with the passing ions of the analyte. Polyatomic interferences have a larger collision cross section than monatomic analytes of interest, therefore increasing the collision frequency of the interferent with the H 2 and/or He molecules in the CRC. The choice of gas determines whether the CRC is operated under reaction mode and/or collision mode. In reaction mode, H 2 gas eliminates interferences in two ways: a. by charge transfer polyatomic interferent molecules collide with H 2 and transfer its charge to the H 2 molecule, thus making it mute to the SEM detector; and b. by mass transfer the polyatomic interferent reacts with H 2 and bonds with one hydrogen atom, increasing its mass by 1 amu (Figure 2a). In collision mode, He eliminates interferences by colliding with polyatomic molecules and reducing their kinetic energy through energy transfer from the interferent to He. This drop in kinetic energy coupled with the positive energy discrimination of the CRC stops the interferent from traversing the cell (Figure 2b). This method of energy bias against the polyatomic interferent is termed kinetic energy discrimination (KED). In addition to utilizing the Octopole Collision/Reaction Cell to reduce interferences, we optimized the instrument under hot plasma (1500 Watts) and cool plasma (600 W) conditions. The cool plasma (~6000 K) reduces the ionization efficiency of elements with high IP-1 (e.g., High IP-1: Ar = ev and As = 9.81 ev) as compared to hot plasma (~8000 K) conditions. In the present study, we document the elimination of plasma- and matrix-based polyatomic interferences with different plasma and CRC settings. 4

12 Fig. 2A. Fig. 2B. Figure 2. Working principle of the Octopole Collision/Reaction Cell in collision and reaction mode, adapted from the Agilent 7500cs Operator s Manual. Polyatomic interference elimination by charge transfer and atom transfer reactions (reaction mode) are shown in Fig. 2.A. During atom transfer reactions, the polyatomic mass interferent ( 40 Ar 16 O + ) reacts with H 2 (reaction gas) and binds one hydrogen atom to the interferent, increasing its mass by one amu. During charge transfer reactions, the polyatomic interferent transfers its charge to a H 2 molecule, thus becoming mute to the SEM detector. Interference elimination in collision mode, utilizing an inert gas like He (collision gas), is shown in Fig. 2.B. Polyatomic interferences ( 40 Ar 16 O + ) have a larger ionic radius (effective nuclear volume) than the monatomic analyte of interest ( 56 Fe), increasing the collision frequency of the interferent over that of the analyte with the collision gas (He). More collisions of the interferent leads to greater loss of kinetic energy for the interferent compared to the monatomic analyte of the same mass. This kinetic energy discrimination against larger polyatomic interferences stops it from traversing the off-axis CRC. Moreover, in collision mode, dissociation of polyatomic interferences upon collision with cell gas (He) also eliminates interference. 5

13 CHAPTER 2 EXPERIMENTAL METHODS 2.1 Reagents, Standards, and Sample Matrix All acids, ICP-MS standards, and samples were prepared using 18.3 MΩ cm MQ water. We used Optima (Fisher ) grade nitric and hydrochloric acid for analyte matrix preparation. Analyte blanks of the water source, acids, and elemental standards were closely monitored throughout the entire experiment. Accurate molarity of each batch of acid was determined by titrimetric methods. Samples were analyzed in three different acid matrixes: 0.44 M HNO 3 for general ICP-MS analysis; 1.0 M HNO 3 for seawater samples prepared using the method from Milne et al (2010); and mixed acid (0.048 M HNO M HCl) for measurements of trace metals and mercury in rainwater samples using the method from Landing et al (1998). In addition, all calibration standards were prepared gravimetrically from High Purity Standards (HPS). Laboratory supplies used in the present body of work were acid cleaned using reagent grade 8.0 M HNO 3 at sub-boiling temperature. 2.2 Mass Spectrometry Quadrupole-ICP-MS Elemental concentrations were determined with an Agilent 7500cs single collector Quadrupole-ICP-MS equipped with an Octopole Collision / Reaction Cell (CRC). Depending on the experiment, the instrument was operated either under hot plasma (1500 W) or cool plasma (600 W) conditions. The sample introduction for both plasma conditions was done with a nominal 100 µl/min self-aspirating concentric PFA nebulizer (ESI TM ), a Scott-type quartz spray chamber, a quartz torch with built-in quartz injector (2.5 mm i.d.), and nickel sampler and skimmer cones. The key differences between the two plasma-operating conditions are the voltage settings on Extraction lens 1 (EL-1) and Extraction lens 2 (EL-2). Under hot plasma conditions, optimal sensitivity was achieved with a slightly positive voltage for EL-1 (3.5 to 4.0 V) and a negative voltage for EL-2 (-160 to -150 V). In cool plasma, optimal sensitivity was obtained in soft extraction mode by applying a negative voltage on EL-1 (-180 to -175 V) and a positive voltage on EL-2 (-5 to 5 V) (Table 2) (Misra & Froelich, 2009). 6

14 Table 2. Instrumental settings of Q-ICP-MS (Agilent 7500cs) for hot plasma and cool plasma operations with collision reaction cell. Instrumental Parameter Hot Plasma (1500 W) Cool Plasma (600 W) Spray Chamber Quartz Quartz Torch/Injector Quartz/Quartz Quartz/Quartz Shield Torch Platinum Platinum Sampler Cone Nickel Nickel Skimmer Cone Nickel Nickel Nebulizer ~100 µl/min Concentric (PFA) ~100 µl/min Concentric (PFA) Spray Chamber Temperature 2 C 2 C Carrier Gas Flow 0.70 to 0.75 L/min 0.60 to 0.65 L/min Make-up Gas Flow 0.30 to 0.35 L/min 0.17 to 0.22 L/min Sampling Depth 6.5 to 7.5 mm 7.0 to 8.0 mm Extraction 1 Lens 3.5 to 4.0 V -180 to -175 V Extraction 2 Lens -160 to -150 V -5 to 5 V Reaction Cell H 2 = 4.6 to 4.9 ml/min H 2 = 4.6 to 4.9 ml/min Gas Flows He = 0 ml/min He = 0 ml/min Collision and Reaction H 2 = 2.5 to 3.0 ml/min H 2 = 2.5 to 3.0 ml/min Cell Gas Flows He = 2.0 to 2.5 ml/min He = 2.0 to 2.5 ml/min For daily operation, the ICP-MS was initially tuned under hot plasma (1500 W) conditions with the CRC disabled. Instrumental sensitivity and stability (%RSD 1.5%) was optimized for masses (m/z) 7 Li +, 24 Mg +, 59 Co +, 89 Y +, 140 Ce +, and 205 Tl +. To minimize the formation of polyatomic interferences, the sampling depth and gas flows were adjusted to have <2% oxide formation (m/z: 140 Ce 16 O + / 140 Ce + or 156/140) and <2.5% doubly charged ion formation (m/z: 140 Ce ++ / 140 Ce + or 70/140). Tuning of the quadrupole (peak resolution, peak shape, and resolution axis) and detector calibration (pulse to analog counting mode linearity) were all performed via auto-tune. For cool plasma measurements, the instrument was first optimized in hot plasma before lowering the forward RF power to 600 W. The instrument was re-optimized in cool plasma after allowing the vacuum pressure to stabilize for ~15 minutes. In both plasma modes, the CRC was operated by using H 2 gas (reaction mode), He gas (collision mode), or both He and H 2 gas (collision-and-reaction mode). For the present study, only reaction mode (RM) or collision-and-reaction mode (CRM) are discussed due to their greater efficiency in reducing plasma- and matrix-based interferences (Feldmann et al, 1999; Iglesias et al, 2002; 7

15 Leonhard et al, 2002). The instrument under collision mode (CM) was not able to provide acceptable signal-to-noise (S/N) ratios for several elements, in particular 56 Fe, 78 Se, and 75 As (Figure 3 A-B). The best detection limit for an element results from a combination of both low blanks and high sensitivity during ICP-MS measurement, represented as S/N in cps-per-µg L -1 / matrix blank. To prolong the operational life of the Octopole CRC, the total H 2 and/or He gas flows under both RM and CRM were kept below 5.0 ml/min. This limits the total amount of H 2 atoms entering the cell, which minimizes the physical corrosion of the Octopole CRC over time. Daily operational gas flows were chosen based on optimization of the S/N ratio for particular elements in varying sample-types and matrices. A typical example of a gas flow optimization of the RM (H 2 only) on 56 Fe, 78 Se, and 75 As is given in Figure 4. Removal of interferences from 56 Fe is a benchmark for CRC optimization due to the sheer magnitude of 40 Ar 16 O + interferences on 56 Fe + (4A). For 56 Fe, ICP-MS operation without RM (H 2 = 0 ml/min) has a background noise of ~91% of the analyte signal (the 1 µg/l Fe standard and matrix blank sensitivities are ~46x10 6 cps and ~42x10 6 cps, respectively). With an operational and optimized RM, the noise is reduced to ~3% (130,000 cps and 4,000 cps, respectively) of analyte signal intensity. Moreover, the optimization of gas flows on 56 Fe offers an excellent S/N for low concentration Fe determination, without decreasing the S/N of other analytes of interest, such as 78 Se (Figure 4B) and 75 As (Figure 4C) High Resolution-ICP-MS A Thermo Finnigan Element XR at the University of Cambridge was used to compare the instrumental capabilities of an HR-ICP-MS with that of a Quadrupole-ICP-MS. The sample introduction setup was as follows: quartz cyclonic spray chamber, platinum injector, platinum sampler and skimmer cones, and a 50 µl/min Savillex C-Flow nebulizer. Measurements in low resolution were made using Faraday counting and pulse mode, while measurements made in medium resolution were performed under analog counting mode. All analyses were performed after sensitivity optimization (~2.5 Mcps / µg L -1 ) on 115 In (Table 3). 8

16 Figure 3. Collision mode (CM) gas flow (He) optimization for 56 Fe (3.A), 78 Se (3.B), and 75 As (3.C) in hot plasma. The volume of He gas flow into the CRC (ml/min) is plotted on the X-axis; the left hand Y-axis denotes analyte sensitivity in matrix blank and standard (1 µg/l) in logarithmic scale; the right hand Y-axis denotes the signal-to-noise ratio (1 µg/l standard sensitivity /matrix blank) in linear scale. Analyses were done in mixed acid matrix (0.048 M HNO M HCl). Circles represent matrix blanks; squares: standard sensitivity; and diamonds: signal-to-noise ratios (S/N). The ranges of optimal gas flow, as defined by low matrix blank and high S/N, is shaded in gray. 56 Fe and 78 Se have plasma based Ar-O and Ar-Ar interferences respectively, whereas, 75 As have matrix-based interference from Ar-Cl. Operation of CRC in CM is inefficient in eliminating plasma-based polyatomic interferences, which is evident from very low S/N (< 5) for 56 Fe and 78 Se at high He gas flow. For 75 As, a better S/N (~25) can be achieved in CM as weaker Ar-Cl bonds are more efficiently broken during collision with He. This inefficiency of CM in removing plasma based polyatomic interferences essentially rules out analyses of elements with Ar based interference via this CRC mode. 9

17 Figure 4. Reaction mode (RM) gas flow (H 2 ) optimization for 56 Fe (4.A), 78 Se (4.B), and 75 As (4.C) in hot plasma. The volume of H 2 gas flow into the CRC (ml/min) is plotted on the X-axis; the left hand Y-axis denotes analyte sensitivity in matrix blank and standard (1 µg/l) in logarithmic scale; the right hand Y-axis denotes the signal-to-noise ratio (1 µg/l standard sensitivity /matrix blank) in linear scale. Analyses were done in mixed acid matrix (0.048 M HNO M HCl). Circles represent matrix blanks; squares: standard sensitivity; and diamonds: signal-to-noise ratios (S/N). The ranges of optimal gas flow, as defined by low matrix blank and high S/N, is shaded in gray. Operation of CRC in RM is efficient in knocking out plasma and matrix based polyatomic interferences as evident from high S/N (> 30) for 56 Fe, 78 Se and 75 As at moderate H 2 gas flow (4.5 ± 0.5 ml/min). Considering Fe (4.A), the sensitivity difference between standard and matrix blank is ~9% (~ 46 x 10 6 cps and 42 x 10 6 cps, respectively) without the CRC in operation, whereas in RM with optimal gas flow, the difference increases to ~97% (130,000 cps and 4,000 cps, respectively). Moreover, optimizing the CRC on 56 Fe in RM ensures that other analytes of interest ( 75 As, 78 Se, 111 Cd) had high ( 30) S/N. In addition, during gas flow optimization the lowest acceptable gas flow is chosen to reduce the amount of H 2 gas entering the Octopole CRC, hence minimizing physical degradation of the CRC. 10

18 Table 3. Instrumental settings of HR-ICP-MS (Thermo Finnigan ElementXR) for low and medium resolution Fe analyses. Instrumental Parameter Spray Chamber Injector Sampler Cone Skimmer Cone Nebulizer Cool Gas Flow Sample Gas Flow Auxiliary Gas Flow Additional Gas Flow Sampling Depth Extraction Lens Focus X-Deflection Y-Deflection Shape Quad 1 Quad 2 Focus Quad Filter Lens Setting Quartz (cyclonic) Platinum Platinum Platinum ~50 µl/min Savillex C-Flow 15 L/min L/min 1.30 L/min L/min mm V -950 V 5.25 V 2.25 V 125 V 2.50 V V V 0.00 V 11

19 CHAPTER 3 RESULTS AND DISCUSSION 3.1 Analytical Figures of Merit Direct comparison of HR-ICP-MS and Q-ICP-MS in terms of their sensitivity, instrumental blanks, and signal-to-noise ratios is limited by the unavailability of these results in published works. In Table 4 we present a comparative study of 56 Fe figures of merit from the two types of instruments. The HR-ICP-MS (Thermo Finnigan Element XR) has high sensitivity and a comparatively high instrumental blank, resulting in a low signal-to-noise ratio (S/N Low Res = ~1; S/N Med Res = 120). The Quadrupole-ICP-MS is an Agilent 7500cs which yields a better signal-to-noise ratio for 56 Fe while utilizing cool plasma and reaction mode (H 2 gas only), thus offering a very low detection limit (S/N Cool Plasma, RM = 210). Table 4. Comparison of 56 Fe matrix blanks (in HNO 3 matrix), sensitivity ([Fe] analyte = 1 µg/l; High Purity Standard), and signal-to-noise (sensitivity/matrix blank) ratios of HR-ICP-MS (Thermo Finnigan ElementXR; University of Cambridge) and Q-ICP-MS (Agilent 7500cs; Florida State University). Q-ICP-MS was operated under reaction mode (CRC with H 2 gas) in both hot plasma (1500 W) and cool plasma (600 W) conditions. Plasma Power Matrix Blanks (cps) 1 ppb (cps) Instrument Resolution Matrix Thermo Finnigan Element XR Low 1250 W 0.1 M HNO 3 ~1x10 9 ~1x10 9 ~1 Thermo Finnigan Element XR Medium 1250 W 0.1 M HNO 3 6, , Signal/Noise Agilent 7500cs Low 1500 W 0.44 M HNO 3 1,300 52, Agilent 7500cs Low 600 W 0.44 M HNO , Sample analyses by Q-ICP-MS included a full-metal scan of 71 elements, ranging from mass 7 Li to 238 U. The Q-ICP-MS was operated under hot and cool plasma conditions, utilizing reaction mode or collision-and-reaction mode, and with samples in three different matrix compositions (0.44 M HNO 3, 1.0 M HNO 3, and mixed acid (0.048 M HNO M HCl)). Table 5 lists the sensitivity in counts-per-second per parts-per-billion (cps/µg L -1 ) per isotope, limit of detection in parts-per-trillion (ng/l), and signal-to-noise ratio of the major analytes of 12

20 Table 5. Analytical figures of merit: sensitivity (cps/µg L -1 per isotope), limit of detection (LoD, 3σ; ng/l), and signal-to-noise ratios (S/N) of analytes of interest ( 51 V, 52 Cr, 55 Mn, 56 Fe, 57 Fe, 58 Ni, 59 Co, 63 Cu, 66 Zn, 75 As, 78 Se, 80 Se, 111 Cd, and 208 Pb) in hot and cool plasma conditions with reaction mode or collision-reaction mode of CRC operation. Experiments were conducted using three different matrices: 0.44 M HNO 3 (5.A); 1.0 M HNO 3 (5.B); and mixed acid matrix (0.048 M HNO M HCl) (5.C). Reported sensitivities are corrected for the percent abundance of isotope of choice. The isotopic abundance correction of sensitivity allows for a more meaningful comparison of blanks and instrumental sensitivity for different elements. Table 5.B. compares the expected minimum (Bruland, 1983; Donat & Bruland, 1995) of a concentrated (20-fold) seawater sample (Milne et al, 2010) with the optimal LoD (shaded gray) for each element. Analyte 51 V 52 Cr 55 Mn 56 Fe 57 Fe 58 Ni 59 Co 63 Cu 66 Zn 75 As 78 Se 80 Se 111 Cd 208 Pb Table 5.A M HNO 3 Hot Plasma Cool Plasma CRC Sensitivity LoD Sensitivity LoD Gas (cps/µg L -1 ) (ng/l) S/N (cps/µg L -1 ) (ng/l) S/N H 2 16, , He + H 2 21, , H 2 16, , He + H 2 29, , H 2 75, , He + H 2 52, , H 2 57, , He + H 2 46, , H 2 59, , He + H 2 45, , H 2 8, , He + H 2 15, , H 2 24, , He + H 2 29, , H 2 3, , He + H 2 16, , H 2 27, , He + H 2 23, , H 2 1, He + H 2 2, , H 2 4, , He + H 2 5, , H 2 4, , He + H 2 5, , H 2 100, , He + H 2 86, , H 2 210, , He + H 2 210, ,

21 Analyte 51 V 52 Cr 55 Mn 56 Fe 57 Fe 58 Ni 59 Co 63 Cu 66 Zn 75 As 78 Se 80 Se 111 Cd 208 Pb Table 5.B. 1.0 M HNO 3 Hot Plasma Cool Plasma CRC Sensitivity LoD Sensitivity LoD Gas (cps/µg L -1 ) (ng/l) S/N (cps/µg L -1 ) (ng/l) S/N H 2 20, , He + H 2 23, , H 2 20, , He + H 2 31, , H 2 93, , He + H 2 57, , H 2 71, , He + H 2 50, , H 2 72, , He + H 2 41, , H 2 10, , He + H 2 16, , H 2 30, , He + H 2 31, , H 2 4, , He + H 2 16, , H 2 28, , He + H 2 22, , H 2 160, , He + H 2 170, , H 2 3, He + H 2 4, , H 2 3, He + H 2 4, , H 2 110, , He + H 2 86, , H 2 250, , He + H 2 230, , Expected Seawater Minimum (ng/l) 23,400 3, , ,

22 Table 5.C. Mixed Acid (0.048M HCl plus 0.045M HNO 3 ) Hot Plasma Cool Plasma Sensitivity LoD Sensitivity LoD Analyte CRC Gas (cps/µg L -1 ) (ng/l) S/N (cps/µg L -1 ) (ng/l) S/N 51 H V 2 16, , He + H 2 19, , H Cr 2 16, , He + H 2 26, , H Mn 2 82, , He + H 2 52, , H Fe 2 61, , He + H 2 46, , H Fe 2 64, , He + H 2 45, , H Ni 2 8, , He + H 2 14, , H Co 2 25, , He + H 2 27, , H Cu 2 3, , He + H 2 14, , H Zn 2 36, , He + H 2 30, , H As 2 1, , He + H 2 2, , H Se 2 6, , He + H 2 6, , H Se 2 6, , He + H 2 6, , H Cd 2 133, , He + H 2 110, , H 2 270, , Pb He + H 2 250, ,

23 interest ( 51 V, 52 Cr, 55 Mn, 56 Fe, 57 Fe, 58 Ni, 59 Co, 63 Cu, 66 Zn, 75 As, 78 Se, 80 Se, 111 Cd, and 208 Pb). The reported sensitivity is corrected for percent abundance per isotope to demonstrate the total cps for each element, allowing a more accurate comparison of sensitivities between elements and isotopes. For example, using this scheme of reporting the two isotope pairs of Fe and Se, 56 Fe & 57 Fe and 78 Se & 80 Se have identical sensitivities, within instrumental uncertainty, despite the major difference in their respective isotopic abundance. In addition, Table 5.B. compares the best detection limits achieved in the present study with the lowest concentrations (Bruland, 1983; Donat & Bruland, 1995) from an expected seawater sample that has been pre-concentrated by a factor of 20 (Milne et al, 2010). The detection limits range from 3 to 3,000 times lower than the expected concentrations from the pre-concentrated seawater extractions. The variability in analyte sensitivity in different acid matrices is due to the difference in viscosities of the sample matrix that affects the sample uptake rate. In addition, elements with low IP-1 and lighter mass (m < 65 amu) have a higher sensitivity and signal-to-noise ratio in cool plasma mode versus the heavier elements (m > 65 amu) (Table 5). Based on this observation and depending on the elemental isotope(s) of interest, the CRC operational mode (RM versus CRM) was decided. For Fe, use of RM is preferred for 56 Fe as it efficiently eliminates 40 Ar 16 O + and thus provides the best S/N. However, 57 Fe measurements are optimal with use of CRM as H 2 reacts with 40 Ar 16 O + and 56 Fe + to create 40 Ar 16 O 1 H + and 56 Fe 1 H +, respectively, which both interfere with 57 Fe. Operation of the CRC in CRM (H 2 and He) instead of RM (H 2 ) reduces the total amount of H 2 in the cell and thus minimizes reactions that lead to formation of H 2 based polyatomics within the CRC. Moreover, in CRM, the 40 Ar 16 O 1 H + and 56 Fe 1 H + molecules upon formation are removed through collision with He, thus resulting in a better S/N for 57 Fe (Table 5). 3.2 Plasma-based Interferences Every isotope of Fe has a plasma-based Ar-polyatomic interference, making it a challenging element for ICP-MS analyses. Figure 5 compares the limit of detection, blanks, and air blanks between hot and cool plasma conditions for 56 Fe with and without the CRC in operation (RM). With the CRC turned off (H 2 = 0.0 ml/min), the hot plasma (1500 W) conditions have a larger matrix blank and limit of detection than the cool plasma (600 W) conditions. Argon has a very high first ionization potential (15.76 ev, versus Fe IP-1 = 7.87 ev), therefore Ar is not as efficiently ionized in the weaker cool plasma compared to hot plasma. Thus, there are fewer Ar based polyatomics in cool plasma with the CRC in RM (H 2 =

24 ml/min), resulting in the 0.44 M HNO 3 blanks being ~40% lower than in hot plasma (Figure 5). With the Ar-based polyatomic interferences drastically reduced in cool plasma, accurate determination of trace Fe in natural samples can be readily accomplished. For example, surface seawater ([Fe] SW 2.7 x mol/l) is concentrated by only a factor of via a cation chelating column in 1 ml of 1.0 M HNO 3 (Milne et al, 2010). Figure 5. Comparison of air blank, matrix blank (0.44 M HNO 3 ) and limit of detection (LoD: 3σ) of Fe in hot and cool plasma mode (red and blue bars, respectively), with and without CRC operation in RM. Checkered bars represent the LoD (cps), solid bars: matrix blank (cps), and slashed bars: air blank (dry plasma). The gray region denotes the utilization of the CRC in RM. In both plasma conditions, the blanks are reduced by 3 orders of magnitude in RM. Cool plasma in RM achieves the lowest matrix blanks and best limit of detection due to fewer Arbased polyatomics forming in the plasma. Argon is not as efficiently ionized under cool plasma (600 W) conditions compared to hot plasma (1500 W) due to its high IP-1 (15.76 ev). Figure 6A shows the comparison between matrix blanks and sensitivity (per µg/l) for 56 Fe under all operating conditions: hot plasma in CRM, hot plasma in RM, cool plasma in CRM, and cool plasma in RM. In addition, the comparison includes the different acid matrices of interest: 0.44 M HNO 3, 1.0 M HNO 3, and mixed acid (0.048 M HNO M HCl). In cool plasma mode, the matrix blanks are generally lower for all acid matrices, especially with use of RM (Figure 6A). Moreover, the Fe sensitivity is always high in cool plasma, regardless of which 17

25 Octopole CRC mode is adopted. The weaker cool plasma not only reduces the formation and ionization of Ar interferent molecules, it also reduces the ionization efficiency of other elements with high IP-1. This preferential ionization of low IP-1elements is due to the inefficient ionization of high IP-1 elements (particularly Ar) diminishing the space charge effect in cool plasma (Misra & Froelich, 2009), resulting in increased sensitivity for low atomic weight elements (m/z ~<65). Based on the comparative evaluation of S/N, we conclude that the optimal operating conditions for 56 Fe (and other elements of low atomic mass) analysis measurements in any acid matrix involves the use of cool plasma with the CRC in RM mode (Figure 6B). While the exact S/N values vary across acid matrices, the trend remains the same. To further highlight the sensitivity comparisons for hot and cool plasma conditions for Fe, standard calibrations of 56 Fe and 57 Fe (Figure 7) demonstrate that sensitivity increases by a factor of ~3 over hot plasma when cool plasma conditions are used. In addition, experiments were performed for 75 As, 78 Se, and 111 Cd (Figure 6C 6H). Unlike 56 Fe, these isotopes exhibit optimal S/N ratios under hot plasma conditions in CRM. Figure 6. Comparison of sensitivities (cps/1 µg L -1 ) and matrix blanks (cps) for 56 Fe (6.A), 75 As (6.C), 78 Se (6.E), and 111 Cd (6.G) in all operating conditions: hot and cool plasma, Reaction Mode (RM, H 2 = 4.7 ml/min) and Collision-and-Reaction Mode (CRM, He = 2.8 ml/min and H 2 = 2.0 ml/min). The black bars denote 0.44 M HNO 3 matrix blank, gray bars: 1.0 M HNO 3, and light gray bars: mixed acid (0.048 M HNO M HCl)). The ratio of the sensitivities and matrix blanks can be represented as signal-to-noise ratios (S/N) (6.B, 6.D, 6.F, 6.H). 56 Fe achieves the best S/N under cool plasma conditions with RM. However, 75 As, 78 Se, and 111 Cd all generally exhibited optimal S/N ratios under hot plasma with CRM. These heavy isotopes (> 65 amu) are generally not as efficiently ionized in cool plasma as compared to 56 Fe. 18

26 19

27 Figure 7. Standard calibration in 0.44 M HNO 3 of 56 Fe (circles with solid lines, y 1 -axis) and 57 Fe (squares with dashed lines, y 2 -axis) operated under hot plasma (red) and cool plasma (blue) conditions with the CRC in RM (H 2 = 4.7 ml/min). Both 56 Fe and 57 Fe slopes increase by a factor of ~3 when cool plasma conditions are applied. There is insignificant deviation from the slope, including data in the low-concentration end of the calibration shown in the dark gray inset. 3.3 Matrix-based Interferences Choice of sample matrix and its purity dictates both the type and magnitude of matrixbased interference formation. For the present study, elements with major matrix-based interferences include, but are not limited to, 51 V ( 35 Cl 16 O + ), 75 As ( 40 Ar 35 Cl + ), 111 Cd ( 95 Mo 16 O + ), and 156 Eu ( 140 Ce 16 O + ). The 95 Mo 16 O + (m/z = 111) interference on 111 Cd is caused by the presence of 95 Mo in the analyte matrix. The relative abundance of Cd and Mo in the sample of interest dictates the severity of this oxide-based interference. For seawater samples, [Cd] sw << [Mo] sw ([Cd] SW 4 x mol/kg; [Mo] SW = 110 x 10-3 mol/kg (Milne et al, 2010; Boyle et al, 2012)), which makes accurate determination of Cd in seawater an analytical challenge. Therefore, Cd is used as one of the examples in this study to demonstrate how matrix-based interferences are overcome using the CRC. Similarly, 75 As has a large 40 Ar 35 Cl + (m/z = 75) based interference for matrices containing Cl (e.g., halites, seawater, or presence of HCl in acid matrix). The extremely small mass difference between 75 As + (m/z = ) and 40 Ar 35 Cl + (m/z = ) requires high resolution (M/ΔM = 10,000) mass spectrometry to achieve quantitative 20

28 peak separation between the two ions. However, inefficient ionization of As in the plasma (high IP ev) coupled with usually low abundance in natural samples makes determination of As by HR-ICP-MS a serious analytical challenge. The application of Octopole CRC quasiquantitatively eliminates the polyatomic interference caused by 95 Mo 16 O + and 40 Ar 35 Cl +, rendering accurate and precise analysis of 111 Cd + and 75 As + possible without significant loss in sensitivity (Figure 8). Figure As standard calibration in mixed acid (0.048 M HNO M HCl) operated under hot plasma (red) and cool plasma (blue) conditions, with RM (H 2 = 4.7 ml/min, circles solid lines) and CRM (He = 2.8 ml/min and H 2 = 2.0 ml/min, squares with dashed lines) employed. Hot plasma with CRM achieves sensitivity that is greater than the other operating conditions by nearly a factor of ~2. There is insignificant deviation from the slope, including data in the low-concentration end of the calibration shown in the dark gray inset. Using single element Mo standards at different concentrations, we performed a series of 95 Mo calibrations under all instrumental conditions: hot plasma and cool plasma, with CRM and RM (Figure 9). Under the different plasma & CRC modes and across the Mo concentration range of 0.1 µg/l to 2.0 µg/l, the intensity on 111 amu ( 95 Mo 16 O) never exceeded 8 ng/l, which is comparable to the detection limit of 111 Cd ( 3 ng/l, Table 5A). The formation of 95 Mo 16 O + does not increase with the concentration of Mo in solution. This lack of mass dependency of the 21

29 95 Mo 16 O + interference on 111 Cd demonstrates the successful removal of matrix-based polyatomic interferences by Octopole CRC. The maximum instrumental sensitivity for both Cd and Mo are achieved in hot plasma conditions using CRM (Figure 9 and 10). However, the next best sensitivity for both elements is achieved in cool plasma with CRM, indicating that the Octopole CRC setting dictates the sensitivity on Mo and Cd more than the plasma settings (Figure 9 and 10). For Cd analyses, the advantage of CRM over RM is that excess H 2 is minimized in CRM. This reduces the loss of analyte sensitivity that can result from reactions between Mo (or Cd) and H 2. Figure Mo standard calibration (circles) in 0.44 M HNO 3 executed in hot plasma with CRM (He = 3.0 ml/min and H 2 = 2.0 ml/min), hot plasma with RM (H 2 = 4.7 ml/min), cool plasma with CRM (He = 2.8 ml/min and H 2 = 2.0 ml/min), and cool plasma with RM (H 2 =4.8 ml/min). Measurements of 111 amu (squares) show complete elimination of 95 Mo 16 O + by the Octopole CRC. The intensity on 111 amu never exceeds 8 ng/l, which is comparable to the detection limit of 111 Cd ( 3 ng/l, Table 4A). The best sensitivities on 95 Mo is achieved from hot and cool plasma with CRM while both cool settings in RM achieve very poor sensitivities, suggesting that the CRC operational mode dominates the sensitivity on Cd more than the plasma setting. 22

30 Figure 10. Comparison of sensitivity calibrations (slopes, cps/µg L -1 ) from a 111 Cd standard in 0.44 M HNO 3, operated in hot and cool plasma (red and blue bars, respectively) with the CRC in CRM and RM (white and gray regions, respectively). The solid bars represent the pure Cd calibrations and the shaded bars are the Cd standards spiked with 1 µg/l Mo. Hot plasma with CRM achieves maximum sensitivity and complete elimination of the 95 Mo 16 O + interference, which is inferred from the identical slopes between the pure and spiked calibration. Cool plasma with CRM also achieves complete elimination of the polyatomic interference, within (~2.0%). However, hot and cool plasma with RM show greater variability (~3% and ~5%, respectively), which supports the conclusion that the CRM has a greater affect on Mo (and Cd) than the plasma setting. To further investigate the efficiency of the CRC to remove the matrix-based 95 Mo 16 O + interferent on mass 111 and thus its effect on 111 Cd determination, the sensitivity calibrations (slopes) of pure Cd standard solutions ( µg/l) are compared to that of Cd standards doped with 1 µg/l Mo. This comparison was performed for different plasma and CRC settings (Figure 10). For the hot and cool plasma modes and RM and CRM settings, the average difference in Cd sensitivity for pure Cd standards and Cd doped with Mo standards ( 2.0 %) is within the instrumental uncertainty of Cd determination (< 1.5 %). The similarity in Cd sensitivity of the two solutions demonstrates efficient elimination of 95 Mo 16 O + interference from 111 Cd +. Similar to Mo, ICP-MS operation under hot plasma with CRM results in best Cd sensitivity, however cool plasma with CRM does not demonstrate similar efficiency for Cd because Cd IP-1 (9.0 ev) is somewhat higher than Mo IP-1 (7.1 ev). Moreover, under both hot 23

31 and cool plasma conditions, greater Cd S/N is achieved in CRM over RM (Figure 10), demonstrating that choice of the CRC operational mode is more important than selection of plasma conditions. 3.4 Long-term Reproducibility To assess the long-term reproducibility of the ICP-MS methods described in this work, an external Standard Reference Material (NIST 1643e SRM) was analyzed over a period of 7 months (Figure 10) under all operational conditions and acid matrices. The NIST 1643e analyses were performed at a 1:100 dilution of the original solution ([Fe] 1 µg/l and [Cd] 0.07 µ/l) to match the analyte concentrations in actual samples. The results from the present study ([Fe] Average = 99.6 ± 7.8 µg/l and [Cd] Average = 6.78 ± 0.83 µg/l; n = 26, ±2σ) are identical, within analytical uncertainty of the instrument, to the certified values ([Fe] NIST-SRM = 98.1 ± 1.4 µg/l and [Cd] NIST- SRM = 6.57 ± 0.07 µg/l) (Table 6). Figure 11. SRM NIST 1643e (Standard Reference Material - trace elements in freshwater) Fe and Cd concentration data collected over a period of 7 months. The SRM was analyzed at a 1:100 dilution in three different acid matrices (0.44 M HNO 3, 1.0 M HNO 3, or mixed acid (0.048 M HNO M HCl), which is not specified in this graph. Data was obtained under all instrumental settings: hot plasma (red) and cool plasma (blue), with the CRC in RM (closed symbols) and CRM (open symbols). The measured averages (black symbols, 2σ error bars) are compared to the certified NIST 1643e values (green symbols, 2σ error bars). 24

32 Table 6. SRM NIST 1643e (Standard Reference Material- trace elements in freshwater) Fe and Cd concentration data collected over a period of 7 months (Figure 11). The SRM was analyzed at a 1:100 dilution in three different acid matrices (0.44 M HN0 3, 1.0 M HN0 3, or mixed acid (0.048 M HN M HCl). The reported data includes operation of the Q-ICP-MS under four different instrumental settings: hot plasma and cool plasma, and CRC operation in RM and CRM. Date J\llle 7th Plasma Mode Cool CRC Mode RM Acid Matrix 1.0 M HN0 3 [Mg] ± 2cr [Fe] ± 2cr [Co]± 2cr [Ni] ± 2cr [As]± 2cr [Se] ± 2cr [Cd] ± 2cr [Sb] ± 2cr [Pb] ± 2cr エ Mセ Hァ O li@ H セ M エァ O li@ H セ M エァ O l I@ H セ M エァ O l I@ H セ M エァ O l I@ H セ M エァ O l I@ H セ M エァ OlI@ H セ M エァ O l I@ H セ M エァ OlI@ n.a n.a. n.a. n J\llle 9th Mixed Acid 8018 Cool rrm 0.44 M HN n.a. n.a n.a. n.a. n.a. n.a. 1.0 M HN n.a n.a. n.a. J\llle 13th Hot Mixed Acid n.a n.a. n.a. RM 0.44 M HN n.a n.a. n.a. 1.0 M HN n.a n.a. n.a. 25 J\llle 14th Hot Mixed Acid n.a n.a. n.a. CRM 0.44 M HN n.a n.a. n.a. Mixed Acid n.a n.a. n.a. July 1st July 19th Cool Hot RM RM 1.0 M HN M HN ± ± ± n.a n.a ± ± 0.22 n.a. n.a. n.a. n.a. I 2 July 20th Hot RM 0.44 M HN ± ± ± 1.0 I ± ± ± ± ± ± Sept. 9th Oct. 14th Oct. 16th Hot Hot Hot RM RM RM 0.44 M HN ± M HN ± M HN ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.26 n.a ± 0.04 n.a ± ± ± ± ± ± ± Oct. 17th Dec. 15th Hot Hot RM CRM 0.44 M HN ± 463 Mixed Acid ± ± ± ± n.a ± 0.70 n.a ± ± I Dec. 16th Hot RM 0.44 M HN n.a Dec. 18th Jan. 16th Average Certified Value Cool Cool RM CRM Mixed Acid 8040 Mixed Acid n.a. n.a ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.20 n.a. n.a