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Microdetermination of Phosphorus Using the SpectraMax Plus Microplate Spectrophotometer: Choice of Microplate, Cuvette, or Test Tube Assay Formats (MaxLine Application Note #24)

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Step 1 Prepare the following stock solutions:
6 N sulfuric acid (dilute 16.7 mL concentrated acid to 100 mL.)
2.5% ammonium molybdate (store at room temperature).
10% ascorbic acid (store for up to 7 weeks at 05 C).

Step 2 Prepare the stock standard, equivalent to 5.0 g Pi/mL (0.161 mM). Weigh accurately 22.2 mg sodium phosphate, monobasic, anhydrous (MW 120) into a 100 mL volumetric flask. Bring to volume with 0.05 N HCl.

Dilute 10 mL of the stock standard to 100 mL with 0.05 N HCl (final concentration: 5.0 g Pi/mL. Step 3 The samples must be aqueous, protein-free and contain 0.1 to 5 g Pi/ mL. If they have been acid-treated (to deproteinize or to ash them), either dilute or neutralize, so that the acid concentration is less than 1 N (See reference 2).

Step 4 Prepare the standard curve by making a serial, 1 to 2 dilution of the stock standard in deionized water to obtain the following concentrations of Pi: 5.0, 2.5, 1.25, 0.625, 0.3125, 0.156 and 0.078 g/mL. For the 0 standard, use deionized water.

Step 5 Prepare the working reagent by mixing 1 volume 6 N sulfuric acid, 1 volume 10% ammonium molybdate, 1 volume 10% ascorbic and 2 volumes deionized water. The working reagent should not be stored for more than 24 hours.

Step 6 Test tube/cuvette method: Pipet 1.0 mL aliquots of each sample and standard into 12 x 75 mm test tubes. For the 0 standard, use deionized water. Add 1.0 mL reagent and vortex briefly to mix. Cover each tube (e.g. with Parafilm) and place in 37 C incubator for at least one hour.

Microplate method: Pipet 150 L aliquots of each sample, standard and blank into the designated wells of the microplate. Add 150 L reagent to each well. Place a lid on the microplate and put the plate in the drawer of the reader and mix for approximately 5 seconds. Incubate the plate at 37 C for approximately on
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