Practical Absorbance and Fluorescence Spectroscopy Chapter 2

Wavelengths UV 10 – 400 nm Visible 400 – 700 nm Near IR 700 – 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 UV-visible absorption spectrometer Rotating Wheel Sample Monochromator Lamp Detector Mirror Reference

Absorbance and Beer-Lambert Law 𝐴= 𝑙𝑜𝑔 10 𝐼 0 𝐼 =ε𝑐𝑙 Extinction Coefficients & Transition Types Π  Π* > 104 CT 103 – 105 d  d 10 – 500 orbital angular momentum forbidden d  d < 10 also spin forbidden

Basic Layout of a Fluorimeter PMT Sample Monochromator Excitation Lamp Spectrum of Emission Monochromator Excitation spectrum should look like absorption PMT Emission

Morgan. T. 2014 Summary of Lamps, www.che-revision.weebly.com 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

Morgan. T. 2014 Summary of Mountings, www.che-revision.weebly.com 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 Resolution Distinguish adjacent features depends on dispersion Purity Amount of stray or scattered radiation Light Gathering Power Improved by power of source, but compromised by narrower slit to maintain resolution

Monochromator Performance 𝑠𝑙𝑖𝑡 𝑤𝑖𝑑𝑡ℎ ∝𝑏𝑎𝑛𝑑𝑤𝑖𝑑𝑡ℎ ∝𝑜𝑢𝑡𝑝𝑢𝑡 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 Houston – 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 𝐷 −1 = 𝑑λ 𝑑𝑥 Lower value = better dispersion dx ~ fdθ (f = focal length) 𝐷=𝑓 𝑑θ 𝑑λ

Resolution Resolving Power – distinguish separate entities etc … 𝑅= λ 𝑑λ where λ = average wavelength 𝑅∝ 𝑤 −1 𝑑θ 𝑑λ where w-1 is effective slit width 𝑓/𝑚𝑖𝑟𝑟𝑜𝑟= 𝑓 𝑐 𝑑 𝑐 𝑤ℎ𝑒𝑟𝑒 𝑓:𝑓𝑜𝑐𝑎𝑙 𝑙𝑒𝑛𝑔𝑡ℎ, 𝑑:𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 𝑜𝑓 𝑐𝑜𝑙𝑙𝑖𝑚𝑎𝑡𝑜𝑟 𝑚𝑖𝑟𝑟𝑜𝑟 Small f/number = greater radiation gathering power

Morgan. T. 2014 Summary of Mountings, www.che-revision.weebly.com Detectors Transducers that converts electromagnetic radiation into electron flow Uses Photoelectric Effect E = hv – w (w = work function) Need to know the different types of detectors Morgan. T. 2014 Summary of Mountings, www.che-revision.weebly.com

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. 𝐼 𝑓 = Φ 𝑓 𝐼 0 𝑥 2.303ε𝑐𝑙 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 Non-Linearity Fluorescence reduces at high concentrations For both emission and excitation

Fluorescence Lifetimes Typical lifetime around 1 – 10 ns 𝐼 𝑡 = 𝐼 0 𝑒 − 𝑡 τ 𝑓 Where τf is fluorescence emission litetime

Fluorescence Quantum Yields Φf = fluorescence quantum yield Fraction of excited state molecules that decay back to ground state via fluorescence photons Between 0 – 1 Polar environments reduce Φf Φf also very dependent on ionisation (switch from fluo to non-fluo etc…)

Stern – Volmer Plot Quenching

Cuvettes EDC Quartz 200 – 2800 nm Optical Glass 300 – 2600 nm ES Quartz 190 – 2000 nm IR Quartz 300 – 3500 nm Therefore for UV <300 nm, need quartz not glass. Plastic can be used in visible (polystyrene is fluorescent; PMMA ‘poly(metyl methacrylate)’ used instead)

Forster Resonance Energy Transfer

Fluorescence Polarisation