I have been checking my sample for Hydroxyl radical scavenging activity. I have

Question

I have been checking my sample for Hydroxyl radical scavenging activity. I have dissolved Hydrogen peroxide in Phosphate buffer. My sample is clear and reagent is also clear but when I have mixed my sample extract with Hydrogen peroxide reagent a precipitant is formed. Control is very less than solution that is kept for reaction to proceed. So I am getting an answer in negatives (230 nm). Is that formation of precipitate affect the absorbance of the solution? Does precipitation increase the OD value compare to clear solution? Mine is mushroom powder. It is extracted with Ethanol. Extract is dried and finally it is dissolved with solvent above said. That has been used for Hydroxyl radical scavenging activity. Hydrogen peroxide 0.01 milli molar reagent is prepared with Phosphate buffer. First i have taken reading for H2O2 and added extract kept for ten mins and took reading. in 230 nm..

Explanation / Answer

The answer to the question as asked is that yes, any precipitate will affect the value that you are reading in your spectrophotometer. The basis of spectrophotometry is that light passes through a sample, and less light emerges than the amount which entered. The amount of light that has "disappeared" is used to deduce something about the solution in the cuvette. Light can be absorbed by chemical groups (this is the basis for e.g. DNA having an absorbance value) or it can be scattered by particulate material (this is the basis for measuring the cell density of a bacterial culture). If you are trying to make a measurement of the first type (absorbance) and you get a precipitate your reading will be distorted by the light scattering effect.

Precipitates are incompatible with spectrophotometry.

As the comments have noted: if you give more details of your protocol someone may be able to help. Phosphates are, of course, notorious for generating unwanted precipitates.

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