Spectrophotometry is a common lab technique for determining solute concentration in a solution. A spectrophotometer uses a prism or diffraction grating to select light of a desired wavelength and pass it through the test sample. A detector on the other side measures the intensity of light striking it. The measurement gives the per cent of transmittance and absorbance when the instrument is properly calibrated. After you've measured the absorbance, finding concentration is a straightforward calculation that relies on a formula called Beer's Law.
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Things you need
- Graphing program such as Excel
Write down the following equation: Absorbance = (absorptivity) * (length) * (concentration). This formula is called Beer's Law. You know the absorbance from your experiment, while the length component is the distance through the "cuvette" -- the container used to hold the sample in the spectrophotometer. For most cuvettes the distance is 1cm, although you should verify it. Finally, molar absorptivity is a constant specific to a given substance, solvent and wavelength. Calculate molar absorptivity using the absorbance of the standard solutions you measured in your experiment.
Enter the absorbance and concentration of at least five standard samples used in your experiment. These standard samples have known concentration, so you can use them to find the molar absorptivity. Skip this step if you are working with a molecule like DNA whose molar absorptivity at 260 nm is already known. For DNA, you can use the calculator under the "Resources" section below.
Use the graphing program to graph your table data with concentration on the x-axis and absorbance on the y-axis. Derive a line of best fit for this data using the program; this equation should be linear. The precise procedure will vary depending on the graphing program you use; consult the user guide for your spreadsheet program or the online help if necessary.
Plug the absorbance value for your unknown into the line-of-best-fit equation from your graph and solve for concentration. Alternately, if you're dealing with a molecule like DNA whose molar absorptivity at a given wavelength is already known, plug the absorbance into the Beer's Law formula you wrote down. Divide absorbance by length and by the molar absorptivity constant. This operation will give you the concentration in moles per litre.
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