Practical absorbance and fluorescence spectroscopy
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Practical Absorbance and Fluorescence Spectroscopy PowerPoint PPT Presentation


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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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Practical Absorbance and Fluorescence Spectroscopy

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Practical absorbance and fluorescence spectroscopy

Practical Absorbance and Fluorescence Spectroscopy

Chapter 2


Wavelengths

Wavelengths

UV10 – 400 nm

Visible400 – 700 nm

Near IR700 – 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

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

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

Rotating Wheel

Sample

Monochromator

Lamp

Detector

Mirror

Reference


Absorbance and beer lambert law

Absorbance and Beer-Lambert Law

Extinction Coefficients & Transition Types

Π Π*> 104

CT103 – 105

d  d10 – 500 orbital angular momentum forbidden

d d< 10also spin forbidden


Basic layout of a fluorimeter

Basic Layout of a Fluorimeter

PMT

Sample

Monochromator

Excitation

Lamp

Spectrum of Emission

Monochromator

Excitation spectrum should look like absorption

Emission

PMT


Radiation sources

Radiation Sources

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


Wavelength selection

Wavelength Selection

Absorption Filters

Combine to select narrow bands of frequencies

Interference Filters

Relies on optical interference


Monochromators

Monochromators

Do you know the different types of dispersive elements?

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


Slits giggedy

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

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 performance1

Monochromator 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

Dispersion

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

Resolution

Resolving Power – distinguish separate entities etc …

where = average wavelength

where w-1 is effective slit width

Small f/number = greater radiation gathering power


Detectors

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

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 detail1

Fluorescence in Detail

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

Results to Non-Linearity

Fluorescence reduces at high concentrations

For both emission and excitation


Fluorescence lifetimes

Fluorescence Lifetimes

Typical lifetime around 1 – 10 ns

Where τf is fluorescence emission litetime


Fluorescence quantum yields

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…)


Quenching

Stern – Volmer Plot

Quenching


Cuvettes

Cuvettes

EDC Quartz 200 – 2800 nm

Optical Glass300 – 2600 nm

ES Quartz190 – 2000 nm

IR Quartz300 – 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 Resonance Energy Transfer


Fluorescence polarisation

Fluorescence Polarisation


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