We had a blue colored solution whose lambda max should be around 750 nm. At low concentrated solution (100ug/ml), absorbance value upto 0.7, the lambda max was observed around 740 nm.
But in the concentrated solution, absorbance value greater than 1, lambda max shifts to 640 nm and further decreases with increasing absorbance value.
What is the reason behind these shift in lambda max at higher concentration?
We are trying to make calibration curve, and wavelength should me measured at lamda max. But, since the lambda max shifts in higher concentration, we are having problem. Should we not consider absorbance value greater than 1?
what may be best solution here?
Peter, Thank you for your answer.
I didn't actually get your answer(Undergraduate student). The spectrum in question is in Visible range. (maximum wavelength at around 750 nm)
I would like to know if the shift observed from 740 nm to 640 nm in increasing the concentration of analyte is normal or abnormal behavior, and should we consider absorbance reading above 1?
We are using Biotec Epcoh gen3 mulitplate reader.
The reaction we carried out is FC method for the colorimetric detection of Phenol, which gives blue colored solution.
Does lambda max depends upon concentration???
We get different lambda max for same analyte at different concentration (from 10- 100 ug/ml). Please find the attached figure for more information.
At first: How did You do blank correction?
To be sure, that Your observation is not an artefact you may use a Spectrometer instead of a platereader, just once to see how you dye behaves.
Question remain about the used plates and potentially other solutions in adjacent wells. Even if You are not looking at fluorescence, it is not impossible that is a Kind of crosstalk.
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