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SPECTROPHOTOMETRY BASIC BASIC PRINCIPLES OF SPECTROPHOTOMETRY 1 Principles of Light Measurement Properties of light and energy (Photons) Wavelength Amplitude (intensity, brightness) Frequency (waves / second) Relationships: E = hf; f = c/! 2 3 1 BASIC SPECTROPHOTOMETRY 4 Energies of atoms and molecules 5 6 2 BASIC SPECTROPHOTOMETRY 7 8 Transmission/Absorption of light Reflection = bouncing Refraction = bending Diffusion = spreading Diffraction = combination of all three wavelength isolation filters Prisms -- non-linear Diffraction gratings -- linear 9 3 BASIC SPECTROPHOTOMETRY 10 11 12 4 BASIC SPECTROPHOTOMETRY 13 Absorption / Emission of light Atoms Molecules Solids 14 15 5 BASIC SPECTROPHOTOMETRY Laws of Absorbance Beer / Lambert Laws Absorbance vs. %Transmittance Formulas 16 17 18 6 BASIC SPECTROPHOTOMETRY 19 20 21 7 BASIC SPECTROPHOTOMETRY 22 23 24 8 BASIC Components SPECTROPHOTOMETRY 25 Spectrophotometer Power supply Light Source -- halogen quartz Collimating lenses -- organize parallel lines Focusing lenses -- narrow or intensify beam Monochromator Band pass Stray light = band width at band height (room light, scratches, dust, fingerprints, wide bandpass) Slit width -- exit slit 26 Sample cell -- quartz or sliica Photodetector Photomultiplier tube: Read-out device -- cathode, dynodes, anode digital, recorder, printer 27 9 BASIC SPECTROPHOTOMETRY 28 29 30 10 BASIC SPECTROPHOTOMETRY 31 32 33 11 BASIC SPECTROPHOTOMETRY 34 35 36 12 BASIC SPECTROPHOTOMETRY 37 Analytical Problems Absorbance Variation Check photometric accuracy with Neutral Density filters Noise and Drift Recorder or oscilloscope Stray Light Check with sharp cut-off filters Linearity Dye solutions Wavelength Filters: holmium oxide or didymium 38 13 ... View Full Document

Basic Principles of Spectrophotometry

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