spectroscopy 2013.pptx

8/10/2019 Spectroscopy 2013.pptx

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FBMS523: Molecular Spectroscopy.

Spectrophotometers.

Absorption Spectrophotometry

Spectrophotometer Components

Radiation Sources

UV Visible

Wavelength Selection

Filters

Monochromators

Optical pathway

Detectors

Single / Dual beam instruments

Spectrophotometer Variants


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Absorption Spectrophotometry...

Definition: A measure of the attenuation of a

test material of a spectrally defined

incident radiation.

Beer-Lambert Law: A = e c l

A = Absorbance, e= molar absorptivity

c = concentration, l = path length

Wavelength of incident radiation is generally in

the range 190-750 nm.

190 - 380 nm Ultraviolet

380 - 750 nm Visible Light

>750 nm Infrared


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The Electromagnetic Spectrum...


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Spectrophotometer Components...

Radiation

Source

Wavelength

Selector

Sample

cuvetteDetector

Signal

processorand output


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Radiation Sources...


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Radiation Sources UV...

Hydrogen / Deuterium Lamp

UV source, range = 340nm down.

Low voltage arc lamps

Arc causes electrical excitation of hydrogen /

deuterium at low pressure

emit a continuous UV spectrum in the region of

160nm to 340nm.

Deuterium lamps are most common

Deuterium lamps give a generally more intense

radiation than hydrogen.


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Radiation Sources Visible...

Tungsten / Tungsten-Halogen Lamp Visible source, range = 350 (320) 760 nm.

Tungsten lamps present in older machines / cheap

visible only spectrophotometers. Output wavelength

is limited by the absorption of the glass envelopesurrounding this type of lamp to 350nm

Tungsten halogen lamps contain a small quantity of

iodine vapour causes re-deposition of vaporised

tungsten on the filament (prolongs lamp life).Quartzenvelope of these lamps permits the source to be

used at lower wavelengths - 320nm.


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Wavelength Selection... Filters

Glass/Gelatine Cheap

Relatively poor bandwidth - approx. 50nm

Visible region only

Interference Moderate cost

Reasonable bandwidth - approx. 10nm

UV, Visible and IR.

Interference Wedges Monochromators

Expensive

Good bandwidth - approx. 1nm

UV,Visible and IR


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Grating and Prism monochromators...


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Monochromator Dispersion...


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Grating Monochromators...


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Sample Holder...

Square cuvettes, 1cm pathlength.

Different internal volumes available.

Available in different materials

Glass - visible light only.

Plastic - visible light only, disposable.

Silica - UV and visible.

Flowcells

permanent holders in the spectrophotometer

samples are drawn in sequentially by a pump.


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Detectors...


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Detectors...

Photovoltaic cells visible range only, max sensitivity @ 550 nm

cheap

amplification problems

Phototubes / Photomultiplier tubes wide response range available

extremely sensitive (photomultipliers)

Photoconductivity transducers

wide response range available

especially good for IR

Silicon photodiodes / Photodiode arrays

full UV/Vis response

rapid scanning spectrophotometers


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Phototubes...


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Photomultiplier tube...


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Photodiode Array Spectrophotometers...


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Single/Dual beam spectrophotometers...


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Single / Dual Beam Instruments...

Single Beam

Compact

Simple

cheap alternative for single wavelength readings

Dual Beam Larger instruments

complex

more expensive

Simultaneous blanking at all wavelengths Two Variants

Dual beam in space

Dual beam in time (more common)


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Variations on a theme...

Turbidimetry

particle counting, best with blue light (420 - 450 nm)

Nephelometry

measures reflected light, detectors at 90 to incident light

laser source, more accurate than turbidimetry

Fluorimetry

excite molecules at one wavelength, detect at another

emmission wavelength usually longer than excitation

measures emitted light at 90 to incident light very specific, individual excitation and emision wavelengths

for different substances.

very sensitive, good for quantitative work

spectroscopy 2013.pptx

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