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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 7 After incubation, read each tube at 820 nm in the SPECTRAmax PLUS cuvette port. For the reference reading, use the 0 standard. Alternatively, transfer each standard and sample to a clean glass or quartz cuvette and read 820 nm. If using a microplate, read it in the SPECTRA-max PLUS.


CHEN et al RESULTS
Color development proceeds gradually until reaching a maximum at approximately 90 minutes ( Figure 6). The reaction is approximately 85%, 91% and 96% complete at 30, 45 and 60 minutes, respectively. The plate could be read as early as 30 minutes, though maximum sensitivity is obtained after 90 minutes incubation, as specified in the original procedure.


Figure 7 compares standard curves prepared and read in a microplate with standard curves read in tubes or transferred to a standard 1 cm cuvette. All curves have excellent linearity from 0.078 g/mL to 5 g/mL. The standards have slightly higher absorbance when read in 12 x 75 tubes than in a cuvette, indicating the tubes are a little more than 1 cm in diameter. The standard curve generated in the microplate is lower, reflecting the shorter pathlengths through the samples in the microplate. Identical results are ultimately obtained, whether the user chooses to perform the analyses in tubes, cuvettes, or a microplate.


SUMMARY
Procedures are detailed for measuring inorganic phosphorus using Molecular Devices SPECTRAmax PLUS microplate spectrophotometer. The first method takes advantage of a commercial kit and is suitable for samples which contain fairly high concentrations of inorganic phosphorus, such as serum or urine. The second procedure is much more sensitive and is useful for samples containing as little as 0.1 g Pi/mL. For either method, Molecular Devices SPECTRAmax PLUS microplate spectrophotometer offers the versatility of making the absorbance measureme
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