How to troubleshoot absorbance analysis when readings fluctuate

Question

I am trying to measure creatinine in fluids by using an enzymatic (creatinine) test. I try to determine the concentration by the difference in absorbance before and after 5 minutes after a reagent is added. Have a look:

diazyme test

My problem is that the measurements ‘jump’ all over the place, but seem centered at the right value. My absorbance is on the order of 0.0010 (the photometer provides 4 decimal places resolution) for my sample material for a concentration of say 60 umol/l. That means a change in the least significant digit in the absorbance gives an error of minimum 10%. I think this could be causing my fluctuating results, because simply rotating the cuvette changes the absorbance by at least 0.0001. I also see that the temperature in the room change 2 degrees during the day. Perhaps it could influence the speed of the reaction.

What is the right way to troubleshoot a situation like this to find out why my measurements are unstable?

Answer 1

Instead of thinking about the instrumental (spectrophotometer) problems, re-think your experiment. You are testing the instrument at its typical performance limit. The absorbance of the method is too low! Let us convert the absorbance 0.0010 to percent transmittance. It is 99.77%

What does that mean physically? You are "requesting" the instrument to differentiate between 100 % transmittance and 99.77% transmittance. No, this difference will be buried in noise (and hence the fluctuations).

The only solution to your analytical problem is to re-make the samples in such a way that the absorbance >0.1 and it should not exceed 1.5.

In short, prepare a 100-fold more concentrated sample. All these suggestion assume that you are not making any mistake in solution or reagent preparation. Re-check your solution preparation as a sanity check.