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Things You'll Need
Instructions
Turn on the spectrometer and zero it by putting distilled water in a cuvet and running it as a sample. Different spectrometers will have slightly different software or means of operation, but each should come with clear instructions for how to calibrate with a blank sample of distilled water.
Fill a cuvet with the first sample of known concentration. Run the sample to determine the absorbance value. Record the sample number, the already-known concentration and the absorbance reading. For example, Sample 1 might have a concentration of 0.05 moles per liter and an absorbance of 400.
Fill a fresh cuvet with the next sample and run it through the spectrometer. Record the sample number, concentration, and absorbance. For example, Sample 2 might have a concentration of 0.5 moles per liter and an absorbance of 4,000. Do the same for the third sample. For this example, assume that Sample 3 has a concentration of 5 moles per liter and an absorbance of 40,000.
Use the Beer-Lambert Law to calculate the molar absorptivity coefficient for each sample. This equation states that absorbance is equal to the concentration of the solution multiplied by the path length (the width of the cuvet) multiplied by the molar absorptivity coefficient: A = ebc, where e is the molar absorptivity coefficient, b is the path length, c is the concentration and A is the absorbance. Multiply the path length in centimeters by the concentration, then divide the absorbance by that number. For this example, assume that the cuvet is 1 centimeter wide. For Sample 1, multiply 1 by 0.05, which equals 0.05. Divide 400 by 0.05 for a molar absorptivity coefficient of 8,000.
Perform the same calculation for the other samples. For Sample 2, multiply 1 by 0.5 for a result of 0.5. Divide 4,000 by 0.5 to yield a molar absorptivity constant of 8,000. The same calculation for Sample 3 also yields a result of 8,000. In real laboratory conditions, each sample will produce a slightly different constant. Record each one.
Average all the calculated molar absorptivity constants by adding them together and dividing the total by the number of known samples you ran. You can now use the resulting molar absorptivity coefficient to estimate the concentration of unknown samples.