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FBMS523

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Molecular Spectroscopy.
Spectrophotometers.

• Absorption Spectrophotometry
• Spectrophotometer Components
• Radiation Sources
– UV
– Visible

• Wavelength Selection
– Filters
– Monochromators




Optical pathway
Detectors
Single / Dual beam instruments
Spectrophotometer Variants

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

..The Electromagnetic Spectrum..

.Spectrophotometer Components. Radiation Source Wavelength Selector Sample cuvette Detector Signal processor and output ..

. .Radiation Sources..

Radiation Sources UV. range = 340nm down. . • Deuterium lamps are most common • Deuterium lamps give a generally more intense radiation than hydrogen.. • • • • Hydrogen / Deuterium Lamp UV source. 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..

• Tungsten / Tungsten-Halogen Lamp • Visible source. Output wavelength is limited by the absorption of the glass envelope surrounding 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).. range = 350 (320) – 760 nm.Radiation Sources Visible..Quartz envelope of these lamps permits the source to be used at lower wavelengths . • Tungsten lamps present in older machines / cheap visible only spectrophotometers.320nm. .

. 50nm • Visible region only – Interference • Moderate cost • Reasonable bandwidth .approx. Visible and IR. 10nm • UV. • Interference Wedges • Monochromators • Expensive • Good bandwidth .approx. • Filters – Glass/Gelatine • Cheap • Relatively poor bandwidth . 1nm • UV.Wavelength Selection.Visible and IR .approx..

.. .Grating and Prism monochromators.

. ..Monochromator Dispersion.

..Grating Monochromators..

• Available in different materials – Glass .. – Plastic .visible light only.UV and visible. 1cm pathlength. . disposable. • Different internal volumes available.visible light only. • Flowcells – permanent holders in the spectrophotometer – samples are drawn in sequentially by a pump.Sample Holder.. • Square cuvettes. – Silica .

..Detectors. .

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 ..

.Phototubes.. .

Photomultiplier tube... .

. ..Photodiode Array Spectrophotometers.

.Single/Dual beam spectrophotometers.. .

..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) .

– very sensitive. detectors at 90º to incident light – laser source.450 nm) • Nephelometry – measures reflected light. best with blue light (420 . good for quantitative work . detect at another emmission wavelength usually longer than excitation measures emitted light at 90º to incident light very specific.. individual excitation and emision wavelengths for different substances. • Turbidimetry – particle counting.Variations on a theme. more accurate than turbidimetry • Fluorimetry – – – – excite molecules at one wavelength..