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

DNA and protein solutions can range between the two extremes. Thus variable meniscus formation can be a source of betweenwell or betweenplate error in microplate analyses, unless steps are taken to ensure that meniscus curvature is identical in all wells. Alternatively, the pathlength may be measured in each well with PathCheck and the absorbance values normalized.


For best precision, the meniscus, whether flat or curved, must be symmetrical, and not skewed up one side of the well (Figure 5B). A sample with an asymmetrical meniscus has a variable and unpredictable optical pathlength. Also an irregular meniscus acts like lens and can deflect the light beam away from the detector, resulting in falsely high absorbance readings.

3 Particulates and bubbles. Dust particles are becoming much more problematic as the beam size and the detector size in the typical instrument become smaller. A particle is not noticeable unless it happens to be in the optical path at the moment of measurement, in which case it has a huge effect. Such particles can cause artifactual spikes in the data up to 0.3 OD above the sample absorbance.

4 Dirty microplates. Microplates can appear perfectly clean, yet have high optical density at wavelengths below 400 nm (the lower limit of detection by the human eye). Fingerprints and residual contamination in reused plates cause errors in UV absorbance measurements.

5 Sample turbidity. Colloidal suspensions scatter light and therefore increase apparent optical density. If the particles are much smaller than the wavelength of the light, the scattering is proportional to (1/lambda)4 and is known as Rayleigh scattering1. Thus the optical density of such a suspension is 10 times greater at 250 nm than at 450 nm. If the particles are larger than the wavelength, light scattering is due to ordinary diffuse reflection (Tyndall scattering).
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