# Spectrophotometry

## Molar Absorptivity

Tartrazine is a yellow colored food dye. If light is passed through a solution of tartrazine, some of the light will be absorbed by the tartrazine molecules. According to Beer's Law, the absorbance of a solution depends upon three factors:

• The concentration ( C ) of tartrazine molecules. The higher the concentration, the more likely a photon is to encounter a tartrazine molecule.
• The cell path length ( L ). The more solution a photon passes through, the more likely the photon is to encounter a tartrazine molecules.
• The intrinsic ability of the tartrazine molecule to absorb a photon.

Once a photon encounters a tartrazine molecule, the photon might or might not be absorbed. The probability the photon is absorbed is characterized by the molar absorptivity ( ε ). In older literature, ε is sometimes called the extinction coefficient. The probability of absorption depends upon the electronic, vibrational and rotational properties of the molecule and upon the energy of the photon. The energy of the photon is often characterized by the wavelength ( λ ) of the light.

Mathematically, Beer's Law may be written as:

A = ε L C

The concentration is a property of the sample solution. The cell path length is a property of the equipment (specifically, the cell) used in the measurement. The molar absorptivity is a property of the molecule (and the solvent in which it is dissolved). Changing the wavelength of the light changes the tendancy of the molecule to absorb light. Thus ε is a function of the wavelength. This means the absorbance of the solution is also a function of the wavelength of the light. Sometimes A and ε are written with a λ subscript to emphasize this connection.

Aλ = ελ L C

Now that Beer's Law has been formulated, let's revisit the spectrophotometric measurements on solutions of tartrazine. Use the virtual experiment to measure the absorbance of a solution of tartrazine. Then use Beer's Law to determine the molar absorptivity of the tartrazine. (Recall that μM represents micromolar. 1 μM = 1 x 10-6 mole/L) This experiment uses only 425 nm light, so you will measure A425 and will then calculate ε425.

Operation of the Spectrophotometer: Select the desired cell path length and tartrazine concentration. Then start the simulation. Once photons begin reaching the detector, start the Data Acquisition. The intensity of light (photons per second) reaching the detector will be displayed. Note that the simulation employs more photons than are shown on the screen.

For a blank (that is, a solution containing no tartrazine), the intensity is I0 = 150. photons/sec . Use the measured intensity and this value for I0 to calculate the transmittance ( T ) and the absorbance ( A ).

T =
I I0

A   =   - log10 T

Questions

1. What is the molar absorptivity of tartrazine at 425 nm?
2. What are the units for the molar absorptivity?

Light Source
Cell
Detector
Concentration

Cell Path Length

Spectrometer

Data Acquisition

photons/sec

MolarAbsorptivity.html version 3.0
© 2000, 2014, 2023 David N. Blauch