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Intense ecological interest in phosphorus stems from its major role in metabolism in the biosphere. In comparison to the relatively rich supply of other major nutritional and structural components of the biota (C, N, O, S), phosphorus is least abundant and commonly limits biological productivity in aquatic ecosystems.

Phosphorus occurs in a number of inorganic and organic compounds in both particulate and dissolved forms. Differentiation of forms is based on their reactivity with molybdate, ease of hydrolysis, and particle size. In this procedure, acidic ammonium molybdate reacts with orthophosphate to produce a yellow phosphomolybdate complex. Ascorbic acid then reduces this complex, giving an intense blue color.

It must be stressed that when one is analyzing for phosphorus in the µg range, laboratory contamination from dust and detergents will often produce higher concentrations than the lake or river water itself. Clean glassware with phosphate-free detergents (i.e. Liqui-Nox). Ideally, all glassware used for phosphate determination should be acid washed and stored in a dilute acid (1-5%) solution.

Because of the reported possibility of phosphorus adsorption onto polyethylene, samples for phosphorus analysis should be collected in acid-washed glass bottles. Samples should be refrigerated immediately, and analysis should be completed within a few hours. If analysis must be delayed, any filtration (to separate soluble reactive phosphorus, PO4, from total phosphate) must be done immediately and samples stored frozen.

Phosphorus and nitrogen are the elements normally in highest demand by aquatic plants and algae, and is usually the element in shortest supply in oligotrophic systems. Oligotrophic systems are generally characterized by PO4-P levels below 10 µg/l while eutrophic systems may have concentrations in excess of 100 µg/l.

Option A. Procedure (CHEMets – ppm – mg/L)

1. Fill the sample cup to the 25 ml mark with sample.

2. Add 2 drops of A-8500 Activator Solution. Cap the sample and shake to mix the contents well.

3. Place the CHEMet ampoule in the sample cup. Snap the tip by pressing the ampoule against the side of the cup. The ampoule will fill leaving a small bubble to facilitate mixing.

4. Mix the contents to the ampoule by inverting it several times, allowing the bubble to travel from end to end each time. Wipe all the liquid from the exterior of the ampoule. Wait 2 minutes for color development.

5. Use the appropriate comparator to determine the level of ortho-phosphate in the sample. If the CHEMet ampoule is between the two color standards, a concentration estimate can be made.

    a. For the low range comparator, place the CHEMet ampoule flat end downward into the center tube of the low range comparator. Direct the top of the comparator up toward a source of bright light while viewing from the bottom. Rotate the comparator until the color standard below the CHEMet ampoule shows the closest match.

    b. For the high range comparator, hold the high range comparator in a nearly horizontal position while standing directly beneath a bright source of light. Place the CHEMet ampoule between the color standards moving it from right to left along the comparator until the best color match is found

Option B. Procedure (Hach reagent pillows)

1. Transfer 25 ml of the sample into a clean container that has not been exposed to detergents. Add the contents of one PhosVer3 Reagent packet to each sample. Immediately cap and shake to mix. A blue color will develop if phosphate is present. Wait at least 2 min for full color development but do not wait more 10 minutes before taking a reading. Prepare the spectrophotometer during this time.

2. Use a USB cable to connect the spectrometer to the computer. Start the LoggerPro software.

3. To calibrate the spectrometer, choose Calibrate → Spectrometer from the Experiment menu. The calibration dialog box will display the message “Waiting .. seconds for the lamp to warm up”. Allow the spectrometer to warm up for at least three minutes. Follow the instructions in the dialog box to complete calibration.

4. Click on the “Configure Spectrometer” icon in the menu bar (rainbow looking icon third from the right). Set Collection Mode to Abs v Concentration. Set wavelength to 890 nm. Change units to μg/L. Click OK.

5. Click on the Data Collection icon in the menu bar (immediately to the right of the Configure Spectrometer icon). Select Events with Entry.

6. Insert the first sample into the spectrometer. Click the green COLLECT button. The spectrometer will take a few seconds to produce a reading. Select KEEP on the top right side of the menu bar. Enter the location of the sample. Then select STOP. Insert the next sample and COLLECT. In the dialog box, select Append To Latest to save the data. Continue for all samples.

7. To determine the amount of PO4‐P in your samples, consult a standard curve constructed by plotting absorbance vs. known phosphate concentrations.

Note ‐ the PhosVer 3 Reagent may cause some turbidity depending on a large number of factors. It is recommended that a reagent blank be run on each lot by adding the contents of one packet to 25 ml demineralized water. This should be read using demineralized water as a blank. The value found should be subtracted from the final test readings.