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Absorbance reading does not match spectra - (Sep/06/2010 )

Hi All

I am using a new substrate so after my assay I ran a spectra (200-800nm) to determine the optimal wavelengh. The spectra looked very good with the peak at 630nm close on 2 absorbance units. I then read the wells at a single wavelengh of 630nm but the readings were in the region of 0.4 Au. Does anyone know why this occurs? And what can I do about it?

Just to add, I read at various wavelengths after that (i.e. 600, 610, 620, 640, 650, 660, 670, 680, 690, 700) as I received the same results - all reading in the range of 0.4 Au.

Any advice on this will be gratefully accepted.

Many thanks,


You have to give more details, what you're writing looks impossible.
The readings from your sample looks as the didn't react, a other possibility is that the reactionproduct breaks down. I,m just guessing by lack of information.


The path length is different between the spectrophotometer and your platereader.