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Practical Absorbance and Fluorescence Spectroscopy Chapter 2 Wavelengths UV Visible Near IR 10 β 400 nm 400 β 700 nm 700 β 3000 nm When electronic bands are at high energy, the choromphore can absorb in the UV but not appear coloured. Absorption and Fluorescence Absorption A single electron being promoted to a higher energy orbital on absorption of a photon. Fluorescence Absorption whereby the energy is lost by emitting a photon rather than through heat. Basic Layout of a dual-beam UVvisible absorption spectrometer Rotating Wheel Sample Monochromator Lamp Detector Mirror Reference Absorbance and BeerLambert Law π΄ = πππ10 πΌ0 = Ξ΅ππ πΌ Extinction Coefficients & Transition Types Ξ ο Ξ * > 104 CT 103 β 105 dο d 10 β 500 orbital angular momentum forbidden dο d < 10 also spin forbidden Basic Layout of a Fluorimeter PMT Sample Monochromator Lamp Spectrum of Emission Monochromator Excitation spectrum should look like absorption Excitation PMT Emission Radiation Sources Morgan. T. 2014 Summary of Lamps, www.che-revision.weebly.com Wavelength Selection Absorption Filters Combine to select narrow bands of frequencies Interference Filters Relies on optical interference Monochromators Do you know the different types of dispersive elements? Morgan. T. 2014 Summary of Mountings, www.che-revision.weebly.com Slits (giggedy) Slits Controls luminous flux from monochromator Also controls spectral bandwidth Spectral Bandwidth Monochromator cannot isolate a single wavelength. A definite band is passed. Long narrow slit with adjustable width allowing selection of bandwidth. Monochromator Performance ο Resolution ο Distinguish adjacent features depends on dispersion ο Purity ο Amount of stray or scattered radiation ο Light ο Gathering Power Improved by power of source, but compromised by narrower slit to maintain resolution Monochromator Performance π πππ‘ π€πππ‘β β πππππ€πππ‘β β ππ’π‘ππ’π‘ πππ‘πππ ππ‘π¦ Houston β we have a problem! Large bandwidth bad Low output intensity also bad Fight for the two! Also small slit width decreases S/N ratio Dispersion Spread of wavelengths in space D-1 : Linear reciprocal dispersion, defined as the range of wavelengths over a unit of distance πΞ» β1 π· = ππ₯ Lower value = better dispersion dx ~ fdΞΈ (f = focal length) π·=π πΞΈ πΞ» Resolution Resolving Power β distinguish separate entities etc β¦ π = Ξ» πΞ» where Ξ» = average wavelength π β π€ β1 πΞΈ πΞ» where w-1 is effective slit width π/ππππππ ππ = π€βπππ π: πππππ πππππ‘β, π: ππππππ‘ππ ππ ππππππππ‘ππ ππππππ ππ Small f/number = greater radiation gathering power Detectors Transducers that converts electromagnetic radiation into electron flow Uses Photoelectric Effect E = hv β w (w = work function) Need to know the different types of detectors Fluorescence in Detail Excited electronic state Fluorescence only occur from v = 0 state of S1 to any sub-level of S0 Ground electronic state Fluorescence in Detail Fluorescence emission photons have lower energy than excitation. πΌπ = Ξ¦π πΌ0 π₯ 2.303Ξ΅ππ Implies that fluorescence intensity proportional to I0. True; but in practise there is a limit! Only true for low concentrations. Inner Filter Effect Results to NonLinearity Fluorescence reduces at high concentrations For both emission and excitation Fluorescence Lifetimes Typical lifetime around 1 β 10 ns πΌπ‘ = πΌ0 π β π‘ Οπ Where Οf is fluorescence emission litetime Fluorescence Quantum Yields Ξ¦f = fluorescence quantum yield Fraction of excited state molecules that decay back to ground state via fluorescence photons Between 0 β 1 Polar environments reduce Ξ¦f Ξ¦f also very dependent on ionisation (switch from fluo to non-fluo etcβ¦) Quenching Cuvettes EDC Quartz Optical Glass ES Quartz IR Quartz 200 β 2800 nm 300 β 2600 nm 190 β 2000 nm 300 β 3500 nm Therefore for UV <300 nm, need quartz not glass. Plastic can be used in visible (polystyrene is fluorescent; PMMA βpoly(metyl methacrylate)β used instead) Forster Resonance Energy Transfer Fluorescence Polarisation