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UV Absorbance Measurements in SpectraMax Microplate Spectrophotometers MaxLine Application Note #32)

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Quartz microplate (Hellma Cells, Inc., Tel: 7185449534, 9166)


CHOICE OF
MICROPLATES
Quartz microplates are commercially available, but expensive (> $1000). There areat least three UVtransparent plastic microplates available. Figure 1 shows the spectra of the abovementioned microplates between 200 and 400 nm.


Quartz is transparent throughout the wavelength range. The CorningCostar UV and Greiner UV microplates have good transmission down to ~220 nm. The Polyfiltronics UV Plate is usable down to 240 nm, though it has a higher background than the Costar and Greiner plates. Standard polystyrene microplates can be used down to approximately 300 nm.

The microplates described above have excellent uniformity in the spectral regions where they have high transmission. At wavelengths near their cutoff values, they may have undesirable welltowell variability in optical density (OD). This source of error can be minimized by prereading the plate with water in the wells. (Reference solvent, usually water, is important because its refractive index influences the apparent background OD of the microplate, resulting in 0.004 to 0.014 lower OD values than dry plates.) SOFTmax PRO will make the wellby well background subtractions automatically. Table 1 summarizes the useful spectral ranges and recommended preread ranges for the 5 microplates.


MICROPLATES VERSUSCUVETTES: OPTICAL PATHLENGTH IS THE DIFFERENCE
In traditional spectrophotometers, samples are read horizontally and the optical pathlength is fixed by the physical dimensions of the cuvette (Figure 2). In micro-plate spectrophotometers, samples are read vertically, so the optical pathlength in each well varies, depending on the volume of fluid in the well and the degree of curvature of the meniscus.
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