# Introduction to Spectrophotometry & Beer’s Law

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

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)

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

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

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.

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

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

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)

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.

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

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