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

The variable light path has made it difficult for people to compare results obtained in microplate readers and spectrophotometers.


The BeerLambert Law states that absorbance of a solute in solution is determined by its absorptivity, its concentration, and the optical pathlength (Figure 3). For a given solute under defined solution conditions, absorptivity (sometimes called Extinction Coefficient) is a constant. In traditional cuvettes, the pathlength is also a constant (typically 1 cm), so the only variable is concentration. In microplates, both concentration and optical pathlength are variables. In order to relate absorbance values in microplates with those in cuvettes, one must measure the optical pathlengths. (SPECTRAmax microplate spectrophotometers and SOFTmax PRO can do the measurements and calculations automatically.)


FACTORS DETERMINING ABSORBANCE VALUES IN MICROPLATE WELLS
1. Total amount of solute in the optical lightpath. With different optical pathlengths, different concentrations of DNA can have identical absorbance values as long as the products (Concentration x Pathlength) are identical. For example, wells containing DNA at concentrations of 1000, 500 and 333 ng/mL will have the same absorbance values if their respective sample volumes are 100, 200 and 300 L; i.e., the total amount of DNA in each well is 100 ng (Figure 4).


2. Meniscus curvature and symmetry. The optical pathlength through a sample in a microplate well is affected by the curvature of the meniscus (which in turn is dependent on the composition of the solution and surface treatment of the microplate). For example, 100 L of water or buffer in an untreated micro-plate (flat meniscus) has a pathlength of 0.32 cm, versus 0.25 cm for 100 L of 0.1% Tween (curved meniscus) a difference of 23% (Figure 5A). Meniscus formation in
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