Practical absorbance and fluorescence spectroscopy
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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

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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 and Fluorescence


A single electron being promoted to a higher energy orbital on absorption of a photon.


Absorption whereby the energy is lost by emitting a photon rather than through heat.

Basic layout of a dual beam uv visible absorption spectrometer
Basic Layout of a dual-beam UV-visible absorption spectrometer

Rotating Wheel







Absorbance and beer lambert law
Absorbance and Beer-Lambert Law spectrometer

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
Basic Layout of a spectrometerFluorimeter






Spectrum of Emission


Excitation spectrum should look like absorption



Radiation sources
Radiation Sources spectrometer

Morgan. T. 2014 Summary of Lamps,

Wavelength selection
Wavelength Selection spectrometer

Absorption Filters

Combine to select narrow bands of frequencies

Interference Filters

Relies on optical interference

Monochromators spectrometer

Do you know the different types of dispersive elements?

Morgan. T. 2014 Summary of Mountings,

Slits giggedy
Slits ( spectrometergiggedy)


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
Monochromator spectrometer 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 performance1
Monochromator spectrometer Performance

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 spectrometer

Spread of wavelengths in space

D-1 : Linear reciprocal dispersion, defined as the range of wavelengths over a unit of distance

Lower value = better dispersion

dx ~ fdθ (f = focal length)

Resolution spectrometer

Resolving Power – distinguish separate entities etc …

where = average wavelength

where w-1 is effective slit width

Small f/number = greater radiation gathering power

Detectors spectrometer

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,

Fluorescence in detail
Fluorescence in Detail spectrometer

Excited electronic state

Fluorescence only occur from v = 0 state of S1 to any sub-level of S0

Ground electronic state

Fluorescence in detail1
Fluorescence in Detail spectrometer

Fluorescence emission photons have lower energy than excitation.

Implies that fluorescence intensity proportional to I0. True; but in practise there is a limit! Only true for low concentrations.

Inner filter effect
Inner Filter Effect spectrometer

Results to Non-Linearity

Fluorescence reduces at high concentrations

For both emission and excitation

Fluorescence lifetimes
Fluorescence Lifetimes spectrometer

Typical lifetime around 1 – 10 ns

Where τf is fluorescence emission litetime

Fluorescence quantum yields
Fluorescence Quantum Yields spectrometer

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 – spectrometerVolmer Plot


Cuvettes spectrometer

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
Forster spectrometerResonance Energy Transfer