How to Make a Molar Absorptivity Spectrum

Molar absorptivity spectra are especially useful in biochemistry.

An absorption spectrum is a little like a fingerprint: it's unique to a given compound. Consequently, you can often identify compounds based on their absorption spectra and figure out the concentration, or the amount of the compound present. The tool you need for this measurement is called a spectrophotometer. Many spectrophotometers are designed to either measure absorbance at a specific wavelength, or to collect absorption data across a spectrum of different wavelengths.

Things You'll Need

• Goggles, gloves, eye protection
• 0.1 molar solution of pure compound
• Micropipette
• Test tube or microfuge tube
• 2 clean cuvettes
• Deionized water

Instructions

1. Take the 0.1 molar solution of your compound and add 200 microliters of it to a test tube or microfuge tube.

2. Change the pipette tip and add 1.8 mL of deionized water to your test tube or microfuge tube. Stir with the pipette tip to mix.

3. Change the pipette tip and transfer 1 mL of the solution you've made to a cuvette. Fill another cuvette with 1 mL of deionized water.

4. Insert the blank cuvette, with the deionized water in it, into the spectrophotometer. Follow the manufacturer's instructions to calibrate or "blank" the machine.

5. Insert the solution-containing cuvette into the spectrophotometer. Follow the manufacturer's instructions to collect the spectrum. (Instructions may vary depending on the model you have in your lab.)

6. Repeat the procedure using a different concentration of the sample if necessary. For example; if you want a 0.05 molar solution of your compound, combine 100 microliters of the solution with 1.9 mL of deionized water.

Tips & Warnings

• Always wear safety gear when working in a lab.

References

• Xavier University of Louisiana: Spectrophotometry and the Analysis of Riboflavin
• "Biochemical Techniques, Laboratory Manual"; Aaron Coleman, et al.; 2010
• "Techniques in Organic Chemistry"; Jerry R. Mohrig, et al.; 2010
• CalPoly ChemWeb: Absorption Spectra of Conjugated Dyes