Lidstrom:Path Length in microwell plates
Back to Protocols
- The Beer-Lambert law states A=εCL
- A = absorbance at a particular wavelength
- ε = molecular extinction coefficient for a particular type of molecule at a particular wavelength
- C = concentration
- L = path length
- The path length in a cuvette is constant, and is usually 1cm.
- The path length in micro plates is not, and depends on:
- liquid volume
- plate geometry
- Most micro plates aren't just cylinder shaped. The cross-sectional area increases with vertical distance from the bottom of the well.
- meniscus shape
- This is affected by the components of the solution.
- In our lab, you can only guess the path length
Path Length Approximations
- Betsy Skorvan used 0.51cm for full-area plates containing 200uL of solution.
- This handy website gives approximate path lengths for several plate types with varying volumes:
PathCheck sounds cool, but is limited
- Our Molecular Devices brand SpectraMax190 plate reader has a feature called PathCheck (see manual), that appears to calculate the path length based on near-infrared wavelengths that few other compounds absorb at. However, there are two kinds of plate readers that use PathCheck. The fancy ones have a spot for a 1cm cuvette to be placed, and the less fancy ones (including ours) do not. With our machine, you can only calculate the path length when it is identical to the path length of a reference well. That means you can't calculate differences in path lengths across a plate due to pipetting variability. In order for the software to calculate differences in path lengths, it would have to know the absorbance property of a reference solution with a known path length. And one can't know the path length without a cuvette!
- I believe you should be able to calculate differences in path length if you have a fancier model that has the option to use a reference cuvette.
- PathCheck applied to measurement of protein solutions in the SPECTRAmax® PLUS microplate spectrophotometer
- UV measurements in microplates suitable for high-throughput protein determination: a paper that uses a SpectraMax Plus reader to do high throughput protein concentration analysis.