Absorbance can also be calculated using the ratio between the intensity of a reference sample and the unknown sample. It is given by the equation A = log10(Io/I). [4] X Research source Intensity is obtained using a spectrophotometer. The absorbance of a solution will change based on the wavelength that is passed through the solution. Some wavelengths will be absorbed more than others depending upon the makeup of the solution. Remember to state which wavelength is being used for your calculation. [5] X Research source
Absorbance between readings can vary due to the concentration of the solution and the shape of the container used to measure intensity. Molar absorptivity compensates for these variations. [7] X Research source
Prepare a solution of known concentration, c, for analysis. Units for concentration are molar or moles/liter. [8] X Research source To find l, measure the length of the cuvette, the piece that holds the liquid samples in the spectrophotometer. Units for path length are measured in centimeters. Using a spectrophotometer, obtain a measurement for absorbance, A, at a given wavelength. The unit for wavelength is meters, but most wavelengths are so small, they are actually measured in nanometers (nm). [9] X Research source Absorbance has no units.
For example: Using a cuvette with a length of 1 cm, you measured the absorbance of a solution with a concentration of 0. 05 mol/L. The absorbance at a wavelength of 280 nm was 1. 5. What is the molar absorptivity of this solution? ɛ280 = A/lc = 1. 5/(1 x 0. 05) = 30 L mol-1 cm-1
Draw a line between each of the points. If the measurements are correct, the points should form a straight line indicating absorbance and concentration are proportional to Beer’s Law. [11] X Research source
The equation for the slope of a line is (Y2 - Y1)/(X2 - X1). The point higher on the line is given the subscript 2, while the lower point is given the subscript 1. For example: The absorbance at a . 2 molar concentration is 0. 27 and at 0. 3 molar is 0. 41. The absorbance values are Y-values, while concentrations are X-values. Using the equation for a line (Y2 - Y1)/(X2 - X1) = (0. 41-0. 27)/(0. 3-0. 2) = 0. 14/0. 1 = 1. 4 is the slope of the line.
Continuing our example: If 1. 4 is the slope of the line and the path length is 0. 5 cm, then the molar absorptivity is 1. 4/0. 5 = 2. 8 L mol-1 cm-1.
