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

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...

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.

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 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).Quartz
envelope of these lamps permits the source to be
used at lower wavelengths - 320nm.

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

Grating and Prism monochromators...

Monochromator Dispersion...

Grating Monochromators...

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.

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)

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