Transcript Calibration and verification of Spectrophotometers Alan

Calibration and verification of
Spectrophotometers
Alan Rielander
INTERCAL
Basic scanning Spectrophotometer
Spectrum
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William Herschel (1738 - 1822) was one of the most important astronomers that ever lived. In 1800 he performed a famous experiment
where he tried to measure the temperature of different colours of the spectrum by placing a thermometer on each colour. He found to
his amazement that the hottest part of the spectrum was in a place where there was no colour at all. It was a spot beyond the red end
of the spectrum. For the first time it was possible to talk about invisible light. This hot light became known as Infrared (below the red)
because it was shown to have longer wavelength than visible light. Apart from its wavelength, Infrared has all the other properties of
light.
After learning about William Herschel's discovery of infrared light, which he found beyond the visible red portion of the spectrum in
1800, Johann Ritter began to conduct experiments to see if he could detect invisible light beyond the violet portion of the spectrum as
well. In 1801, he was experimenting with silver chloride, which turned black when exposed to light. He had heard that blue light caused
a greater reaction in silver chloride than red light did. Ritter decided to measure the rate at which silver chloride reacted to the
different colours of light. He directed sunlight through a glass prism to create a spectrum. He then placed silver chloride in each colour
of the spectrum and found that it showed little change in the red part of the spectrum, but darkened toward the violet end of the
spectrum. Johann Ritter then decided to place silver chloride in the area just beyond the violet end of the spectrum, in a region where
no sunlight was visible. To his amazement, this region showed the most intense reaction of all. This showed for the first time that an
invisible form of light existed beyond the violet end of the visible spectrum. This new type of light, which Ritter called Chemical Rays,
later became known as ultraviolet light or ultraviolet radiation (the word ultra means beyond). [2]
The spectrum covered by UV-VIS spectrophotometers is from 180nm to 1100 nm
The ultraviolet spectrum is from 180nm to 340nm and the visible spectrum is from 340nm to 1000nm with some spectrophotometers
going as far as 1100nm in the near infrared range.
The light sources are provided by a Tungsten-Halogen Lamp for the visible and a Deuterium lamp for the ultraviolet range. The
Deuterium lamp provides Emission lines at 486 and 656.1, useful for instrument wavelength calibration and validation
The lamps provide a broad spectrum output and we need to split the spectrum into individual wavelengths to make measurements at
precise measurements.
Manipulating the light source to our needs
Diffraction of light using a prism
Diffraction grating
The principles of diffraction gratings were discovered by James Gregory, about a
year after Newton's prism experiments, initially with artifacts such as bird
feathers. The first man-made diffraction grating was made around 1785 by
Philadelphia inventor David Rittenhouse, who strung hairs between two finely
threaded screws. This was similar to notable German physicist Joseph von
Fraunhofer's wire diffraction grating in 1821.
• Originally, high-resolution gratings were ruled using
high-quality ruling engines whose construction was a
large undertaking. Henry Joseph Grayson designed a
machine to make diffraction gratings, succeeding with
one of 120,000 lines to the inch (approx.
47 000 per cm) in 1899. Later, photolithographic
techniques allowed gratings to be created from a
holographic interference pattern. Holographic gratings
have sinusoidal grooves and may not be as efficient as
ruled gratings, but are often preferred in
monochromators because they lead to much less stray
light. A copying technique allows high quality replicas
to be made from master gratings of either type,
thereby lowering fabrication costs.
Filter wheel
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Gratings produce second order spectrum which can interfere with our sample as they are not
at the same wavelength we want to use. A filter wheel is positioned behind the slit to block the
unwanted wavelengths of light getting through. These are order sorting filters and the filter
wheel I controlled so that the relevant filter is in place for the portion of the spectrum we are
working with.
Calibration
• Wavelength calibration
Spectrums of Holmium, Didymium and Samarium
Photometric accuracy in Visible
• In 1970 the American National Bureau of Standards initiated the search
for a standard using the following criteria. We want to use materials that
are transparent in the range of interest usually between 200 and 800 nm,
have a transmittance independent of wavelength(optically neutral), have
a spectral transmittance independent of temperature, have low
reflectance and free of interferences, be non fluorescent, be stable
homogeneous and be free of strain, have mechanical stability for the size
used, be easy to fabricate by conventional techniques used in optical
shops, be simple to use in conventional spectrometers and be readily
available and relatively inexpensive.[5]
• Originally Corning 8364, Chance ON-10 and SCHOTT NG-4 Glass were
initially selected from many glass types. SCHOTT NG Glass was finally
selected since this material exhibits the best optical neutrality.
• This glass is usually calibrated at 440.0nm, 465.0nm. 456.1nm, 590nm and
635nm at room temperature of 20.5°C and 25.5°C to check stability.
Neutral Density filters – Visible
Spectrum
Neutral Density filters
Potassium Dichromate - UV
The use of potassium dichromate solvated in dilute perchloric
acid is an established and well recognised method for the
validation of the absorbance scale and linearity of a
spectrophotometer in the UV region.
Metal on Quartz Filters - UV
• Some manufacturers have produced metal on
Quarts coatings for photometric accuracy
testing in the UV. NIST SP 260-68 outlines the
production of these filters. I have used these
filters in the past but have found that the
coating as very sensitive and can deteriorate
easily.
Stray Light
• Stray light can be described as an indication by the instrument of
transmitted light at wavelength the monochromator is set to, when
in reality there is no light being transmitted through the sample.
This apparent transmission is caused by light of other wavelengths
than that established by the monochromator being sensed by the
detector, and usually results in non-linearity of an absorbance to
concentration relationship. The poorer the stray light performance
of an instrument the lower the absorbance value at which this
correlation begins to deviate from a straight line. Stray light can be
a problem at any wavelength but energy throughput of an
instrument decreases, for example as you move into the UV region
apparent stray light will become increasingly problematic.
• The grating produces second order spectrum which is attenuated by
the filter wheel. This light would interfere with the sample reading
if allowed to pass through. This is just one possible source of stray
light.
Stray Light Solutions
Alignment
• The alignment of the cell holder is critical especially when using flow cells.
Some manufactures use a10mm beam width and some use a focused
beam. If the cell is out of alignment then some of the energy will be
attenuated. This is especially critical with flow cells. In addition there are
two standard beam heights. 8.5mm and 10mm. this is known as Z
dimension. This is your beam height from the base of the cell.
Manufacturer
Z Dimension
Beckman®
8.5 mm
Bio-Rad
8.5 mm
Hewlett Packard®
15 mm
Hitachi®
8.5 mm
Perkin-Elmer®
15 mm
Pharmacia
15 mm
Shimadzu®
15 mm
Spectronics®
8.5 mm
Varian®
15 or 20 mm
Shimadzu
15 mm
Thermo Spectronic
8.5 & 15
Resolution standards
• Ratio table:
SBW (nm):
• 0.5 1.0 1.5 2.0 3.0
• Ratio:
• 2.5 2.1 1.6 1.4 1.0
Scan the 252-262 nm region, and observe the spectral changes with
SBW.
The 253.49 nm and 259.56 nm peaks should be visible at a SBW of 0.2
nm or less.
Energy in the visible Spectrum
Energy in the UV Spectrum
Relationship between Energy and Gain
Scan speed