The use of a spectrometer and the principles of beers

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spectrophotometry. The use of a spectrometer and the principles of Beer’s Law, allow one to plot a graph of Absorbance vs. concentration of various colored solutions and obtain calibration curves (also known as standard or working curves or Beer’s Law Plots). It is simple matter to then use these curves and determine unknown concentrations of these solutions. Beer’s Law states that the  absorbance (A) of a solution is dependent on three factors: (1) the  molar absorptivity (a) , the value of which depends on the absorbing species and on the wavelength used, (2) the path length (b) of the solution through which the light must past, and (3) the  concentration (c) of the solution. A = abc What is the absorbance? To define it, we must first need to define the transmittance (t) which is simply the ratio of the intensity of the light transmitted by a sample (I) to the intensity of the light incident on the sample (Io):
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T = I/I o %T = I/I o x 100% If  all the light is absorbed by the sample, %T = 0; if  no light is absorbed the %T = 100%. The absorbance is then defined by the equation: A = log 10(1/T) = log 10 (I o /I) We can see, therefore, that when T = 0.10 (%T = 10%), A = 1.0, and that when T = 100 (%T = 100%) that A= 0.00. The most  accurate results are when the absorbance readings are in the range of A = 0.10 to 1.0 (or %T = 80% to 10%). This experiment makes use of a single colored species at a fixed wavelength and all of the cuvettes have the same path length; therefore, a and b are constant. Thus, according to Beer’s Law, a plot of the absorbance versus concentration should be linear with a y-intercept of zero. Of course, small deviations from this are to be expected. That is why knowing the R or R2 value is helpful…it indicates how closely the substance adheres (follows) Beer’s Law.
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