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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)

using microplates). If necessary, create a new Cuvette Set or Plate Section by selecting New Cuvette or New Plate from the Experiment menu.

Step 2 Set up the Instrument Settings dialog box as shown in Figure 1 for a cuvette (upper) or for a plate (lower). Select to perform an end-point read at 820 nm. DO NOT SELECT PATHLENGTH CORRECTION (i.e., leave the PathCheck checkbox unchecked). The phosphomolybdate absorption band extends into the near IR and interferes with the measurements needed for pathlength calculation.

Note: The Sigma kit package insert recommends 660 + 40 nm. However, the broad absorbance band of the reduced phosphomolybdate complex is actually maximal near 820 nm, and the latter wavelength gives approximately 25% higher sensitivity.

Step 3 Create a template showing where standards and unknowns will be located in the Cuvette Set or the Plate Section.

Step 4 Click the Reduction button in the Cuvette Set or the Plate Section tool-bar to display the Reduction dialog box. Set the wavelength combina tion to L1 and the data mode to Absorbance as shown in Figure 2.


Prepare the reagents and samples
Step 1 Dissolve 1.0 g Fiske & Subbarow Reducer in 6.3 mL deionized water. The Reducer solution can be stored for 1 month at room temperature, stored in a tightly-capped amber bottle. The acid molybdate reagent is supplied at a ready-to-use concentration in the kit.

Step 2 Prepare the standards by diluting the stock as specified in the package insert, except prepare only 2.5 mL (not 5.0 mL) of each dilution and use 12 x 75 mm glass tubes to hold the dilutions. The inorganic standard supplied with the Sigma kit contains potassium phosphate, equivalent to 20 g Pi/mL. Because the kit is intended to be used with serum samples, a one to ten dilution of the sample is factored in and the standard curve is expressed in mg/dL of
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