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Ammonium
- Manual Chemistry
Introduction Ammonia nitrogen, in the form of NH3 and NH4 , is the product resulting from the biochemical decomposition of nitrogenous matter. The so called indolphenol blue method (first reported by Berthelot in 1859) between ammonia, phenol and hypochlorite in an alkaline medium. The resulting blue color is similar to that of indolphenol dyes and its intensity is proportional to the ammonia concentration. The indolphenol colorimetric reaction was modified by introduction of the catalyst nitroprusside, which accentuates the blue color at room temperature (Lubochinskwy and Zaltal, 1954; Mann, 1964).
Notes
The following method is specific to ammonia. The technique is a sensitive modification of the phenol hypochlorite method in Standard Methods 1985, p 382 by Bowden and Finlay.
Capabilities
Range 0.1 - 10 um /liter (using 5 cm cells)
General Comments
Safety Phenol breaks down plastic so use glass to store this reagent. Phenol can cause sever burns and is readily absorbed through the skin. It should be handled in a hood and precautions should be taken to avoid any skin contact (i.e. goggles, gloves, and lab coats should be worn). The phenol reagents should be added to the samples in a hood. Make sure waste and old reagents are stored in the proper labeled container, including the waste that runs through the spec.
Contamination & Problems
Always use ammonia-free water for all reagents. Get the water from the Nanopure unit immediately prior to use. Make absolutely sure the DI used to make the standards is also used for the blank. This is vital in determining where any errors are being introduced. Acid wash all glass and plastic ware in acid bath. Following the acid bath, immediately rinse 3-5 times with fresh distilled water. Use dedicated glassware for each step of the procedure, including the same containers for the same standards. Rinse with DI several times before making daily standard but do not acid wash unless flask has been contaminated. Handle all flasks and sample tubes with care; fingers are a great source of contamination. The best way to keep blanks low is to pre-react tubes the day before running the analysis. However, this is unnecessary if samples are being run every few days. Sample tubes should initially be designated for each nutrient being analyzed since PO4 regents contain (NH4)6 Mo7 O244H2O and not subsequently mixed. Sample tubes should be pre-reacted prior to being used for the first time each year. When tubes and glass ware are in regular use they should be rinsed 3 – 5 x with DI but not acid washed after use. Then placed in drying oven hanging upside down so no edges contact. Acid baths should be changed on a regular basis (i.e. weekly for NH4+, and every 2-3 weeks for PO4,) Ammonia nitrogen (NH3) is a gas and can be easily lost from samples at high pH, especially if they are frozen. The pKa of ammonia is about 9.
Accuracy When making the standard dilution series, make sure the pipettes are calibrated properly. It is important to get very accurate dilutions when working w/ such low concentrations. Check calibration by weighing the desired amount on the balance and adjust pipet accordingly.
Reagents
Apparatus & supplies
Test tube racks (147919C) (holds 40) Test tubes 13x 100 mm (25 ml) (14957J ) case of 72 5 ml pipette for aliquoting of sample Re pipette set to .3 ml for phenol solution Re pipette set to .250 ml for oxidizing solution Re pipette set to .2 ml for sodium nitroprusside solution 1 liter graduated cylinder (Phenol solution) 200 ml graduated cylinder required to make sodium hypochlorite (5% NaOCl) 500 ml liter volumetric (sodium nitroprusside solution) 1 liter amber bottle (Phenol solution) 1 liter amber bottle (sodium hypochlorite) 500ml amber bottle (sodium nitroprusside solution) 200 ml graduated cylinder (Phenol solution) 200 ml graduated cylinder required to make oxidizing solution (sodium hypochlorite) 100 ml graduated cylinder required to make sodium nitroprusside solution Drying oven to dry tubes and glassware 125 ml quorpack wide mouth bottles to store standards in
Preparation of Stock reagents
It is suggested that all reagents be made fresh each day. To save time once each week phenol can be weighed out and sodium nitroferricyanide re - crystallized.
1. Phenol Solution *Handle with care, read MSDS for Phenol prior to use* Once each week weigh out enough crystalline reagent grade Phenol for the all the runs that week and place the crystals in scint vials wrapped in foil. Each scint vial should contain 15 g of phenol.
2. Sodium Hypochlorite reagent (5% NaOCl or Bleach) A new bottle should be opened at the start of each week since a large head space allows oxidation which increases the pH. Squeeze the bottle to reduce the head space after opening. Stock solution (enough for 1 run
of 500 samples each)
To 100 ml of water add 50 ml 5% NaOCl solution (commercial bleach). Adjust pH to 6.5 to 7 with HCL (~ 75 ul 1 N HCL).
3. Sodium Nitroprusside Solution (sodium nitroferricyanide)*Handle with care, read MSDS for prior to use* Re-crystallization of Nitroprusside
Dissolve 5 g Nitroprusside in 10-12 mls of DI water. Add 25 mls of 95 % EtOH. Freeze solution over night to reform crystals, then filter and dry crystals. Re-crystallized sodium nitroprusside will give a lower reagent blanks.
Stock solution (enough for 5 runs
of 500 samples each)
Once each week make the stock solution for the all the runs that week. Dissolve 1.75 g of re-crystallized sodium nitroprusside in 500 ml of deionized water. Store in an amber bottle. This solution is stable for at least one week.
Working solutions: Set out each day for use.
1. Phenol solution (300 ul /sample) re-pipette contains enough for 500 samples Dissolve 15 g of crystalline reagent grade Phenol in 150 ml H20, mix thoroughly and while cooling with tap water or ice if necessary, add 3.75 g NaOH. This is slightly different from the Bowden method in which they added .032 g/ ml. It is a return to the original standard methods recipe of .025 g/ ml. It s hoped that this solves some of the problems with the short shelf life of the phenol that occurred in 1997. 2.
Sodium Hypochlorite (250 ul / sample) re-pipette contains enough for 500
samples.
In a clean, calibrated re pipette To 100 ml of water add 50 ml 5% NaOCl solution (commercial bleach). Adjust pH to 6.5 to 7 with HCL (~ 75 ul 1 N HCL).
3. Sodium nitroprusside (200 ul /sample) re-pipette contains enough for 500 samples aliquot 100 ml Re-crystallized Nitroprusside solution into clean calibrated re-pipette
Sample bottle preparation
Sample bottles should be acid
washed, rinsed 3x with Nanopure DI,
and filled prior to being brought
into the field.
Sample collection
1. Rinse syringe 3 x with water to be sampled. 2. Attach filter to end of syringe (either a GF/F or .2 um membrane filter) and filter 10-30 ml though filter to rinse filter. 3. Dump DI from sample bottle and rinse with 30-40 ml of filtrate. 4. Re-fill syringe and fill sample bottle with filtrate.
Blanks
Duplicates should be run for all blanks, with reagent blanks run at the beginning and end of each sample set. Three types of blanks should be run with each set of samples. 1. Trip blanks (DI blanks that are carried out to the field and back). These would DI from a sample bottle that is acid washed, filled with DI, and brought into the field and then returned. 2. Field blanks (DI blanks filtered in the field as if they were samples). 3. Reagent blanks (Fresh DI from the Nanopure run as a sample).Three types of blanks should be run with each set of samples.
Aliquoting samples
1. Fill cup with fresh DI from Nanopure system. 2. Rinse pipet tip 3 x with DI. 3. Aliquot 5 ml of sample into test tube. (repeat if running duplicates) 4. Begin at step 2 for next sample.
Procedure
Add all reagents
in the hood
1. Aliquot 5 mls of filtered water/sample for each blank or sample. 2. add 0.3 ml Phenol solution 3. Vortex 4. add 0.250 ml Sodium Hypochlorite solution. solution 5. Vortex 6. add 0.2 ml Sodium nitroprusside 7. Vortex 8. Cover with para-film and place samples in Dark for at least 1 hour but no more than 24 hours. (Color formation should be complete after 10 min). Samples should be ready between 1-2 hr. Make sure darkness is complete.
Reading on the Spectrophotometer
1. Make sure Spec is set to sip 3ml. 2. Set wavelength to 630 nm and make sure the spec is reading absorbance. 3. Make sure the waste tube enters the ammonium waste container. 4. Sip DI water and 0 the reading. 5. Sip standards so low standards are read first (start w/ blanks and work way up) After each standard, sip water through and re zero if necessary. Sip water through between high standard and first sample to remove any residual NH4. Re zero if necessary. 6. Before re zeroing, sip again to make sure all residual material is removed and the reading is stable. 7. Wipe the sipper tube on spec w/ kimwipes to dry off the outside of the tube in between samples so that contamination is minimized. 8. Wear gloves when reading standards and samples on the spec. Ammonium from hands could be left on sipper tube, and reagents are harmful. 9. If absorbance is higher than highest standards then you will need to dilute your sample.
Standard Preparation
Stock A Solution-1000mM NH4-N
Dissolve 0.06607g of dry (dried in oven opvernight) (NH4)2SO4 (m.w.= 132.14) in approximately 900ml of deionized water contained in a 1 L volumetric flask. Dilute the solution to the mark with deionized water and mix it well. Transfer this solution to an amber bottle. Refrigerate when it is not in use.
Stock B Solution-20mM NH4-N
Prepare this solution daily. Using a volumetric pipet or a calibrated automatic pipet, add 2.0ml of Stock A to approximately 90 mL of deionized water contained in a 100ml volumetric flask. Dilute the solution to the mark with deionized water and mix it well.
Working Standards
Prepare these solutions daily. Use adjustable, microliter pipets to add the designated volumes of stock A or B listed in the following table. Calibrate the pipet for each required volume. (NOTE: The standards for each day are presented in bold typeface. The other concentrations are included for reference, if needed.) Prepare each standard by adding the required amount of stock to the required volume volumetric flask containing deionized water. Dilute each to the mark with deionized water and mix well. Keep these solutions tightly sealed.
Record the absorbance for each standard and spike sample in the nutrient log book. Determine the standard curve by plotting absorbance (y-axis) versus standard concentration (x-axis). We have created an Excel workbook, entitled nutrients.xls, for data input. Put a copy of the daily Excel worksheet on a diskette and a copy of the curves on the door to the spec room.
Method Detection Limit
The detection limit for this method should be determined daily. If the detection limits are consistent for a couple of weeks, then it will be necessary to perform this task weekly.
Spike DI water with 2-3 times the estimated instrument detection limit . This should be around 0.2 to 0.3 mM. (Use the 0.2 or 0.3 mM standard as the spike.) Run 7 spikes as samples after the standard curve has been run. Calculate the method detection limit (MDL) by MDL= [t(7, 0.01) * s]
where t=t statistic for 7 reps
(t=3.14) with 99% confidence and s=standard deviation
of the calculated concentration.
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