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

ate if samples are not read immediately.
Run the blanks (buffercontaining wells) in triplicate, at least, and note that their absorbance values fall within the expected range (to avoid accidental use of dirty plates or incorrect plates).

3. Automix briefly before reading to encourage symmetrical menisci.

4. Avoid bubbles. If samples are prone to foaming (e.g. contain detergent, protein, or nucleic acids) use reverse pipetting technique.

5. If samples have low absorbance, maximize optical pathlength by using increased sample volumes (e.g. 250 L to 300 L in 96well microplates).

6. Run samples in duplicate or triplicate.


SUMMARY

Absorbance measurements made through microplate wells are subject to path-length variability and are vulnerable to interference from surface effects. The need to avoid bubbles has long been obvious and the effect of meniscus curvature on pathlength is predictable and may be normalized by employing PathCheck. Microplates are essentially open vessels exposed to dust and packing debris even in the cleanest of laboratories. Dust particles have not been a serious problem in the past because microplate readers had large optical beams and therefore were relatively insensitive to occasional particles in the light path. Modern microplate spectrophotometers, however, have optics designed for microplates with smaller wells and are very susceptible to particles in the light path. All of the above factors contribute to the need for careful attention to technique, especially in the UV spectral range, in order to get accurate, reproducible absorbance results in microplates.


REFERENCES

1. Jenkins, F.A. And H.W. White. 1976. Fundamentals of Optics. McGrawHill, New York. Pp. 466468.


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