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Absorbance reading does not match spectra


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#1 Brightside

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Posted 06 September 2010 - 01:01 AM

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,
B

#2 Gerard

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Posted 06 September 2010 - 11:54 AM

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.
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#3 sgt4boston

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Posted 07 September 2010 - 06:19 AM

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




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