I want to apply spectrophotometer via Beer-Lambert law to determine changes in concentrations of a mixture of coloured organic compounds. How do I go about it?
Take an absorbance spectrum of the mixture in a 1-cm pathlength quartz cuvette, being sure to blank the spectrophotometer with the solvent that the mixture is dissolved in. Dilute the mixture to the point that the maximal absorbance in the spectrum is about 1. Pick one wavelength to measure, preferably the wavelength of a peak in the spectrum. The absorbance of the mixture at that wavelength will be directly proportional to the concentration of the absorbing substances, according to the Beer-Lambert law, as long as the mixture is a true solution, i.e. that it is clear, not turbid at all.
The actual concentration of the absorbing substance can be determined if you measure the extinction coefficient. This is done by measuring the absorbance of several known concentrations, plotting the linear absorbance versus concentration standard curve, and measuring the slope. Since this is a mixture, you can't measure the molarity, only the mg/ml, so the units of the extinction coefficient will be ml mg-1 cm-1.
you make a scan in the field of the visble of each dye to determine its maximum asorption wavelength Lmax1, Lmax2, Lmax3, ....
then you take a solution of your dye1 and you make several dilutions C1, c2, c3, C4 .... that you will measure their intensities I1, I2, I3, I4 .... at this wavelength Lmax
you then trace a calibration curve C = f (I).
you repeat the same steps for each dye.
afterwards, you pass your mixture of dyes in the UV spectrometer to measure the intensities of your wavelengths Lmax1, Lmax2 (second dye), Lmax3 (third dye), and from the calibration curves you can deduce the concentrations of each dye in the mixture.
good luck.
You may use Beer-Lambert Law to calculate, which is A=lg(1/T)=Kbc:
A is the absorbance, T is the transmittance (transmittance), is the outgoing light intensity (I) than the incident light intensity (I0). K is the molar absorption coefficient, which is related to the properties of the absorbing material and the wavelength λ of the incident light. c is the concentration of light absorbing substance, b is the thickness of absorption layer.
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