Calibration Approach for Gaseous Oxidized Mercury Based on Nonthermal Plasma Oxidation of Elemental Mercury

Atmospheric mercury measurements carried out in the recent decades have been a subject of bias largely due to insufficient consideration of metrological traceability and associated measurement uncertainty, which are ultimately needed for the demonstration of comparability of the measurement results. This is particularly challenging for gaseous HgII species, which are reactive and their ambient concentrations are very low, causing difficulties in proper sampling and calibration. Calibration for atmospheric HgII exists, but barriers to reliable calibration are most evident at ambient HgII concentration levels. We present a calibration of HgII species based on nonthermal plasma oxidation of Hg0 to HgII. Hg0 was produced by quantitative reduction of HgII in aqueous solution by SnCl2 and aeration. The generated Hg0 in a stream of He and traces of reaction gas (O2, Cl2, or Br2) was then oxidized to different HgII species by nonthermal plasma. A highly sensitive 197Hg radiotracer was used to evaluate the oxidation efficiency. Nonthermal plasma oxidation efficiencies with corresponding expanded standard uncertainty values were 100.5 ± 4.7% (k = 2) for 100 pg of HgO, 96.8 ± 7.3% (k = 2) for 250 pg of HgCl2, and 77.3 ± 9.4% (k = 2) for 250 pg of HgBr2. The presence of HgO, HgCl2, and HgBr2 was confirmed by temperature-programmed desorption quadrupole mass spectrometry (TPD-QMS). This work demonstrates the potential for nonthermal plasma oxidation to generate reliable and repeatable amounts of HgII compounds for routine calibration of ambient air measurement instrumentation.


Foreword and scope of the method
The calibration method is based on generation of a known amount of gaseous elemental mercury (Hg 0 ), its quantitative oxidation to gaseous oxidized mercury species (Hg II ) by NTP in the presence of reaction gas, Hg II thermal reduction back to Hg 0 , and its determination by cold vapor atomic fluorescence spectrometry (CV-AFS). The method is designed to be suitable for calibration of any commercially available atmospheric mercury speciation unit. In the presented SOP, the medium used for Hg II collection is a sorbent trap (in the following sections named "plasma trap"; see Figure 1 of the article's main body for more information). Instead of a sorbent trap, commonly used atmospheric Hg speciation methods often utilize denuders or sorbent membranes. Although these Hg II collection methods can be used for calibration instead of sorbent traps, their compatibility with our calibration was not validated in our work. The instructions provided in this SOP are fit-for-purpose regardless of the method used for Hg II collection and regardless of the method used for mercury detection.
The intended amounts of Hg II species produced using this method for calibration of ambient concentration levels of Hg II are in the order of 50-100 pg. Therefore, ultra-trace analysis protocols have to be applied to prevent contaminations: keeping the laboratory clean, providing appropriate ventilation, and adequately washing glassware, tools, and containers.

Safety precautions
Follow universal precautions. Wear gloves, a lab coat, and safety glasses whilst handling chemicals. For handling the non-thermal plasma and its power supply unit, the safety rules for working with electrical equipment should be followed: turn off the power supply of the equipment before inspecting it; use only tools/equipment with non-conducting handles; avoid contacting the setup with wet hands and wet materials; do not store highly flammable chemicals near the electrical equipment; call expert electrician if the power supply or instrument keeps causing burn-out of fuses.

Preparation of reagents and materials
10% w/v SnCl2 in 10% v/v HCl:  Weigh accurately 10 g of SnCl2 into a clean glass beaker using a plastic spatula (beaker and spatula are used only for SnCl2).
 Add 10 mL of concentrated HCl directly to the SnCl2 and transfer to a 100-mL volumetric flask. Mix and wait for complete dissolution of SnCl2.
 Add Milli-Q water to the mark (100 mL).
 Purge the SnCl2 solution with Hg-free nitrogen gas for two hours in order to obtain a mercury-free solution.
16% w/v NH2OH · HCl solution:  Weight 16 g of NH2OH · HCl in a suitable glass bottle, add Milli-Q water to a 100-mL mark, and mix well.
 Add 20 μL of SnCl2 solution and purge for 2 hours with Hg-free nitrogen gas.
10% w/v KOH:  Dissolve 10 g of KOH in Milli-Q water to the final volume of 100 mL.
 Purge the KOH solution with nitrogen for two hours in order to obtain a mercury-free solution.
10% w/v Na2S2O3:  Dissolve 10 g of Na2S2O3 in Milli-Q water to the final volume of 100 mL.
 Purge the Na2S2O3 solution with nitrogen for two hours in order to obtain a mercury-free solution.
S-3 0.5% w/v KMnO4 solution:  Dissolve 0.5 g of KMnO4 in Milli-Q water to the final volume of 100 mL.
Bromine monochloride (BrCl) oxidizing solution:  Weigh 15 g of KBrO3 and 11 g of KBr into a clean 1-liter glass bottle.
 Add 200 mL of Milli-Q water.
 Add 800 mL of concentrated HCI. The dilution has to be carried out in a well-ventilated fume hood to prevent exposure to toxic fumes released during dissolution of KBrO3.
 Keep the bottle wrapped with aluminum foil.
 The prepared solution can be kept for an unlimited time if stored in darkness at 5 °C in a tightly closed Teflon or glass bottle.
Gold-coated corundum and gold trap preparation:  Gold-coated corundum: Dissolve 1 g of HAuCl4·xH2O in 10 mL of Milli-Q water and add 10 g of Al2O3. Evaporate the solution in an automatic rotary evaporator under reduced atmospheric pressure and then heat the remaining material at 500 °C for 4 h in an argon atmosphere.
 Gold trap preparation: Insert a small piece of quartz wool at the end of the longer part of the column. Then insert about 2 cm of gold-coated corundum. It is recommended to weigh the goldcoated corundum in order to obtain a better reproducibility between the traps. Insert a larger piece of quartz wool. Precondition the new trap by heating it at least 4 times before use.
Plasma trap preparation: shown in the main body of the article, in Figure 1.
Drying columns preparation: similar as for gold trap preparation, except that the gold-coated corundum is replaced with soda lime.

Mercury standard solutions (SRM NIST 3133)
Standard stock solution, 99.54 µg g −1 of Hg in 5% HNO3: Important note: record the mass after each volume addition or subtraction step, otherwise the Hg mass concentration cannot be evaluated correctly. Fill a weighed 100-mL glass flask with 5% HNO3 solution exactly to mark of 100 mL and remove 1 mL of the solution. Gently shake an ampoule of Standard Reference Material 3133, Mercury (Hg) Standard Solution for 2 minutes and open it according to the certificate. Take 1 mL of SRM solution with a pipette directly from the ampoule to the prepared glass flask containing acid solution. By doing so, a solution with an approximate Hg concentration of 99.54 µg g −1 is obtained (the exact value depends on the mass weighing). Cap the flask, shake it well and leave it on room temperature for 1 hour before further dilutions.
Intermediate and working standard solutions, 0.9954 µg g −1 of Hg in 5% HNO3 and 0.9954 ng g −1 of Hg in 5% HCl: Prepare an intermediate Hg standard solution with the concentration of 0.9954 µg g −1 by 100-fold dilution of standard stock solution in the same manner as for standard stock solution. At the end, prepare a working Hg standard solution with the concentration of 0.9954 ng g −1 by 1000-fold dilution of the intermediate standard. The last dilution is made with 5% HCl solution instead of a 5% HNO3 solution.
Calculate the exact concentrations of the Hg standard solutions by taking into an account the exact mass of pipetted aliquots.

S-4
Prior to use, thoroughly wash all laboratory glassware following the procedure:  Allow the glass vessels to soak overnight in 2% Micro-90 detergent solution.
 Rinse the vessels thoroughly, first with tap water, and then with Milli-Q water.
 Rinse with water until the color of the KMnO4 solution is no longer visible.
 Rinse three times with Milli-Q water.
 Fill the vessels with 1% HCl solution and store in mercury-free storage facilities.
 Vessels should be emptied just before use for sample processing and allowed to dry at 60 °C.

NTP calibration protocol
As previously stated, the calibration method is comprised of three main steps: i) Hg 0 generation, ii) NTP oxidation of Hg 0 to Hg II species in the presence of a reaction gas, and iii) thermal reduction of Hg II species to Hg 0 with subsequent CV-AFS analysis. The second step has three different setup variations depending on the used reaction gas (O2, Cl2 or Br2).

Hg 0 generation
Similar setup is used as in Figure 2a of the manuscript's main body.
 Provide a clean 250-mL glass impinger ("bubbler") and add 100 mL of Milli-Q water and 3 mL of 10% w/v SnCl2 in 10% v/v HCl.
 Connect a drying column (soda lime trap) to the impinger gas exit and a gold trap downstream of the drying column.
 Add 100 pg of Hg from the working Hg standard solution. In the case of concentrations listed in the section 3.4, this means that 100.5 µL of working standard solution (Hg concentration of 0.9954 ng g −1 ) needs to be pipetted directly to the impinger.
 As soon as the Hg standard is added, quickly close the impinger and connect it to a N2 flow of 1 L min −1 for 10 minutes to purge the Hg 0 to the gold trap. Note: lower total volume of the solution in the impinger can be used together with a lower N2 flow and shorter purging time since these parameters mostly depend on the shape, size, and design of the impinger.
 After 10 minutes, remove the Hg-loaded gold trap from the impinger and use it for the second step.

NTP oxidation of Hg 0 to Hg II species
Similar setup is used as in Figure 2a of the manuscript's main body. Three different Hg II species can be produced via NTP oxidation: HgO with O2 reaction gas, HgCl2 with Cl2 reaction gas, and HgBr2 with Br2 reaction gas. Therefore, we can have three setups that marginally differ from each other.
 Assemble the following parts of the setup (similar as in Figure 2a in the manuscript's main body, going downstream in consecutive order): Hg-loaded gold trap, T-split for the introduction of reaction gas, plasma trap, and impinger with solution for reaction gas reduction. The reaction gas is obtained from i) O2 gas cylinder for O2 reaction gas and ii) electrolytic reaction for Cl2 and Br2 reaction gases (electrolysis described in section S4 of the Supplementary Information). The solution for reaction gas reduction is 50 mL of 10% w/v KOH for Cl2 reaction gas and 50 mL of 10% w/v Na2S2O3 for Br2 reaction gas. In the case of O2 reaction gas, the reduction of the reaction gas is not needed. The final flow of the gas mixture (He + reaction gas) should be 370 mL min −1 , where He represents >99% of the gas mixture and reaction gas represents <1% of the gas mixture.
 IMPORTANT NOTE: Cl2 and Br2 reaction gases are strong oxidants on their own (even without NTP). Therefore, it is important to release them into the system only when every part of the setup is gas-tight. Always connect the reaction gas the last (to avoid unnecessary reactions), after the whole setup is already on the flow of He gas.
 When the setup is assembled and under gas-tight He flow, connect the copper electrodes as shown on Figure 2a in the main text of the article, turn on the plasma driver and set it to these parameters: power applied to the electrodes of 180 µW, radiofrequency of 20 kHz, effective voltage of 345 V.
 After plasma is homogeneous, release the reaction gas flow and after approximately 10 seconds start heating the gold trap (e.g., using a heating coil) to 400 °C to release the Hg 0 from the trap.
 After heating is finished, wait for 60 s to ensure complete release of Hg 0 and then close the reaction gas flow and turn off plasma by turning off the plasma driver.
 After the reaction gas flow is closed and plasma driver is turned off, the setup can be safely disassembled. The produced Hg II that is needed for further calibration is trapped on the plasma trap.

Thermal reduction of Hg II species to Hg 0 and subsequent CV-AFS analysis
Similar setup is used as in Figure 2b of the manuscript's main body.
 Connect the plasma trap that is loaded with Hg II species to a gold trap downstream.
 Connect both traps to the He flow of 370 mL min −1 .
 Heat the whole plasma trap (both KCl crystal part and Al2O3 catalyst) to >600 °C. The temperature should be achieved as fast as possible to avoid desorption of Hg II and its re-deposition on cold spots of the system. For compete thermal reduction of Hg II to Hg 0 (without Hg II desorption), the plasma trap should be heated to >600 °C in under 20 s.
 After the heating is completed, wait for 60 seconds to ensure complete downstream transport of Hg 0 to the gold trap.
 The gold trap is now ready for the determination of mercury by double amalgamation CV-AFS measurement. 1

S3 -Calculation of the decay time-corrected peak areas for samples and standards and the calculation of oxidation or thermal reduction efficiency
The peak area of the sample and standard activity had to be corrected for the decay since the start of irradiation. From the corrected peak areas for samples and standards, the oxidation or thermal reduction efficiencies were calculated as shown in the following paragraphs.
Eq. (1) was applied for calculation of both A0, s (sample peak area at reference time) and A0, std (standard peak area at reference time). If we insert the A0, s and A0, std values from Eq. (1) into the Eq. (2) (used for oxidation or thermal reduction efficiency calculation), we obtain Eq.
where i is either sample (s) or standard (std).
Where: A0, s is the sample peak area at reference time t=0, A0, std is the standard peak area at reference time t=0, As is the sample peak area at the time of measurement, Astd is the standard peak area at the time of measurement, t1/2 is the half-life of 197 Hg [s], tpassed, s is the time passed since reference time t=0 till the start of the sample measurement [s], tmeasurement, s is the time passed during the sample measurement [s], η is the oxidation or thermal reduction efficiency [%], Fdil is the dilution factor, Frep is the repeatability factor (value of 1).

S4 -Electrolytic production of reaction gases Cl2 and Br2
Since Cl2 and Br2 are notoriously difficult to handle, they were produced in the laboratory just for the needs of our experiments. We used electrolysis of 1 M NaCl solution to produce Cl2 and electrolysis of 1 M KBr solution to produce Br2. 4.5 V battery was used as a power source for electrolysis. NaCl infused agar gel was used as a salt bridge to provide a closed electric circuit. The experimental setup for electrolytic production of Cl2 and Br2 is shown in Figure S1. Figure S1. Experimental setup for electrolytic production of reaction gases Cl2 and Br2.
As Cl2 and Br2 are both water-soluble, the impinger solution containing the anode was always purged with He. This increased the amount of Cl2 and Br2 in the gas stream. As Br2 immediately dissolves in the solution, it is not released into the gas stream until enough Br2 is produced and its vapor pressure is sufficiently high.

S-9
S5 -All replicates for the Hg II thermal reduction and production of Hg II species by NTP.

S6 -Calculation of the combined standard uncertainty for the proposed Hg II species calibration
The uncertainty of the developed calibration was estimated according to the GUM and Eurachem guidelines. 3,4 The model used for uncertainty evaluation was shown in Eq. (3). To make the model clearer, we substituted the exponential terms: This way, the model can be re-written as: The equations for the standard uncertainty of the exponential terms (e.g. EFp,std) are as follows: Note that we assumed constant tpassed, std and tpassed, s to obtain Eq. (9) and Eq. (10) and constant t1/2 to obtain Eq. (11) and Eq. (12). This assumption was made because in Eq. (9) and Eq. (10), the relative standard uncertainties of tpassed, std and tpassed, s were negligible in comparison to the relative standard uncertainty of t1/2 (three orders of magnitude smaller relative standard uncertainties for tpassed, std and tpassed, s than for t1/2 ). Similar assumption was made for Eq. (11) and Eq. (12); in this case, the relative standard uncertainty of t1/2 was negligible in comparison to the relative standard uncertainty of tmeasurement, std and tmeasurement, s.
The equation for obtaining standard uncertainties for As and Astd is calculated according to Poisson distribution with some adjustments due to background correction for the peak areas. The exact equation used for calculation is provided by the Genie 2000 gamma analysis software tool and is calculated automatically by the software, using Eq. (13). 5 Since 197 Hg has two doublet peaks in the activity measurement spectrum, the equation can be applied for both peaks, giving us two uncertainties u(A1) and u(A2). The standard uncertainty of the combined peaks (no overlapping of the peaks for our case) is calculated using Eq. (14). Where: Ai is the sample peak area for the observed peak, Bi is the background peak area for the observed peak, Wp,i is the spectral width of the sample peak region for the observed peak, Wb,i is the spectral width of the background peak region for the observed peak.
The last uncertainty components are present in Fdil and Frep. u(Fdil) originates from two pipetting steps, one for the standard (5 mL pipette, u(Vstd)) and one for the sample (1 mL pipette, u(Vs)). Both u(Vstd) and u(Vs) are calculated from uncertainty due to temperature influences (VT) and uncertainty due to repeatability (Vrep); 10 replicate measurements) with Eq. (15). By combining both pipetting uncertainties, u(Fdil) is calculated with Eq. (16). Frep value equals 1, but has to be included due to the components of the measurement procedure that influence the repeatability.
The expanded standard uncertainty is obtained by multiplication of the combined standard measurement uncertainty with a coverage factor, k (k=2) with Eq. (18).

= ( ) • (18)
S-12 Expanded standard uncertainty for the calibration of gaseous HgCl2 by NTP oxidation is therefore 0.07 (k=2). Similar as for gaseous HgCl2, we applied the above calculation for the uncertainty evaluation for gaseous HgO and gaseous HgBr2. The expanded standard uncertainties amounted to 0.05 (k=2) for HgO and 0.09 (k=2) for HgBr2.