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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)


INTRODUCTION
Most spectrophotometric methods for inorganic phosphorus (Pi) analysis are based on the color formed upon the reduction of a phosphomolybdate complex. The classic (and still widely-used) method is that of Fiske and Subbarow1, in which the reduction is carried out with sulfite and aminonaphtholsulfonic acid. Over the years, modifications have been offered with various reducing agents and differing concentrations of reactants. One of the most sensitive and well-characterized procedures is that of Chen, Toribara and Warner2 in which the reducing agent is ascorbic acid.

This application note details two methods for measuring inorganic phosphorus using Molecular Devices SPECTRAmax PLUS microplate spectrophotometer. The first method uses a commercial kit obtained from Sigma Diagnostics, based on the original Fiske-Subbarow chemistry, and offers the convenience of ready-made reagents. The second method2 is approximately 10 times more sensitive, and thus is useful for samples containing 0.1 to 5.0 g Pi/mL . Whichever method is used, the SPECTRAmax PLUS microplate spectrophotometer offers the versatility of making the absorbance measurements in microplates, in standard 1 cm cuvettes, or in 12 x 75 mm test tubes.


SIGMA KIT MATERIALS

1. SPECTRAmax PLUS microplate spectrophotometer system

2. Test tubes (12 x 75 mm, glass), or 1 cm cuvettes, or transparent microplates (e.g., polystyrene)

3. Trichloroacetic acid, (20% w/v) if samples need deproteinization

4. Pipettor and tips

5. Inorganic Phosphorus kit (Sigma Chemical Co., catalog # 670-C)


SIGMA KIT METHODS
Setting up the instrument and the software

Step 1 Using SOFTmax PRO, open either a Cuvette Set (if using test tubes/ cuvettes) or a Plate Section (if
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