Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NO_x

1. Solution Preparation

1. Before sampling, prepare the solutions, calibrate the NO_x analyzer (either luminol or chemiluminescence), and check that the system is working properly and that new filters are installed.
2. Make sampling solutions using 1 M potassium permanganate (KMnO₄) stock solution and 10 M sodium hydroxide (NaOH)¹⁰, and then dilute the solution with ultrapure water to the correct volume.
   NOTE: Purchase premade solutions because they tend to contain lower "blank" NO₃^– concentrations than other forms.⁴
   1. Prepare 10 M NaOH.
      1. Weigh out 200 g of solid NaOH and pour it into a 500 ml volumetric flask. Add ultrapure water (18.2 MΩ·cm at 25 °C) to the meniscus line of the volumetric flask and allow the NaOH to dissolve.
   2. Because this process exudes heat, place the volumetric flask in a room-temperature (~22 °C) water bath and allow it to cool as it dissolves, usually taking 1-2 hr. Store 10 M NaOH in 500 ml amber plastic bottles for up to 1 month.
   3. Prepare a sampling solution of 0.25 M KMnO₄ and 0.5 M NaOH in a 500 ml graduated cylinder (450 ml solution volume).
      1. Add 112.5 ml of 1 M KMnO₄ and then fill with ultrapure water up to 405 ml.
      2. Add 22.5 ml of the 10 M NaOH solution prepared in step 1.2.1 to the graduated cylinder and fill to the 450 ml line with ultrapure water.
   4. Store the solutions in 500 ml amber glass bottles and label each solution with the date (use letters to distinguish each bottle).
   5. Once the solution is made, take a laboratory blank. Remove 25 ml of the solution and record from which solution bottle it came. Store blanks in 60 ml amber glass bottles.
      NOTE: Each solution bottle should yield 8-11 samples (35-50 ml each) and one field blank (25 ml) after the laboratory blank is taken.

2. Field Setup

1. Choose a sampling location (such as a rooftop) and install the system (if using the stationary system). For the mobile laboratory, pack all instrumentation into a typical passenger vehicle. See Figure 1 for a diagram of the automated system.
2. Change all filters labeled in Figure 1 before sampling to ensure that they are working most effectively and efficiently.
   NOTE: There are three types of filters in the system: a PTFE particle filter (1.0 µm; 47 or 25 mm use the larger surface area in more polluted air) to remove particles that may contain NO₃^–, a nylon membrane filter (1.0 µm) for removal of HNO₃ gas, and a hydrophobic filter (10.0 µm) to protect the vacuum pump and the critical orifice from solution droplets. With new filters at the start of a sampling period, the particulate filter and the NO₃ filter will not need to be changed for a couple of days, except in highly polluted or dusty conditions. The hydrophobic filter should be changed every 4-6 hr for as long as sampling is continuously done.
3. For installation of the system, connect the system and the instruments to polytetrafluoroethylene (PTFE) tubing (1/4-inch outer diameter) and aim the inlet, also PTFE tubing, in the direction of the desired air collection.
   NOTE: The mobile laboratory is for taking on-road measurements, whereas the stationary laboratory is for taking ambient urban air and near-road measurements.
4. Set up the 'mobile laboratory', consisting of the NO_x collection system, an NO_x box, a CO₂ analyzer, a global positioning system (GPS) unit, and a marine battery.
   1. Pack the system and all instrumentation into the car. Power the system with a 12 V marine deep-cycle battery for ~12 hr, similar to the maximum duration of one day of mobile measurements. Recharge the battery at the end of the sampling day to prepare for the next day.
      NOTE: Use a separate battery so that there is no need to hardwire to the car battery nor to keep the car running to make measurements. Use two batteries if sampling will be close to or more than 12 hr in order to avoid stopping for a few hours to recharge the battery.
   2. Connect the instruments to the PTFE inlet tube closer to the inlet than to the collection system, because the vacuum pump for the system operates at flow rates of 3-5 L/min, much larger than the flow rates for the NO_x box (~1.5 L/min) or the CO₂/H₂O analyzer (<1 L/min).
   3. Secure the PTFE inlet tube to the front of the car on the roof, pointing towards the front of the car, a position that is the longest possible distance from the exhaust pipe, to avoid capturing self-emissions from the car exhaust pipe. For example, in the mobile laboratory using a midsized sport utility vehicle, the inlet was located on the roof of the car, 2 feet from the driver's side door, placing it 1.6 meters above the road and 2.54 meters from the back bumper.
      1. Alternatively, use an electric or other zero emission vehicle.
   4. Record geolocation and vehicle speed data every second using a GPS unit (if this data is of interest). Synchronize the laptop computer recording the NO_x and CO₂ data with the GPS time prior to measurements.
   5. Turn on the instrumentation at the beginning of the sampling day and turn them off at the end of the sampling day, even when the collection system is not running (the instruments require warm-up time, so leave it running throughout the day to avoid multiple warm-up times).
   6. Load the NOx box with luminol solution when it is turned on at the beginning of the sampling day, and then flush it with water at the end of the day, before the instrument is turned off, as directed by the manufacturer. Store the luminol and solution samples in a cooler in the mobile laboratory to avoid degradation of the solutions. Store the luminol solution in a refrigerated unit overnight.
   7. Calibrate the NO_x box-a luminol-based NO₂/NO analyzer¹¹ and a differential, non-dispersive infrared (NDIR) CO₂/H₂O analyzer using a commercial gas dilution calibrator and following the manufacturer's instructions. The NO_x box has a response time of ~5 sec, which is better equipped to resolve on-road NO_x emission plumes.
5. Set up the 'stationary laboratory', consisting of the NO_x collection system and a chemiluminescence NO_x concentration analyzer.
   1. Fix PTFE tubing to a surface and point it in the direction of the air to be collected.
   2. Split the PTFE tubing at the inlet with a tee fitting to connect both the NO_x analyzer and the automated collection system.
   3. Connect the stationary system to a power outlet (120 V alternating current).
   4. Run the NO_x concentration analyzer continuously throughout the sampling period, even when the collection system is off or switching samples. Use the valve built into the collection system to isolate it during that time so that the NO_x analyzer is sampling ambient air.
   5. Calibrate the chemiluminescence NO_x concentration analyzer. This is used for the stationary measurements, as it has a slower response time (>30 sec), which is better for ambient air measurements.
      1. Calibrate based on the manufacturer's instructions using a gas dilution calibrator. Dilute a standard of 25 ppmv NO in N₂ with zero air to achieve approximately seven calibration points between 0-200 ppbv NO. Using an ozone titrator, calibrate the NO₂ concentrations across the same range (0-200 ppbv NO₂).
   6. If using the mobile laboratory, calibrate the NO_x box-a luminol-based NO₂/NO analyzer¹¹ and a differential, non-dispersive infrared (NDIR) CO₂/H₂O analyzer using a commercial gas dilution calibrator and following the manufacturer's instructions. The NO_x box has a response time of ~5 sec, which is better equipped to resolve on-road NO_x emission plumes.

3. Sample Collection

1. Perform tests on the system to ensure that the flow meter, the syringe pump, the computer software, and the vacuum pump are all working. Turn on each component and check that it is working properly. With the computer software, complete the sampling protocol once or twice to make sure that everything is working properly.
2. Turn on the system so that air is bubbling through the solution and bubbles are visible.
   NOTE: The computer program automates the movement of the solutions throughout the system, but the diaphragm pump and the vacuum pump are operated manually. The user must select the amount of time for the collection of a sample (between 30 and 120 min) that collects enough NO_x for the concentration of the sample to be above the blank nitrate concentration of the solution. Atmospheric NO_x concentrations of 50-100 ppbv near sources such as vehicles require only a 30 min collection time. For ambient urban NO_x concentrations (5-30 ppbv), samples should be collected for up to 120 min. Using the equations provided in steps 6.5 and 6.5.1, the user can back-calculate the collection time to achieve the desired sample concentration in solution.
   1. Use a collection system with a syringe pump to automatically move the solution from the reservoirs into the gas-washing bottle and from the gas-washing bottle to the waste. The four electronically actuated valves and the syringe pump are controlled by a computer program specifically written for the collection system, which has four modes: 1) dispense new solution, 2) clean the tubing, 3) collect the sample, and 4) clean the gas-washing bottle, as follows:
   2. To automatically dispense new solution, aspirate 35 ml of solution (V_S) into the syringe pump from the solution reservoir and dispense it into the gas-washing bottle. The gas-washing bottle frit causes the solution to bubble when the vacuum pump is turned on and the gas sample is introduced.
      NOTE: Choose a solution volume between 25-35 ml based on the NO_x concentrations sampled and the collection times desired.
   3. Clean the tubing between the syringe pump and the gas-washing bottle by automatically pulling the residual solution in the tube back into the syringe and depositing it into the waste container.
   4. Once the sampling solution is in the gas-washing bottle, manually turn on the pump. When the desired amount of time for sampling has been achieved, manually turn off the pump.
   5. After sampling is done, collect the solution by opening the automated valve under the gas-washing bottle to drain the solution into a collection vial and cap via gravity. When the sample is done collecting NO_x, collect the solution in a 60 ml amber glass bottle and manually remove the bottle. The program waits ~2 min for the solution to fully drain and then automatically moves on to the next step.
   6. Once the sample is done draining, automatically close the valve and clean the gas-washing bottle by aspirating ultrapure water into the syringe pump and dispensing it into the gas-washing bottle via a spray nozzle to clean the sides of the gas-washing bottle. Extract this wastewater from the gas-washing bottle by aspirating it into the syringe pump and discard it into the waste reservoir. Store the frit with 25 ml of nanopure water.
   7. Repeat steps 3.2.2 to 3.2.6 to dispense the next solution sample.
3. Take field blanks during the collection for each solution bottle (labeled with the letters A-Z before the beginning of collection) that is used by sending 25 ml of solution through the system without turning on the vacuum pump to collect air. Collect the solution immediately after it is put into the system.
4. Record the volumetric flow rate every 5 min using a flow meter during each collection, along with the air temperature (T) and pressure (P) on the flow meter, to derive the standard flow rate. Flow rates of 3-5 L/min are achieved with a diaphragm pump (30 L/min capacity) and a critical orifice to reduce the flow rate.
   1. Set the flow rate at the beginning of sampling to measure the flow approximately every 1 sec. After 5-10 sec, change the flow measurement frequency to 5 min.
   2. Collect the flow-rate data every 5 min for the duration of the sampling period.
      NOTE: If the flow rate drops significantly, the sample should be collected for longer than initially expected. Small changes (<25%) in the initial flow rate are to be expected. The hydrophobic filter should be checked to see if it is coated to the point that it is clogged.
   3. Before the sampling is stopped, change back to 1 sec flow-rate measurements and collect flow data for 5-10 sec before turning off the sample.
      NOTE: The solutions may be stored for up to (maximum) seven days before step 4 must be performed.

4. Sample Reduction

NOTE: Reduce the samples to remove the KMnO₄ within 7 days of collection. The original method⁴ suggests that this must be done within 24 hr of sample collection. Below are results that suggest that the samples can be stored for up to seven days prior to reduction.

1. The permanganate in solution is a strong oxidizer. Reduce the sample to stop the oxidation reaction of permanganate with ambient NO_x or with other N species that could potentially lead to interference if they are oxidized to nitrate⁴.
2. Label two 400 ml beakers, one for blank solutions and one for samples. Acquire two stir rods, one for each beaker. Also, acquire a 500 to 5,000 µl pipette and pipette tips.
3. Weigh each sample bottle while it contains the solution and record the mass of the full glass bottle. After the solution has been poured into the beaker, weigh the empty glass bottle.
4. Pour the solution from one sample into the sample beaker and one blank solution into the blank beaker.
5. For the sample beaker, slowly introduce 10 ml of hydrogen peroxide (H₂O₂) in 5 ml batches to the sample, stirring vigorously while adding each 5 ml of H₂O₂. This volume is for sample solutions of 35 ml. For each 25 ml of sampling solution (either blank or sample), add 5 ml of H₂O₂. Adding the full 5 ml for less than 25 ml of sample solution is recommended to ensure full conversion, and adding more H₂O₂ will only result in the dilution of the solution.
   1. Introduce the first 5 ml of H₂O₂ above the beaker, so that the tip does not touch the beaker, the stir rod, or the solution.
   2. Add the second batch to the side of the beaker and around the side, as more is added, in order to wipe down the sides of the beaker, ensuring that all of the sample solution is reduced. If more than 5 ml of H₂O₂ is added, perform this step on the last 5 ml introduction of H₂O₂ and follow step 4.5.1 for the intermediary introductions of H₂O₂.
   3. Change the pipette tip after each sample is changed to avoid any cross-contamination.
6. For the blank solution beaker, add only 5 ml of H₂O₂. Add roughly half of the above the solution and add the other half around the sides of the beaker. Change the tip after each blank.
7. Check to ensure that the solution above the precipitate is clear or pale yellow. If purple or blue colors remain, add more H₂O₂ to verify that it is fully reduced.
8. Pour the entire contents of the beaker, both the liquid and the brown precipitate that forms into 50-ml centrifuge tubes that have been labeled according to the sample or blank number or letter.
9. Once all solutions are reduced (the H₂O₂ is added), load the centrifuge in batches of 20, ensuring that the centrifuge is balanced. A benchtop centrifuge can typically accommodate 20 centrifuge tubes at a time.
   1. Operate the centrifuge at 3,220 x g for 15 min with each batch of centrifuge tubes.
10. While the centrifuge is running, weigh the empty glass bottles and record their masses. Additionally, label 60 ml amber plastic bottles (previously cleaned by leaching in ultrapure water) and weigh the empty plastic bottles. Record their masses as well.
11. Once the centrifugation has finished, pour the supernatant liquid into the amber plastic bottle (with the solid remaining in the tube) and properly dispose of the centrifuge tube.
12. Weigh the sample bottles, now full, and record their masses.

5. Sample Neutralization

NOTE: Perform the neutralization of samples and blanks (reproduced here from Fibiger et al.⁴ with updates). Note that this step is required for the colorimetric quantification of the nitrate concentration in the solution; this may not be necessary with other concentration techniques.

1. Perform neutralization manually or with an automatic titrator.
2. For manual neutralization, use 12.1 M hydrochloric acid (HCl) and introduce it into the amber plastic solution bottle with a pipette. Take extreme care (eyewear, lab coat, fume hood, etc.) when handling HCl, particularly at a 12.1 M concentration.
   1. Calculate the volume of 12.1 M HCl that is to be added to the sample solution to neutralize it using the following equation:
      
      where V_HCl is the volume of HCl added, mass_{full glass bottle} is the mass of the glass bottle the solution was collected in with the solution, and mass_{empty glass bottle} is the mass of the glass bottle the solution was collected in without the solution. Assume that the densities of the solutions to be 1.00 g/cm³, as they are dilute solutions.
      NOTE: Add 85% of this volume first in 0.20 ml increments. Depending on the air that is being sampled, other species that are collected can lower the original solution pH and change how it responds to the addition of acid.
   2. Add the 85% volume of HCl to the bottle using a pipette and a disposable tip in 0.2 ml increments. Cap and shake the bottle between each 0.2 ml addition to ensure the acid has mixed with the solution. Check the pH using litmus paper by removing 20 µl of solution and pipetting it onto the litmus paper.
   3. If the pH is between 4 and 10, label the sample as neutralized and record the pH. Repeat steps 4.2.1 and 4.2.2 for all other samples being processed. While the colorimetric concentration analysis can proceed with samples that have a pH as low as 4 or as high as 10, get as close to 7 as possible in order to yield the best results.
   4. If the pH is still above 10 after the 85% volume addition, add HCl in smaller increments than 0.2 ml of HCl (0.10 or 0.05 ml), shake the bottle to homogenize, and check the pH with litmus paper and 20 µl of solution after each addition of HCl.
   5. Once the pH is within the desired range, label the sample as neutralized in the same way as before and set it aside.
   6. If the pH is below 4, use 10 M NaOH to bring the pH up to the correct range. Add increasingly small amounts of NaOH and check the pH after each addition using litmus paper and the same method as before.
   7. Record the amount of HCl (V_HCl) and NaOH (if needed) added to each sample bottle, along with the final pH.
3. For the automatic titrator method, use an automatic titrator.
   1. Dilute 12.1 M HCl to 4 M with ultrapure water and introduce it to the titrator (0-25 ml of hydrochloric acid are possible) according to the instructions of the instrument. 4 M allows the titrator to be precise enough without adding large volumes to the samples.
   2. Set the automatic titrator to titrate to a pH of 7.
   3. Record the final pH and the volume of HCl added. Label the sample as neutralized and record the final pH.
   4. Use the same beaker for each titration. In between each sample, wash the beaker, pH probe, and stirrer with ultrapure water at least 3 times and dry.

6. Sample Measurement

1. Measure the concentration of each of the samples (C_B or C_S) using a spectrophotometric nutrient analyzer that utilizes colorimetric chemistry to make concentration measurements.
2. Prepare the samples and put them into the instrument according to the manufacturer's instructions.
3. Generate a standard calibration curve from 0-15 µM nitrate (7 calibration points) from a 30 µM stock KNO₃ solution.
4. Prepare and run quality controls of 8 and 10 µM nitrate along with the samples. Calculate the pooled standard deviation of QCs across multiple runs to estimate the concentration uncertainty. Typical pooled standard deviations of ±0.4 µM are observed (across 7 runs, N = 27 data points).
   1. Convert the NO_x concentration determined by the colorimetric concentration analyzer from μM to ppbv using the following mixing ratio equation:
      
      where MR_NOx is the mixing ratio of NO_x (the value reported in ppbv), n_NOx is the number of nmol of NO_x collected, R is the ideal gas constant in , T is the temperature (in Kelvin), P is atmospheric pressure (in atm), and V is the volume of air (in L) collected. The total volume of gas sampled is determined by numerical integration of the flow rate time series (equivalent to the area under the flow rate curve as a function of time).
   2. Calculate the number of moles of NO_x using the following set of equations:
      
      for the sample,
      
      for the blank, and
      
      where C_S is the concentration of the sample measured by the colorimetric concentration analyzer, in µmol; V_S is the volume of the sample, in ml; C_B is the concentration of the blank, in µmol; V_B is the volume of the blank, in ml; V_aliquot is the volume of what was neutralized, in ml (typically the entire volume of the solution); and V_HCl is the volume of HCl added to neutralize the aliquot, in ml.

7. Nitrogen Isotope Ratio Preparation

NOTE: Quantify the nitrogen isotopic composition based upon the denitrifier method. Details of this method are published elsewhere in their entirety, and users should consult these publications for full method instructions^12,13. The method utilizes denitrifying bacteria to convert liquid NO₃^– samples into gaseous nitrous oxide (N₂O) for isotopic determination. Users who do not have the denitrifier method readily set up may have samples analyzed for isotopic composition by external facilities. Users should consult these facilities to ensure that the appropriate data corrections are consistent with those in step 8.

1. Based upon the concentration determined for each sample and blank, inject the appropriate volumes into pre-prepared, capped vials with bacteria^12,13. Target a specific size for injection by dividing the target size, such as 20 nmol N, by the concentration (µmol/L) of the sample or blank to determine the number of ml to inject into each vial via a syringe.
   1. Inject nitrate reference materials (e.g., IAEA-NO-3 and USGS34) at least in triplicate with each set of samples to be run for isotopes. These reference materials are used to correct the final data to standardized, internationally accepted values¹⁴.
   2. Fill two beakers with ultrapure water for rinsing the syringes between the injections of samples and reference materials. Obtain an empty bottle for waste solution.
   3. Dip the tip of the syringe being used for injection in the first beaker of water and dry it off. Rinse the full volume of the syringe with ultrapure water and discard the water as waste. Repeat three times.
   4. Following a similar procedure to step 3.2.3, fill the syringe with a small amount of sample to pre-rinse the syringe. Discard it as waste. Refill the syringe with sample and gently knock to remove any air bubbles so that an accurate volume is measured.
   5. Inject the sample into the sample vial.
      1. If there is more than 3 ml being injected into the sample, use a second "vent" needle to relieve pressure in the vial. Push the syringe with the sample into the rubber septa and begin injecting the sample. After 0.5 ml of the sample has been injected, insert the second "vent" needle. Leave the "vent" needle in until 1 ml is left to be injected, and then remove the "vent" needle. Continue injecting the last of the sample.
   6. Store the vials overnight in a warm (~24 °C) area. The following morning, inject 0.1 to 0.2 ml of 10 M NaOH into each sample to lyse the bacteria.

8. Isotope Ratio Determination

NOTE: Once the bacteria are lysed, the samples are ready to be run on the isotope ratio mass spectrometer (IRMS).

1. Inject each sample with three to four drops of antifoam before setting them up to run on the mass spectrometer.
2. Determine the isotopic composition by ion ratio mass spectrometry. Use a mass spectrometer interfaced with a modified system for the automated extraction, purification (removal of CO₂ and H₂O), and isotopic analysis of N₂O at m/z 44, 45, and 46^{12, 13}.
3. Run a blank with only media solution and no sample in the vial at the beginning of each run.
4. Calibrate the raw isotope ratios from the mass spectrometer using reference materials (e.g., IAEA-NO-3 and USGS 34) treated in the same manner as the samples, based on the correction scheme in Kaiser et al. (2007)¹⁵. This puts them in a form that is usable for data comparisons with other laboratories in the N isotope biogeochemistry community.
5. Due to concerns about the linearity of the mass spectrometer values, if the area of the injected sample is outside of ±10% of the target area, adjust the concentration using that percentage and re-inject the sample, following the above procedure.
   1. To finalize the sample data for δ¹⁵N of NO_x (the delta notation is defined using the following equation for δ¹⁵N: ("delta 15-N"): δ¹⁵N = [(¹⁵N/¹⁴N_sample/¹⁵N/¹⁴N_standard) – 1] x 1,000‰ and the standard used for the nitrogen samples is atmospheric N₂ gas), correct the δ¹⁵N for the contribution of the nitrate blank that is found in the KMnO₄ solution:
      
      where δ¹⁵N_{total, measured} is the value determined for the sample from the mass spectrometer run, δ¹⁵N_{blank, measured} is the value determined for the blank, and the Sample and Blank Concentrations are the values determined from the colorimetric analyses. This equation removes the effect of the blank from the isotopic ratio, so that the isotopic ratio is now representative of the NO_x collected in situ.
      NOTE: The isotopic composition and concentration of the blank solution nitrate is measured with every batch of solution used. This blank is different from any potential blank that might be encountered with the denitrifier method alone (which is also quantified with every run). Any blank associated with the denitrifier method is true for all samples and reference materials; however, the permanganate solution blank is only applicable to samples and therefore must be quantified and corrected for (by mass balance) separately.