Question

A technologist is performing a colorimetric assay using 96?well ELISA microtiter plates.   The ELISA plate reader measures the absorbance of the solution in each well (cuvet) by recording the light passing through the solution from the top to the bottom of each well.  The assay’s color, when tested in a standard spectrophotometer, is linear to an absorbance of 4.0 however the ELISA reader has a range of linearity which only extends to an absorbance of 1.2.  When a microtiter plate is placed in the ELISA reader, the technologist notes that the solution in several wells has an approximate absorbance of 1.5.  The assay material is very expensive and each assay takes one day to perform.   Considerable effort is made to get as much data as possible from each run.  Therefore, in order to measure the absorbance of the solution in these wells, the technologist dilutes the samples by adding a volume of buffer equal to the volume in the well.  To her surprise, the absorbance values remain the same.

1. Considering Beer’s law, why didn’t the absorbance value change?

2. Are there any steps that can be taken to bring the absorbance within the linear range?

3.    Calculate the concentration of the compound using the values below. Show all calculations

Dilution 1:2

Abs std 0.45

Abs unk 0.78

Conc std 4 g/dL

Answer 1

1. You have to repeat the analysis this may be an instrument handling errors. You have gone for the dilution of the well concentration this is not good way. You have to dilute the stocks solutions up to 10 times for the good reading. The absorbance value will not same, it will follow the Beer’s law.

1. Most of the nonlinearity results have been found in the high concentration of molecules. This usually happens due to electrostatic interactions between molecules in close proximity.

Further you have to also ensure about the following factors of the sample and molecules. There are some limitations of the Beer-Lambert law which are as follow:

• Sometimes scattering of light happen due to particulates in the sample
• Ensure the fluoresecence or phosphorescence nature of the sample
• If sample has property of changes in refractive index at high concentration of molecules in the sample solution.
• If shifts in chemical equilibria as a function of concentration.

1. For the calculation of concentration of molecules first you have to plot a calibration curve. Then you can get value of unknown concentrations in the sample.