I am in the lab and trying to perform the molybdenum blue test for phosphorus using a photometer. It appears common speak in colorimetric lab sheets that
Absorbance of a mix of two concentrations is the sum of the absorbances of the individual concentrations
This is often exemplified by statements such that: “You can zero your photometer on the reagent and subtract the absorbance of the sample to just measure the absorbance due to the reaction”. I tried to test if this logic is correct, but I think it is false and performed the following experiment by preparing 4 curettes to find out:
- I zeroed my instrument on a cuvette with distilled water.
- I prepared 1ml of the molybdenum reagent
- I prepared 1ml of the molybdenum reagent to which I add 10 ul phosphorus sample
- I prepared 1ml of distilled water to which I add 10 ul of phosphorus sample.
I measured the absorbance of the four cuvettes within about 1350 milliseconds of mixing and they were 0, 0.172, 0.072, 0.402. I measured again after 10 seconds and got similar absorbances.
As 0.172 + 0.072 = 0.244 and not 0.402, I conclude that the logic cited above is false.
QUESTION: Am I correct in jumping to this conclusion? What is the reason that the mixed substance does not have the absorbance of the sum of the individual concentrations?