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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..

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what is your sample and how is it stored ? Is it DNA dissolved in TE/water or cell extracts ? –  WYSIWYG Jun 3 '13 at 9:04
    
also.. please describe your protocol so that the problem can be understood better –  WYSIWYG Jun 3 '13 at 9:38
    
this question would be much more suited to Chemistry.SE –  MattDMo Jun 3 '13 at 14:50
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@MattDMo UV spectroscopy is a very common technique in the biolab, so this is certainly on-topic here. Migration is rather disruptive, so we try to avoid it for questions that are not actually off-topic on the source site. –  Mad Scientist Jun 3 '13 at 15:52
    
Perhaps you could help us by explaining the basis of the assay? Am I correct in thinking that UV promotes OH radical formation? And that hydrogen-bonded OH radical absorbs at 230 nm? So is the UV beam from the spectrophotometer both promoting radical formation and also allowing the measurement of how much there is of it? And finally, do you get the precipitate if you leave out the peroxide? –  Alan Boyd Jun 3 '13 at 16:29

1 Answer 1

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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Thank you for response. 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.. –  venkata krishna Jun 3 '13 at 13:46

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