1. Introduction to Spectrophotometry & Beer’s Law
AP Chemistry
Mrs. Weston

2. What is spectrophotometry?
A type of spectroscopy that studies the transmission or reflection of different frequencies of the electromagnetic spectrum (specifically visible, near-IR and near-UV ranges) by a sample of matter (usually a soln)

3. Other types of spectroscopy
Atomic absorption spectroscopy
Atomic emission spectroscopy
Mass spectroscopy
Nuclear magnetic resonance (NMR)
Fourier transform infrared (FTIR) spectroscopy

4. Spectrophotometer
Measures absorbance or transmittance of light as a function of wavelength
Sample is placed into cuvette
Light of selected wavelength is passed through sample
Amount of light absorbed is measured

5. Because other compounds in a solution (or the solvent itself) may absorb the same wavelengths as the compound being analyzed, the absorbance of the sample is compared to a reference blank.
Ideally, the reference blank should contain everything found in the sample solution except the substance being analyzed.
When using a Spec 20, every time the wavelength is changed, the instrument needs to be re-zeroed.

6. How it works
Transmission = I/Io
Absorbance = log T

7. How to choose wavelength
We want to analyze samples using the wavelength at which the most light is absorbed – Wavelength of maximum absorption (λmax)

8. Beer’s Law
Amount of light absorbed is proportional to the concentration of the solution
A = abc
A = absorbance
a = proportionality constant
(ε = molar absorptivity)
b = path length (same for entire experiment)
c = concentration (M)

9. What does this tell us?
There is a direct relationship between absorbance and concentration.
When we prepare solutions of known concentration and analyze them at λmax, we can plot absorbance as a function of concentration.

10. The concentration of the unknown can be determined by finding its absorbance and plugging it into the equation for the best fit line.
y = mx + b

11. Uses of spectrophotometry
Determining concentration of any colored solution (Fe, Cu, Co, Ni, MnO4, etc)
Biology
Biochemistry
Forensic science