Ultraviolet Visible Spectroscopy (U.V.)
What is UV spectroscopy: UV spectroscopy is the measurement
of the attenuation of the beam of light passing through a sample or after
reflection from the sample surface.
Ultraviolet light: Wavelength
between 190 nm to 400 nm
Visible
light: Wavelength between 400 nm to 800 nm
Principal
of UV: UV spectroscopy is absorption spectroscopy. It is based upon the
phenomenon of electronic excitation so when UV radiation are passed through the
sample the atoms or molecules absorbs the energy and moves from ground state to
the excited state. If it occurs the residual radiation. It is passed through a
prism here is a spectrum with gap in it and it is called an absorption
spectrum. The excitation is detected by the detector amplified and recorded.
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Lambert's beer’s law: When
a monochromatic light pass through in a transparent medium the rate of
intensity is decreases with the thickness of medium. It is proportional to the
intensity of light as well as the concentration of solution.
Instrumentation of UV spectroscopy:
Components of instrument are light source monochromator, sample holder,
detector, amplifier and recorder. This is the schematic diagram about
instrumentation of UV spectroscopy. The basic requirement is light source which
passes polychromatic radiation to monochromator than monochromator converts
polychromatic radiation into monochromatic radiation.Only the light of single
wavelength is then passed to the sample holder, light passed from the sample
holder go to detector where it gets detected amplified and then sent to the
recorder. Electric signal in to form of graph so now let us study about each component
of instrument in detail.
Light
source: Light source are most of the deuterium lamp. It
is used if we need radiations of UV region that is from 200 to 400 nm and the
second is tungsten halogen lamp which is used for the visible region radiation
having the range of 400 to 750 nm.
Monochromator device: It is used to resolve wide band of polychromatic radiation into narrow
band of monochromatic radiation. There are three types of monochromator filters
prisms and gratings.
Sample holder: Sample holder is
made of quartz.
Detectors: It is
detect and convert light energy into electrical signals that are displayed on
the readout device. There are following types of detectors.
Barrier layer cells photo tube
Photo-multiplier tube which is also known as PMD
Thermocouple bolometer etc
Various types of detectors are used but most
commonly used detector is photo-multiplier.
Amplifier and recorder:
Amplifier signal coming from detector and recorder to records them which is
displayed on readout device.
Calibration of UV: Calibrating UV photometer using potassium dichromate kit. Turn
on the spectrophotometer and let it heat up for 45 minutes let's go ahead and
choose a wavelength to calibrate for potassium dichromate there are four
different wavelengths you can choose. Set your machine for 235 nm now we have
to insert the perchloric acid blank. When we insert the blank get a reading zero.
Check the below parameter as par table.
Concentration mg/L
|
Wavelength in nm
|
Uncertainty
|
|||
235
|
257
|
313
|
350
|
||
20
|
0.2186
|
0.2635
|
0.0917
|
0.2114
|
±0.0034
|
40
|
0.4534
|
0.5407
|
0.1832
|
0.4119
|
±0.0020
|
60
|
0.7239
|
0.8544
|
0.2861
|
0.6417
|
±0.0020
|
80
|
0.9794
|
1.1388
|
0.3844
|
0.8523
|
±0.0020
|
100
|
1.2291
|
1.444
|
0.4855
|
1.0766
|
±0.0020
|
Absorbance (A)
|
Double beam spectroscopy:
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It uses two beams of light simultaneously. The
components are a light source optical components like mirrors or collimating
mirrors, gratings, selector mirrors, multiple sample holders and a detector.
The light source is a halogen tungsten lamp operating in the visible region of
EMR and a deuterium lamp which produces UV radiations. The intensity of these
blackbody sources can be controlled by varying the voltage applied to them. The
next components are optical elements which are responsible for collimating
light radiations and focusing them on diffraction gratings. The diffraction grating
separates the wavelength using Bear's law. By changing the angle theta of light
one can obtain a particular wavelength lambda if the grating distance is fixed.
One can obtain required wavelength by rotating the grating. The monochromatic
light exiting the grating assembly is split into two parts using a selector
mirror the light beams travel in two directions and pass through the reference
cuvette and sample cuvette respectively the final component is a photocell or
it can also be a PMT.
PMT stands for photo-multiplier tube it detects even smallest
photo electrons as it has internal amplification assembly. Even for weakest
light intensities good amount of measurable photo current is generated by the
PMT. Now let's understand the working of a double beam spectrophotometer. The
advantage of using a double beam spectrophotometer is that the error due to
intensity variations in source of light is eliminated. The monochromatic light
exiting the grating assembly travels in two different paths one passes through
a reference sample or standard sample and the other one passes through the
analyte under test both the solutions absorb part of light falling on them and
transmit the remaining light. The transmitted light is picked up by the
detector and readings are recorded since the reference and sample under tests
are recorded simultaneously any fluctuations in the light source are eliminated
the concentration of analyte can be determined from the readings of the
intensities. Presently, sophisticated instruments are available which directly
give the concentration of analyte. Since measurements are done simultaneously
these instruments operate at high speeds some instruments have multiple sample
holders so that all the samples are loaded once and the experiment is conducted
the variations in the lab output are compensated automatically spectral scan of
an analyte is possible using double beam spectrophotometer and the changes in
detector sensitivity is also compensated.
Question: why potassium dichromate use in UV
calibration?
Answer: 1-potassium dichromate especially
useful in the visible range but also useful in UV.
2- Potassium dichromate itself is stable and available in high purity.
3-In dilute perchloric acid
solution, it has linear response with good temperature stability and also
stable as solution.
4-The peak are well defined, molar absorptivity at 350 nm is accurately
known.
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