Presentation on theme: "Introduction to Spectrophotometry & Beer’s Law"— Presentation transcript:
1 Introduction to Spectrophotometry & Beer’s Law AP ChemistryMrs. 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 spectroscopyAtomic emission spectroscopyMass spectroscopyNuclear magnetic resonance (NMR)Fourier transform infrared (FTIR) spectroscopy
4 SpectrophotometerMeasures absorbance or transmittance of light as a function of wavelengthSample is placed into cuvetteLight of selected wavelength is passed through sampleAmount 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 worksTransmission = I/IoAbsorbance = 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 LawAmount of light absorbed is proportional to the concentration of the solutionA = abcA = absorbancea = 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)BiologyBiochemistryForensic science