1. Spectrophotometry
A method to determine concentration of a species exploiting the absorption of EMR.

2. Five components of a spectrophotometer:
a stable source of radiant energy
a wavelength selector (“monochromator”)
one or more sample containers (cuvette)
a radiation detector, which converts radiant energy to a measurable electrical signal
a signal processing and readout unit usually consisting of electronic hardware and a computer

3. Components of a Spectrophotometer

4. Beer’s Law
the attenuation of light by an absorbing species is directly proportional to the concentration of the species in the solution, and to the path length through which the absorbance is being measured.

5. Beer’s Law
Light (or other EMR) is passed through the sample which is in a cuvette. The amount of light that passes through the solution is detected on the other side of the solution (= “transmittance”). The higher the concentration of the solute, the lower the transmittance.
Transmittance  Absorbance

6. Beer’s Law A = abc where A = absorbance
a = the molar absorption coefficient (also called the molar absorptivity)
b = path length (in cm)
c = concentration of the light-
absorbing species (moles/liter)

7. Absorbance
the logarithm of the ratio between the initial power of a beam of radiation Po and its power after it has traversed an absorbing medium P,
A = log (Po/P)
where P = intensity of light transmitted through the sample solution
Po = intensity of light transmitted
through an appropriate blank
T = transmittance = P/Po (%T = 100 T)

8. Application of Beer’s Law
Construct a calibration curve of absorbance vs. concentration, using standards of known concentrations. From this data a linear regression equation can be calculated.
The accuracy of solution preparation and dilution can also be determined by analyzing the line of best fit or regression equation.
Friday’s lab