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

Evelyn McGown, Ph.D.
Molecular Devices Corporation. 7/99


In 1994, Molecular Devices introduced SPECTRAmax, the first microplate reader equipped with a monochromator, rather than bandpass filters, for wavelength selection. It was also the first microplate reader capable of measuring absorbance below 340 nm. These new features transformed the microplate reader from a limited colorimetric filterbased instrument to a true UVVis spectrophotometer.

Despite the huge popularity of microplates, many people experience difficulty when adapting assays to them. One source of confusion is the fact that samples read in a cuvette and in a microplate do not have identical raw absorbance values because of differing optical pathlengths. Also, microplates are susceptible to surface effects including floating particulates, foaming and variable meniscus formation. The susceptibility to particulates has increased in recent years because modern microplate spectrophotometers such as SPECTRAmax are designed to accommodate microplates with smaller wells and thus have smaller beams than older instruments. All of these factors cause microplates to demand more attention to careful technique to get accurate absorbance results. Especially in the UV spectral range, appropriate procedures should be followed to avoid poor reproducibility and unsatisfactory sensitivity. This Application Note provides guidelines for optimizing UV measurements in SPECTRAmax microplate spectrophotometers, particularly measurements of proteins at 280 nm and nucleic acids at 260 nm.


MATERIALS
1.
SPECTRAmax microplate spectrophotometer (Molecular Devices Corp.)

2. UVtransparent microplates; e.g.
UVPlate (CorningCostar Cat. No. 3635, Tel: 18004921110)
UVStar (Greiner Cat. No. 655801, E & K Scientific Products, Tel: 408378 2013)
UVMax (Polyfiltronics) fro
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