Phosphorus Analyzer Procedure Last updated: 8.7.15 A. Introduction The phosphorus analyzer can be used to measure soluble reactive phosphorus (SRP) aka orthophosphate (PO 3-4 ) only. Additionally, this result when subtracted from the total phosphorus (TP) from the ICP, can calculate total dissolved phosphorus (i.e. soluble plus insoluble). This is a colorimetric technique where the ammonium molydbdate reacts with the sample to form a blue phosphomolybdenum. The wavelength can then be read by a spectrophotometer inside the machine. B. Set Up Lab Equipment/Supplies P Analyzer Vials KH 2 PO4 salt Glassware Deionized water Pipette tips for making 5 N sulfuric acid (H 2 SO 4 ) Ammonium molybdate salt Ascorbic acid salt (vitamin C) Potassium antimonyl tartate salt Dowfax 2A1 surfactant (lubricant) Gloves Lab coat and safety glasses (for handling acid) C. Procedure Overview 1. Prepare samples for analysis. 2. Empty waste bottle. Refill other bottles. 3. Turn on machine and start pumps. 4. Prepare sequence table with standards, method blanks, and samples. 5. Warm up analyzer. 6. Prepare reagent solution. 7. Run sequence. 8. End sequence and export data to personal folder. 9. Run cleaning solutions through analyzer. 10. Shut off pumps and turn off machines. Soil & Water Lab, Phosphorus Analyzer Procedure Page 1
D. Making Stock Solution and Serial Dilutions Corresponds to Step 11 in Section F. Running the Machine. 1. Add 0.439 g of KH 2 PO4 salt into 100 ml of DI water to make 1000 ppm stock solution. Top off with DI water and mix well. 2. Dilute accordingly using a 100 ml flask: Concentration Amount to Add (ml) Using this Concentration 0 ppm 100 DI water 0.005 ppm 1 0.5 0.05 ppm 0.5 10 0.5 ppm 5 10 5 ppm 0.5* 1000 10 ppm 1 1000 * The 0.5 ppm concentration is used as a check standard throughout the run so make sure you have enough volume to fill a few extra vials. 3. Top off the flasks with DI water and mix well. 4. Pour into storage-appropriate containers and label with date. Refrigerate until use E. Making the Reagent Solution Corresponds to Step in Section F. Running the Machine. Note! Usually making 100 ml is enough for up to 50 samples. The fully mixed regent solution only lasts about 4 hours so be sure to watch the timing. 1. Prepare to mix the reagent solution under the hood by retrieving 5 reagents. 2. 5N sulfuric acid is kept in the acid cabinet below the hood. Use gloves when handling. If needed make 5N sulfuric acid. Stock is typically 36N. Pour this in the hood and wear protective glasses and a lab coat. Be careful when you rinse glassware after use. All rinse water should be neutralized before dumping it down the drain. To make 5N, mix 140 ml into ~800 ml of DI water and fill to 1 L. 3. Potassium antimonyl tartate is kept refrigerated. To make add 0.3 g potassium antimonyl tartate to 100 ml DI water. Mix well. 4. Ammounium molybdate is kept refrigerated. To make add 4 g ammounium molybdate to 100 ml of DI water. Mix well. 5. Ascorbic acid is kept refrigerated, but for best results it should be made the same day. It will break down in about 1 week. To make, add 1.8g ascorbic acid salt from the fridge and add 100 ml of DI water. Mix well. Final produce is 0.01 M ascorbic acid. Return the salt to the fridge. 6. Dowfax lubricant is located above the bench top across from the analyzer. 7. When ready, mix the reagent in the following order (order is important!): Soil & Water Lab, Phosphorus Analyzer Procedure Page 2
Reagent ml to add Full Run (approx. 100 samples) Small Run (approx.. 50 samples) 1. 5 N sulfuric acid 100 50 2. Potassium antimonyl tartate 10 5 3. Ammonium molybdate 30 15 4. Ascorbic acid 60 30 5. Dowfax lubricant 0.5 (15 drops) 0.25 (8 drops) Final color reagent volume, ml 200 100 8. Swirl the color reagent together in a beaker. It should be slightly yellow but not blue. Blue indicates contamination. 9. Store all extra liquids separately in the fridge. F. Running the Machine 1. Prepare samples for analysis by pouring >5mL of sample into P analyzer tubes. See Section G for recommendations on filtering and preserving samples before analysis. 2. Empty the waste bottle next to the machine into the acid waste jar found in the sink. Neutralize with baking soda. Add baking soda until the solution stops reacting when you add it. You can also test the ph to make sure that it s around 7 before dumping it down the sink. Sometimes the baking soda will react and the foam will overflow the acid waste jar. To prevent harm to the sink and pipes, be sure to run the tap water while dumping it down the sink and also while adding the baking soda. 3. Refill the DI and DI+dowfax bottles to the top. To make the DI+dowfax solution, mix 2 ml (60 drops) of dowfax into 1 L of water. 4. Remove the clear plastic dust cover from the autosampler. 5. Turn the machine on by flipping the two switches on the right, back of the instrument (top and bottom). One works the auto-sampler and the other works the main machine. The auto-sampler arm should move up and down when you do this. 6. Turn the computer on and enter the user (administrator) and password (syd-8-ney). 7. Click on the P Analyzer Winflow icon to open the program. 8. Turn on the pumps. Pumps > Turn On> All Pumps. 9. Engage the platens (black domino shaped pieces). It might take a firm push to get them into place. Be sure not to spin the silver knobs as they control the flow of the liquid through the tubes. 10. Click the sequence button in the upper left hand corner (looks like a spreadsheet) to make a sequence. Save the file in your personal folder. See sample sequence in Section H.. Standards 1 through 6 should go in the first 6 slots. Then the controls follow; the standard check is the 0.5 ppm standard. If you have a lot of samples then slots 7-10 should be filled with the standard check (0.5 ppm) but if you only have ~40 samples then just need to fill slots 7 and 8. Samples should start at slot number 11. If you wish to intersperse with check standards and also repeat all the standards at the end this is an option. It is also a good idea to program in a blank (with repetitions) between each sample to avoid carryover between samples. It is recommended to put a known check standard (mid-range concentration) after every 10 samples so you can calculate the instrument s drift factor. 11. Load the vials full of filtered sample into the auto-sampler tray Soil & Water Lab, Phosphorus Analyzer Procedure Page 3
12. Click the schedule button on the left side of the screen (looks like a series of sin waves) to open the sequence that you just saved. If it asks for a operator ID, just type the name of the folder that you saved the sequence to (e.g. Sheila). 13. Press the play button and wait for the line on the plot to stabilize. This takes approximately 40 minutes. The machine is set up to run for 1 hour before advancing automatically to the first line in your sequence table. 14. While the machine is stabilizing, mix your reagent. See section E. Making Reagent Solution. Rinse out the reagent bottle (and tube if necessary) with DI water and fill it with the newly mixed reagent. 15. Once the signal looks relatively stable or during the last 20-30 minutes of the recommended 1 hour take the tube that s attached to the *R slot and move it from the DI+dowfax bottle the brown reagent bottle. 16. Wait until the noise of signal is no more than 200 microam (about 30 minutes). Right click on the plot and select Full Scale to zoom out. To zoom in, right click and drag a rectangle around the plot area you wish to zoom to. 17. When the signal looks stable press the fast forward button to start the run. 18. Watch the peak of the 10 ppm sample as it goes through. This is the SYNC line of the sample table. The peak should be symmetrical with one peak that top at 260,000 to 280,000 microam.. This will take about two minutes to see on the plot. If the peak doesn t appear or reach the suggested range then there is something wrong with the patens, your reagent solution, or another part of the machine. If something is wrong and you would like to stop the machine, press the stop button, fix the problem, press the rewind button, run will not be saved, and then restart by pressing play and then the fast forward button. If peak is good then continue to run the sequence. 19. Watch the calibration curve R squared value. It should be as close to 1 as possible, ideally reaching at least 0.99900 If it s not then stop the run, remake your standards, and start the run again. If it s fine then keep the run going. 20. During the run periodically check that the DI cup next to the autosampler is filled above the bottom of the probe. 21. When the run is finished the results file will save as a.rls. You can find it in the same folder that you saved your sequence in. Export the results by selecting View > Reports. A window with results should pop up. Save these to your folder as a.txt file by selecting File > Export Report. Type in the file name making sure to erase the asterisk that is automatically placed before the extension. 22. Now clean the machine. First switch over the reagent tube back to the DI+dofax bottle and let the system flush out 5 minutes to flush out any remaining samples and acid. 23. Run the cleaning solutions through the machine for all sample types and all runs. It is especially important to clean the machine when you know you have dirty samples (i.e manure runoff, etc.). Cleaning the machine helps us keep the result for our blanks at ~0.02 ppm (+/- 0.01 ppm). After flushing out the reagent in step 22, unscrew the tube on the R* port (DI+dowfax bottle) and connect it to the 1M NaOH solution for 20 minutes. Reconnect to the R* port for 5 minutes to flush out the NaOH and then connect to the 5N H 2 SO 4 solution for 20 minutes. Finally, reconnect to the R* port in the DI+dowfax bottle and run for 15 minutes so the whole system has a chance to be flushed out. 24. Turn off all the pumps (Pumps > Pumps Off > All Pumps) and shut down the computer. Be sure to switch off the auto-sampler and the sensor as well. Soil & Water Lab, Phosphorus Analyzer Procedure Page 4
G. Other Notes and Recommendations Filtering Samples All samples should be filtered with 0.45 um membrane filter before using the P Analyzer. Filters may contain P; therefore if low level accuracy is needed, filters must be pre-washed before P work. Standard Methods (Method 4500) give a procedure for this. Basically, using an empty volumetric flask to catch the flow through water, you should filter a known volume of DI water through the filter before filtering your sample. Keep the filter paper but replace the flask with the centrifuge tube used to store your filtered sample and filter your sample as you would normally. It is best to also run samples to compare unfiltered DI water with filtered DI water during your run (replicates of ~3). Preserving Samples Refrigerate samples immediately and analyze them within 48 hours for best results. Freezing samples can cause ions to precipitate out. This precipitate may be difficult to see but is detrimental to the machine and can alter your results. Shree recommends not freezing samples but instead adding approximately 100 ul of concentrated H 2 SO 4 (or enough so ph = 2) into each full 50 ml centrifuge tube immediately after filtering your samples. If you have already frozen your samples, please re-filter the thawed sample before running them through the P Analyzer. Use a 0.45 um filter. Diluting Samples If you expect your samples to be greater than 0.5ppm, it is recommended that you dilute your samples. For example a common dilution is 1mL sample : 9mL DI water. Unknown Samples For samples where the concentration is unknown and potentially greater than 0.5ppm, add additional blank reps in between samples. This is done by changing the 1 in the R column of the blank line to 2 or 3. Soil & Water Lab, Phosphorus Analyzer Procedure Page 5
H. Example Sequence Table Cup refers to the position on the autosampler. Name is your sample ID. R refers to the reps or number of times the machine will sample from the cup before moving on to the next line. Dil and Wt are not utilized in our set up. Cup Name Type R Dil Wt Vial Comment 6 Sync SYNC 1 1 1 0 Carryover CO 6 1 1 0 Baseline RB 1 1 1 1 Cal 0.00 ppm C 1 1 1 2 Cal 0.005 ppm C 1 1 1 3 Cal 0.050 ppm C 1 1 1 4 Cal 0.50 ppm C 1 1 1 5 Cal 5.00 ppm C 1 1 1 6 Cal 10.00 ppm C 1 1 1 0 Blank BLNK 7 1 1 7 ICV W 0.5 ppm ICV 1 1 1 7.1 0 Blank BLNK 3 1 1 0 Read Baseline RB 1 1 1 11 DI U 3 1 1 12 Sample #1 U 1 1 1 13 Sample #2 U 1 1 1 After 10 samples, the following lines should be added after a blank is run. Use the Comments column to keep track of the number of times a cup is drawn from. The maximum is five times per cup. Cup Name Type R Dil Wt Vial Comment 7 CCV W 0.5 ppm CCV 1 1 1 7.2 0 Blank BLNK 3 1 1 0 Read Baseline RB 1 1 1 Soil & Water Lab, Phosphorus Analyzer Procedure Page 6
The end of the sequence should have the following lines. Cup Name Type R Dil Wt Vial Comment 1 0 ppm check U 1 1 1 2 0.005 ppm check U 1 1 1 2 0.05 ppm check U 1 1 1 2 0.5 ppm check U 1 1 1 0 Blank BLNK 2 1 1 2 5 ppm check U 1 1 1 0 Blank BLNK 2 1 1 2 10 ppm check U 1 1 1 0 Blank BLNK 2 1 1 X CCV W 0.5 ppm CCV 1 1 1 X.X 0 Blank BLNK 1 1 1 0 Read Baseline RB 1 1 1 I. Tubing L refers to the left side of the pump and R refers to the right side of it Tube Color of the Top end Bottom end spot tubing L1 Green Waste bottle TO WASTE port L3 Red Waste bottle Top port of the clear bubbler L4 Red CARRIER IN port DI only bottle R1 Orange Reagent port on DI Dowfax bottle or Reagent bottle Port no. 3 R4 black DI Dowfax bottle Port no. 9 J. Instrument Model: O.I. Analytical 3000 K. Credits: Sheila Saia; Bahareh Hassanpour Guilvaiee; Chelsea Morris Soil & Water Lab, Phosphorus Analyzer Procedure Page 7