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

Turbid samples analyzed in the typical laboratory likely contain various particle sizes so that their optical densities are a a combination of Rayleigh and Tyndall scattering (Figure 6). It is a common practice to correct A260 measurements for background turbidity by subtracting an absorbance value taken at a longer wavelength (e.g. 320 nm). Although such a subtraction may be better than nothing at all, Figure 6 indicates that it is likely an undercorrection.


6 Adsorption to well surfaces. Adsorption of analyte to the walls of the microplate wells removes the analyte from the interrogating light beam and thus decreases absorbance. Adsorption can be an especially severe problem with low concentrations of analyte.

7 Well geometry. Microplates with halfarea wells have twice the optical path-length per unit volume than standard flatbottom microplates. Samples in roundbottom wells have a slightly longer pathlength than the same volume in flatbottom wells. In quartz plates, the wells are cylindrical and the walls are vertical, whereas in plastic microplates, the walls are tapered slightly outward from bottom to top. Because of differences in well geometry, a given sample may have different absorbance values in different microplates. All of these differences can be eliminated by measuring the pathlengths and normalizing the results with PathCheck.


SUGGESTIONS FOR BEST QUALITY UV ABSORBANCE RESULTS

1. Use clean microplates that are UVtransmissible.
Protect the bottom surface from fingers and hard surfaces.
Do not reuse disposable microplates.
Blow out dry microplate with clean dry air.

2. Avoid dust particles in samples:
Filter buffers (e.g. 5 filter) to remove ambient particulate contamination.
Cover micropl
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