Lab Manual for Local Ambient NO2 Monitoring in Kumasi Using Diffusion Tubes

1.62 W 1.59 W 6.66 N 6.68 N 6.7 N 6.72 N LAB MANUAL for local ambient NO2 monitoring in Kumasi using Diffusion Tubes For students and lab staff Issue 1 April 2025 Air Quality lab Kwame Nkrumah University of Science and Technology A collaboration of: Table of Contents 0. Introduction 1 1. Local Production of Diffusion Tubes 2 1.1. Tube 2 1.2. Stainless steel grids 3 1.3. Caps 3 1.4. Holder 4 1.5. Cost 5 2. Preparation of the tubes 5 2.1. Cleaning the components 6 2.2. Preparation of TEA solution and coating of the grids 6 2.2.1. Pipetting Method (20% TEA in deionised water) 6 2.2.2. Dipping Method (50% TEA in Acetone) 8 3. Exposure of the tubes 10 3.1. Selection of measuring sites 10 3.2. Best practices on how to hang the tubes 11 3.3. Duration of monitoring programme 13 4. Analysis of tubes 13 4.1. Preparation of reagent solution 14 4.2. Preparation of standard stock solution 15 4.3. Preparation of calibration standards 16 4.4. How to use the spectrophotometer 17 4.5. Calibration curve 19 4.6. Analysis Schedule 21 4.7. Disposal recommendations for reagent 23 5. The future of the AQ lab at the KNUST 24 6. References 25 Appendix A: Inventory of the Air Quality lab 26 Appendix B: List of Sites 29 Appendix C: labelling and ID Numbers 30 Appendix D: lab Setup 31 Appendix E: Working with the Excel files 33 0. Introduction This laboratory manual documents the specific aspects of measuring NO2 in the Air Quality (AQ) lab at the KNUST in Kumasi, Ghana. It does not intend to present scientifically proven methods but rather a practical documentation of how the lab works. The lab was built by adapting the description in the practical guide of Targa (2008). The manual touches upon the local tube components and preparation, exposure, and the local analysis in Ghana. For more details and comparison with the standard procedure in the EU the practical guide should be consulted (Targa, 2008). There are different methods of monitoring NOx concentrations in ambient air. The Air Quality lab in KNUST uses passive samplers, locally built Palmes-type diffusion tubes (PDTs), for Local Air Quality Management purposes because it is a low-cost, low-tech technique that has been developed by Palmes et al. (1976) and since then has widely been applied in the global north. The grey boxes describe procedures that are advised to be followed carefully in order to ensure reproducible results The creation of the AQ lab at the KNUST was a project that was initiated by Dr. Bas Mijling from the Koninklijk Nederlands Meteorologisch Instituut (KNMI) together with Dr. Benjamin Afotey, Head of Department of Chemical Engineering. The project's goal was to create the first independent, locally run AQ laboratory in Kumasi that is capable of preparing and analysing diffusion tubes without reliance on foreign labs. The lab was built in collaboration with three teaching assistants from Chemical Engineering and three master students from the TU Delft. Feel free to make changes and adjustments to this file in the future. You can access it via the drive of the KNUST Air quality lab: Email: [email protected] Please request the password from AQ lab Staff or Dr. Benjamin Afotey. In case of questions, reach out to one of the contacts below. Senior Prof. Chem. Eng. Dr. Benjamin Afotey [email protected] Project Initiator KNMI Dr. Bas Mijling [email protected] Current AQ lab Staff Emmanuel Yourkuu [email protected] Philip Darko [email protected] Phiona Amakah [email protected] Page 1 1. Local Production of Diffusion Tubes For producing the Ghana-made diffusion tubes, a UV-resistant tube, a stainless steel grid, and two end caps are needed. It is important to notice that all parts can be reused if cleaned properly after use (see 2.1. Cleaning the components). Component Dimensions Specifications Tube Length=17.6 cm; øin=1.4 cm Green PP-R, UV-opaque Grids 100 microns; ø=1.7 cm Stainless steel Top end cap øin=2.1 cm PVC, opaque blue Bottom end cap øin=2.1 cm PVC, white, removable 1.1. Tube For the tube, it is important that it is resistant to UV light and heat to slow down degradation. The tube should also be opaque to UV light. If UV light is not blocked, the NO2 concentration might be overestimated. Do not use any tubes that are cracked, damaged, or have uneven ends, which may prevent the end caps from sealing properly. All tubes should have the same dimensions in terms of length and internal/external diameter. Page 2 The tubes are made of Polypropylene Random Copolymer (PP-R) piping with a 2 cm outer diameter (½ in) and 1.4 cm inner diameter. They are typically green and commercially used for hot and cold water and therefore can be found at many plumbing stores in Kumasi e.g, the plumbing store at Tech Junction (Cletus Hardware, 0244658527) or the plumbing store next to the China mall (Hamus Enterprise, 0591932278). One pipe (4 m) costs around 40 cedi and does suffice for 20 tubes with a length of 17.6 cm (with small leftovers). Manufacturing of tube The tubes have to be cut straight. You can use a handsaw, but the best tool to do this was found to be a pipe cutter. To mark your tubes consistently, a small jig can be built (see right image). The ends need to be smooth-ended and straightened using sanding paper and/or a utility knife. The outer diameter needs to be enlarged by 1 mm to make sure that the end cap fits tightly. Therefore, use an electrical tape (pvc insulating tape ¾ in) and wrap it around the tube’s end 4 to 5 times. Ensure a tight press fit by closing one end with a cap and blowing in it. 1.2. Stainless steel grids Each tube requires two stainless steel grids with a grid size of approximately 100 microns (4x4 mm2). A suitable mesh was not found in Ghana and had to be imported from the Netherlands. In case the imported stock runs low, local solutions have to be considered as replacements. Manufacturing of grids The diameter of each circular grid should be around 1.7 cm. This is slightly larger than the inside diameter of the tube so that the grids are fixed firmly in place when the tube is assembled and cannot fall out during exposure. However, they are also slightly smaller than the outer diameter so that they do not need to be pressed into the endcap and are hard to get back out. The cutting of the grid sheet can be done with a normal pair of scissors. Page 3 1.3. Caps End caps are used on both ends. The top one that holds the grids should be opaque plastic. Sunlight can degrade the nitrite complex formed when NO2 combines with the TEA: dark opaque caps minimize this. End caps may be reused but must be discarded when they have cracks or start to degrade from the sunlight. The caps are made of PVC and are commercially used as end caps for half-inch PVC piping (2.6 cm outer diameter and 2.1 cm inner diameter). They are typically white and can be found at the same stores as the PP pipes. When initially bought from the store the caps need to be cleaned with soap and a toothbrush thoroughly as they are most often dirty. The end cap with grids was spray-painted blue to distinguish it from the removable closing cap. One cap costs 2 cedi. 1.4. Holder To facilitate easy tube replacement, a permanent holder is required. The open end of each tube must remain exposed to free circulation of air. Therefore, tubes should not be directly fixed to walls or poles. Use a spacer block of at least 5 cm between the surface and the tube. We used an L-shaped wooden holder sourced from the carpenter at KNUST or the wood village at Anloga Junction. The wood for each holder costs around 12.5 cedi. Manufacturing of holder The holder is made from two pieces of wood (around 20 cm long) nailed together in an L shape. The top part has two 2 cm diameter holes that were drilled in the workshop of mechanical engineering to accommodate two tubes. The lower part, mounted to the poles, includes two holes for zip ties to secure it in place. Zip ties can be found e.g. at the China Mall. Page 4 1.5. Cost The total local material cost of one locally made diffusion tube is calculated as 6.5 cedi (2 PVC caps, PP-R tube, and electrical tape). One holder costs approximately 13 cedi (wood, nails). Usually, per site, two diffusion tubes are mounted in duplos, making one site cost not more than 26 cedi. It is important to note that all tube components can be reused as long as they are structurally intact and properly cleaned after each campaign. This makes the cost to produce a tube a long-term investment. 2. Preparation of the tubes To prepare the tubes before exposure, three steps are required: 1. Cleaning the tube components 2. Preparation of TEA solution 3. Coating the grids Page 5 2.1. Cleaning the components Before the tubes are assembled and prepared, the components need to be properly cleaned to reduce cross-contamination and ensure reusability. Cleaning of components Chemicals: - Deionised water - Soap - HCI (37%) (optional) Equipment: - Empty buckets/container - Paper towel - Drying rack (optional) Procedure: 1. Soak the components (tubes, caps, grids) in a solution of (warm) water and soap for 30 minutes. For a better handling of the components, this can be done in separate containers. 2. Rinse thoroughly with water. 3. Do a final rinse using deionised water. 4. Let the components air dry. Tip: To test the effectiveness of the cleaning, a few grids can be submerged in the colour reagent solution to check for any observable colour change. Glassware and equipment used in the preparation of solutions and analysis have to be cleaned using the same procedure. For deep cleaning, it is also possible to prepare a 10% HCl solution diluted in deionised water. This solution is used as the cleaning agent and is able to get rid of all traces of nitrate and other contaminants. After using the HCI solution, the components need to be rinsed with deionised water as a last step. 2.2. Preparation of TEA solution and coating of the grids TEA is an organic compound with the chemical formula N(CH2CH2OH)3. It is a colourless and viscous liquid. It is both a tertiary amine and a triol (has 3 alcohol groups). The TEA solution is the absorbent solution present in the metal grids responsible for absorbing NO2. There are two different ways of coating the grids. The first method is to pipette 50 µL of the solution on the grid. The second method is dipping the grid in a solution. The following describes both methods. However, uneven coverage of the grid was experienced with the pipetting method. Therefore the grids for the following campaigns were prepared with the dipping method. Page 6 2.2.1. Pipetting Method (20% TEA in deionised water) The amounts of TEA solution needed are the following: TEA solution needed per tube: 50 µL For a batch of 45 tubes: 2.3 mL Accounting for spare solution: 2.5 mL To prepare 2.5 mL of 20% TEA solution by weight (0.567 g TEA and 2 g deionised water): These masses are obtained by multiplying their densities by their volumes (20% TEA and 80% water). Preparing the 20% TEA and water solution Chemicals: - TEA: triethanolamine - Deionised water Equipment: - 10 mL beaker - Pipette - Analytical scale - (Magnetic) stirrer Procedure: 1. Place a clean 10 mL beaker on a scale. 2. Using a pipette, transfer 0.567 g of TEA into the beaker. 3. Add to the same beaker 2 g of deionised water (by zeroing the scale). 4. Mix the components using a magnetic stirrer. Note 1: This solution should be freshly prepared on the day that it is to be used. Note 2: Quantities can be adjusted to make smaller or larger volumes as required. Tip 1: It is easier to handle if allowed to reach room temperature before use. Tip 2: It can be easier to prepare slightly larger volumes to ensure proper mixing. Impregnating the grids with TEA solution is done following the pipetting method. In this method, 50 µL of the solution is pipetted on the grids with a micropipette once they are placed inside the cap. It is important that the micropipette is accurate and the operator is confident in using it. Try out how it works beforehand with water. If operated correctly the first pressure point sucks the dialed in volume. Pressing further will blow out all the remaining fluid in the tip. To ensure accurate results, first get acquainted with this tool! Page 7 4.1. Preparation of reagent solution As a color reagent a Saltzman-/Griess reagent is used. Sulphanilic Acid, the reaction product of sulphanilamide and an acid (in our case HCl), reacts with the NO₂to form a diazonium salt. The diazonium salt reacts with the NEDD to form an azo-dye. CAUTION! Preparing the reagent involves handling strong acids and hazardous chemicals. Mixing should only be done by trained professionals with appropriate Safety Equipment (lab coat, glasses, gloves, and breathing protection) and safety facilities (fume hood necessary). Inform yourself about the safety information in the MSDS beforehand! Prepare the Reagent under a fume hood in the Process Development lab; avoid breathing in fumes. Avoid mixing on days with frequent lights out. The reagent solution is composed of two separate solutions, Part A and Part B, mixed in a 1:1 ratio. Part A contains sulphanilamide and hydrochloric acid in deionised water. Part B contains NEDD in deionised water. Preparing the Griess Reagent Chemicals: - Sulphanilamide - Hydrochloric acid (37%) - NEDD - Deionised water Equipment: - Glass pipette with balloon - Analytical scale - Magnetic stirrer - Spoon - Tunnel - 2x 100 mL Glass beaker - 2x 250 mL Glass beaker - 2x 500 mL Brown glass bottle - 2x 250 mL Volumetric flask - PPE Procedure (for 250 mL Part A and 250 mL Part B): 1. For the preparation of the reagent solution, it is important to have clean equipment and glassware. To ensure no contamination during the mixing process, soak your glassware in a 10 % HCL solution for 30 minutes, rinse it with deionized water, and let it dry. 2. In a 250 mL beaker, dissolve 5 ±0.01 g of sulphanilamide in 150 mL of deionised water, mixing it well. 3. Add 12.5 mL of hydrochloric acid carefully with the glass pipette and mix well again. Transfer this solution to a volumetric flask using the tunnel. Make up to 250 mL with deionised water. This makes up Part A. Store in a clearly labelled, sealed dark glass bottle. Page 14 4. Dissolve 35 ± 1 mg of NEDD in about 150 mL of deionised water. Make up to 250 mL with deionised water. This makes up Part B. Store in a second clearly labelled, sealed dark glass bottle. Note 1: The two above preparations (Part A and Part B) should now be kept separately. Immediately before use, the required quantity of mixed reagent should be prepared by mixing the above solutions in a 1:1 ratio. The finished Saltzman-/Griess reagent appears clear or slightly pink in color. Once mixed, the colour reagents should be used the same day, not stored. Note 2: The above procedure makes 250 mL of each solution, but if a different volume is required, the quantities of reagents indicated above can be adjusted accordingly. If stored separately in a cool and dark place, the two solutions have a shelf life of approximately 6 months. In contrast to the Practical Guide (Targa et al., 2008), hydrochloric acid is used instead of phosphoric acid (85%). Following the original recipe has resulted in a significantly pink colored reagent. After some experiments, NOx traces were found in the phosphoric acid that led to the premature production of diazonium salt. So far, with replacing phosphoric acid with HCl, the reagent seems stable and colour-sensitive to nitrite. Further side effects might arise and have to be examined. 4.2. Preparation of standard stock solution A set of standard solutions with accurately known concentrations is necessary in order to calculate accurately the mass of nitrite extracted from the grids. For that, first, a standard stock needs to be prepared (1000 µg/mL). Preparing standard stock solution Chemicals: - Sodium Nitrite (analytical quality, min 99% purity) - Deionised water Equipment: - Analytical scale - Magnetic stirrer - Glass petri dish - Spoon - Oven - Desiccator - 500 mL Volumetric flask - 1x 250 mL Glass beaker Procedure: 1. The sodium nitrite must be dried before the preparation of the solution. Place 1 – 1.2 g of sodium nitrite in a petri dish and dry in an oven at 102 ± 2 °C for 1 – 2 hours. Using gloves, remove the petri dish and contents and place it in a Page 15 desiccator with the lid on to cool down. Allow the sodium nitrite to cool for between 30 minutes to 1 hour before use. 2. The mass required to prepare a 500 mL solution of strength 1000 µg nitrite /mL (1 mg/mL) is: 0.5 x 68.995/46.006 = 0.750 g (46.006 g being the molar mass of NO2and 68.995 g being the molar mass of NaNO2.) 3. Weigh out 0.750 ± 0.001 g of the dried sodium nitrite (NaNO2) into a glass beaker or weighing bottle. 4. It is normal to discard any unused dried sodium nitrite rather than return it to the original bottle to avoid the risk of contamination. 5. Put 100 – 300 mL deionised water into the 500 mL volumetric flask; transfer all the sodium nitrite by washing and make the solution up to the 500 mL mark using deionised water. Shake well. The concentration of nitrite ion (NO2-) in the resulting stock standard solution will be 1.000 g NO2per litre. Note: The quantities above can be scaled up or down if a larger or smaller volume is required. 4.3. Preparation of calibration standards To calibrate the spectrophotometer before the analysis of diffusion tubes, a range of nitrite calibration standards is necessary. These are prepared by dilution of the stock standard solution. This is done by preparing a range of intermediate solutions of different strengths, then pipetting an identical small volume (50 µL) of each one into a set of tubes. These tubes are then treated in the same way as real exposed samples: the same volume of reagent mix will be added to them, and they are analysed in the same way as exposed samples. The range of concentrations required for calibration will depend on the likely amounts of nitrite captured by the tubes that are being analysed. This, in turn, will depend on the anticipated ambient NO2 concentration at the monitoring location and the length of the exposure periods. The concentrations are, therefore, examples and can be varied as required. Preparing calibration standards Chemicals: - Standard stock (1000 ug/L) - Deionized water Equipment: - Micropipette (1000-5000 µL) - 5x 10 mL Volumetric Flask Procedure: Using a calibrated micropipette (50 – 2000 µL), measure small volumes of the stock standard solution into a set of 10 mL volumetric flasks. Dilute to 10mL using deionised Page 16 water. The example below shows the dilutions for a range of solutions from 15 – 120 µg/mL: A – 120 µg/mL prepared as follows: 1.2 mL of stock solution measured out using a calibrated micropipette, and made up to 10 mL in a volumetric flask. B – 90 µg/mL prepared as follows: 0.9 mL of stock solution measured out using a calibrated micropipette, and made up to 10 mL in a volumetric flask. C – 60 µg/mL prepared as follows: 0.6 mL of stock solution measured out using a calibrated micropipette, and made up to 10 mL in a volumetric flask. D – 30 µg/mL prepared as follows: 0.3 mL of stock solution measured out using a calibrated micropipette, and made up to 10 mL in a volumetric flask. E – 15 µg/mL prepared as follows: 0.15 mL of stock solution measured out using a calibrated micropipette, and made up to 10 mL in a volumetric flask. F - Blank. 0 mL of stock solution. Having prepared the series of diluted solutions, pipette 50 µL of each one into clean mounted tubes clearly labelled (i.e., Standard A, Standard B,…etc.). These will contain the following total masses of nitrite (remember that these are examples and can be varied as required): A – 6.0 µg of NO2 B – 4.5 µg of NO2 C – 3.0 µg of NO2 D – 1.5 µg of NO2 E – 0.75 µg of NO2 F – 0.0 µg of NO2 For calibration, these tubes should not contain grids or TEA (to avoid introducing uncertainties related to extraction). Before the calibration standards can be analysed to create the calibration curve, the Spectrophotometer has to be turned on and accuracy and repeatability (see section 4.5) 4.4. How to use the spectrophotometer The Air Quality lab at the KNUST is equipped with a HACH DR 5000 with a sipper module. It is important to note that this machine only works with this module and does not function with single cuvette assays. Run through the following steps to ensure stable results: Using the Spectrophotometer Procedure: 1. Turn on the DR two hours before you want to start the analysis to warm up the lamp. This reduces the drift significantly. Page 17 2. In the first hour, you can clean the sipper cuvette with a microfiber cloth. 3. Open the lid and set up the sipper module by putting the white flexible tubing through the tube retainers and around the peristaltic pump (see instruction pictures below or check the manual of the spectrophotometer). Close the tubing clamp. Note: the white flexible tube has to be changed over time, as it gets porous (check the device manual for that). 4. Place the waste tubing into a designated waste collection container for proper disposal of the Griess reagent. Do not dispose of the reagent down the sink! 5. Have the sipper running multiple cycles with deionised water to flush the system. Tip: You can manually increase the sipping time for this purpose. 6. After an hour, turn on the drift check (Instrument Setup > Lampcontrol > Drift Check > 542 nm) to confirm low drift (Range 0.0010 - 0.0080 AU). 7. After the drift check is finished, ensure that the sipper module is constantly sipping 4.5-5 mL in 3s. For that you can add 5 mL water in a beaker and adjust the pump adjusting screw until it sucks the entire 5 mL. Confirm multiple times. Tip: If this is not the case, you most likely have to tighten the adjustment screw of the pump module a bit. 8. Now zero the device on deionised water and measure the absorbance of deionised water a few times. The results should not differ more than shown in the drift check done before. Now, you can start to make the calibration curve and the analysis. Tip 1: The sipper module requires a decent amount of liquid to clean out the previous sample. To maximize the liquid to clean the tube, the inlet tube should be as short and thin as possible. Tip 2: Sometimes, if the cuvette is inserted the entire way, the machine gives the error of “Too much ambient light”. You can bypass this error by repositioning the cuvette 1-2 mm upwards. CAUTION! The sipped solution might contain color reagent or hazardous chemicals. The waste tubing outlet should be connected to the adequate waste container. See section 4.6 for disposal recommendations. Page 18 4.5. Calibration curve A calibration curve has to be done before every analysis schedule and after mixing a new reagent. It should be based on at least six points if calibration is required beyond 1 AU. Points always include a zero standard. This calibration is carried out using an appropriate range of calibration standards, prepared as in section 4.2 above. If diffusion tubes are used, they should not contain TEA. You can also use 10 mL test tubes. Making the calibration curve Chemicals: - Standard solutions (A, B, C, D, F) - Reagent Part A - Reagent Part B - Deionised water Equipment: - Micropipette (50 µL) and tips - Test tubes (3x standard solution) - Volumetric flask - Tube rack Procedure: 1. Clean and prepare 3 test tubes for each calibration standard you use by rinsing them with deionised water and letting them dry. (In total 18 tubes) 2. Prepare reagent 1:1 solutions Part A and B into a volumetric flask (the amount should be enough for the calibration and analysis). Page 19 3. Add 50 µL of each calibration standard in the tubes with a micropipette. Change disposable tips after each set of standards. 4. Add 3 mL deionized water into each of the test tubes. 5. Add 3 mL of reagent into each of the test tubes. 6. Close with cap and shake shortly. 7. Allow to stand for 10 minutes for color formation. 8. Make sure the Spectrophotometer (542 nm) is set up according to the procedure above. 9. Start to zero the spectrophotometer with a zero blank, then measure with increasing concentration of standards. 10. Measure the absorbance per set of calibration standards (3x) and record in the Excel book. 11. A trendline (ideally R2=0,999) is automatically created in the Excel book. Tip 1: It is good practice to label the test tubes according to each calibration standard solution prepared (e.g. A, B, C, D, E, F; as exposed in section 4.2.). Tip 2: For step 3, it is recommended to proceed by each set of standards, starting from the lowest concentration and moving to higher concentrations each time. 4.6. Analysis Schedule It is important to take your time for the analysis to avoid mistakes (approx. one day). Further, doing the analysis with two people makes the analysis and documentation easier. Make sure the UPS is charged, or choose a day when the power seems stable. It is important that the spectrophotometer does not turn off during the analysis. Analyse the tubes in manageable sizes and include blanks every 10th sample. This ensures that all tubes are handled the same way and easy correction for instrument drift. The analysis procedure varies slightly depending on the method used to coat the grids. If the tubes are prepared using the pipetting method, the TEA solution does not evaporate Page 20 completely from the cap after exposure. Therefore, the grids have to be mixed with the color reagent while still inside the tube. If the tubes are prepared using the dipping method, the grids can be extracted and mixed with the color reagent solution in the test tubes. Before starting the analysis, make sure that the Griess reagent solution is ready by mixing Part A and Part B in a ratio of 1:1. Analysis of tubes prepared using the dipping method Chemicals: - Griess reagent solution (Part A and Part B mixed) - Deionised water Equipment: - Test tubes (1 per diffusion tube) - Tube racks - Clean tweezers - Micropipette (3 ml) and tips Procedure: 1. Sort the collected tubes with counting numbers upwards. 2. label the same amount of test tubes with the same tube numbers. Tip: you can also put a line of tape on the table and transfer the numbers there. Just keep track which sample is which! 3. Transfer the grid from the (blue) cap to their corresponding test tubes using tweezers (avoid touching the grids with your hand/ or let them fall on a dirty surface). Note: This only works if you have used the dipping method! With the pipetting method, there might be TEA left in the cap that has absorbed some NO2. 4. Note down the tube number and exposure time in the analysis Excel. Make sure to place a blank after 9 tubes. 5. Place the test tubes in the rack according to the noted order in the Excel. 6. Add 3 mL of deionized water and let it stand for 30 min. 7. You can make the calibration curve in the meantime. Ensure the Spectrophotometer is set up correctly. 8. Add 3 mL of reagent and close with (white) caps. 9. Shake each test tube shortly and let the color develop for 10 minutes. 10. Analyse the samples, making sure to follow the order in the Excel Book. (There should be at least 1 mL left in the tube to make a dilution in case the absorption is too high.) 11. Samples should be analyzed within two hours after adding the reagent. Note 1: Samples that exceed the calibration curve have to be diluted. Therefore, extract 1 mL from the sample in a fresh test tube and add 4 mL of deionised water. Add the dilution factor to the table in Excel. Page 21 Note 2: Slight drift over time is usual. General practice is to account for this by measuring blanks as every 10th sample. In the end, the average drift is subtracted from the absorbance value of all samples. Analysis of tubes prepared using the pipetting method Chemicals: - Griess reagent solution (Part A and Part B mixed) - Deionised water Equipment: - Test tubes - Tube racks - Micropipette (3 mL) and tips Procedure: 1. Sort the collected tubes with counting numbers upwards. 2. label the same amount of test tubes with the same tube numbers. 3. Note down the tube number and exposure time in the analysis excel. Make sure to place a blank after 9 tubes. 4. Place the test tubes in the rack according to the noted order in the Excel. 5. Open the end cap and add 3 mL of reagent into the (diffusion) tubes using a pipette. Let the color develop for 10 minutes. 6. Add 2 mL deionised water into the content of the tube. 7. Close with the yellow end cap and sake. 8. Transfer the volume of each tube into their corresponding test tube. 9. Rinse the diffusion tube with 1 mL deionised water and add it to the content of the corresponding test tube. Let it stand for 30 minutes. 10. This process has to be done for every diffusion tube. The Excel sheet already calculates the mass of nitrite and the corresponding ambient air concentration. Still, it is important to understand the calculation behind it (See Appendix E: How to work with the NO2 Excel book). The mass of nitrite is calculated using the linear equation that results from the calibration curve. To transform that value into ambient NO2 concentration, the following formula is derived from Fick’s first law of diffusion: 𝐶= 1 "𝑠.𝑟𝑎𝑡𝑒" ×𝑚𝑡 C = Concentration of NO2 in the ambient air ug/m3 “s.rate “ = Sampling rate in ug/s Page 22 m = Mass of nitrite in tube in ug t = Exposure time in seconds "𝑠.𝑟𝑎𝑡𝑒"=𝐷273 × 𝑎 𝑙 D273 = Diffusion coefficient of NO2 at 273 K = 0.1361 cm2s-1 a = Area of the tube opening l = Diffusion path (length of tube) Since the diffusion coefficient (D) is representable at 273 K, it is common practice to adjust its value to the temperature conditions of the exposure period. Research has found that D is proportional to the temperature to the power of 1.81. Example: The average temperature in Ghana is 28°C at 1008 hPa, which is 301K. D301 can be calculated using the formula below 𝐷301= 𝐷273×301 273 ( ) 1.81=0.1624 𝑐𝑚2𝑠−1 In addition to that, it is important to take into account the dimensions of the tube (1.5 cm inner diameter and 17.6 cm length). For comparison with WHO limits or AQS Objectives, the concentration has to be reported at 293 K and 1013 hPa. Therefore, a temperature and pressure correction is necessary. 𝐶293,1013=𝐶301×301 293 ×1013 1008 If the attached Excel table is used to store the absorbance Data, it automatically calculates the ambient NO2 concentration, adjusting it to the standard of 293K at 1013 hPa (101325 Pa). 4.7. Disposal recommendations for reagent DO NOT dispose of unneutralized or colored reagent in the environment or open drains as it contains NEDD, which is toxic to aquatic life and is carcinogenic (can cause or promote the development of cancer and the substances may damage DNA). Always collect the Griess reagent and its corresponding chemicals in a separate waste container. Once the container is full, the content must be disposed of safely and responsibly. This chapter is a recommendation, there is no guarantee on the information. Please always contact the person responsible for hazardous chemical disposal. In contexts like Ghana, where hazardous waste infrastructure may be limited, it is essential to first neutralize and degrade the reagent’s harmful components before disposal. One possible way to neutralize the Griess reagent is described below. Work safely and Page 23 Appendix C: labelling and ID Numbers Each batch of tubes consists of 44 tubes: 40 for field deployment at 20 sites (installed in pairs) and 4 reserved as lab blanks. Tubes numbered 10, 20, 30, and 40 serve as lab blanks to improve the robustness of the analysis, accounting for slight drift in the spectrophotometer over time (see Section 4.5. Analysis Schedule). labelling System Ghana-made Tubes (P0X:0X) ● First Batch: labelled P01:01, P01:02, … P01:10 (Blank) ○ P = Prototype ○ 01:01 = Batch 01, Tube 01 ○ 01:02 = Batch 01, Tube 02 ● Second Batch: labelled P02:01, P02:02, … P02:10 (Blank) ○ P = Prototype ○ 02:01 = Batch 02, Tube 01 ○ 02:02 = Batch 02, Tube 02 Buero Blauw (Netherlands) Tubes (B0X:0X) ● labelled B01:01, B01:02, … B01:10 (Blank) ○ B = Blauw ○ 01:01 = Batch 01, Tube 01 ○ 01:02 = Batch 01, Tube 02 This labelling system ensures clear tracking of tube batches and their specific roles in the study. Page 30 Appendix D: lab Setup The lab is divided into two main areas: 1. Cleaning station 2. Analysis station Cleaning station The cleaning station is equipped with a sink and drying rack. Additional equipment are tube racks and paper towels. Analysis station Page 31 The analysis station is equipped with a spectrophotometer (HACH DR 500), an UPS and a sink with a waste disposal container connected to the spectrophotometer’s outlet. Additional equipment are tube racks and test tubes. It can be set up as shown in the pictures below. Set up for Calibration Curve Set up for Tube Analysis The tubes can be traced and identified by labelling the bank where the tube racks stand. Page 32 Appendix E: Working with the Excel files To facilitate the analysis of the tubes and the data, evaluation excel files have been created. All data is stored on a Google Drive that can be accessed via the following credentials: Email: [email protected] Please request the password from AQ lab Staff or Dr. Benjamin Afotey. In the folder 02_Data you can find a subfolder in which the data of the sites (Name, GPS Data, Description of the site) is saved, folders for each measuring campaign (Format: start yymmdd_cycleXX), and two Excel templates (results_cycleXX.xlsx and analysis_cycleXX.xlsx). These templates are empty and should always be copied into a new campaign folder. Before you start the analysis download the excel files that are needed and open them in Excel, not google sheets! After going to the field to collect and place new tubes, use the file results_cyclexx.xcel file to record the start exposure time and end exposure time with the corresponding tube. DO NOT CHANGE THE FORMAT OF THE TIME! The batch numbers should be counting up and every 10th tube should be a blank. Open the analysis_cycleXX.xlsx file and copy the start and end date of the exposure in the corresponding fields. Do a quick calculation to validate if the exposure time is correct. Also, fill in the average temperature during the exposure and the average pressure (find this data on openmeteo.org). Proceed to use this file to make the calibration curve on the according field. For that, just enter the absorbance values that are measured - automatically, a linear curve will appear, and the R2 value can be read off. Proceed with your measurements of the samples in the order that they are written down, and note the measured absorbance. Automatically, the Mass of Nitrite and the ambient NO2 Concentration are calculated. You can copy back the values of the ambient NO2 Concentration to the results excel. Page 33