Ultraviolet Visible Spectroscopy (U.V.)

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.