I have a newly synthesized fluorescent dye, so before I can use it in my experiments I am supposed to calulaute the molar extinction coefficient exaclty. Otherwise I will not be able to calculate the exact concentration. Does anyone have an idea of doing that. I know I have to prepare a set of concentrations and within the certain limitations of Beer-Lambert law, but first of all how can prepare the stock solution of the accurate concentration that I can start with to prepare other solutions by sequential dilution and measure the absorbance. I hope that I clarified the issue to you. Thanks in advance.
Something to consider is the solvent used for the absorbance measurement. The extinction coefficient will depend on the solvent. If the solvent is water, the pH may also affect the extinction coefficient. So make sure to state the solvent when reporting the extinction coefficient. Also, make sure not to let the absorbance exceed the linear range of the spectrophotometer.
Dear Adam, thanks for your answer. Actually, I use toluene for some of them and chloroform for the others. And I totally agree with you, the solvent does an effect. However, I just need to know how to start preparing the stock soln of an exact concentration based on the information provided (molecular weight, and exact mass) and then it will be easier to me to make the appropriate dilution bearing in mind the deviations from Beer's law.
I would typically start by making a 10 mM stock solution, and dilute that. For 1 ml of a 10 mM solution, weigh out in mg 1/100 of the formula weight (includes the mass of the parent molecule + any salt ions and water). So if the formula weight is 500, weigh out 5.0 mg and dissolve it in solvent to a volume of 1 ml. Then prepare dilutions from that.
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