UV SPECTROSCOPY by S Shrivastava & Dr S Nayak

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Slide 1: 

Bansal College of Pharmacy, Bhopal www.bansalpharmacy.com AN OPERATIONAL OVERVIEW UV INSTRUMENTATION & METHODOLOGY Shobhit Shrivastava Assistant Professor Dr. S. Nayak Principal

Slide 2: 

Electromagnetic Radiation: Atomic Spectra Molecular Spectra Beer Lambert Law I= Io-IT -dI/db=kI A=log Io/IT =abc Electronic Transitions Sigma to Sigma* (Methane) antibonding to Sigma*(Alcohol, Ether) Pi to pi*(K-band, Aromatics) antibonding to pi*(R-band, Saturated ketone)

Slide 3: 

Chromophore Auxochrome

Instrumentation : : 

Instrumentation : Introduction Have a look at this schematic diagram of a double-beam UV-Vis. spectrophotometer;

Slide 5: 

Components Sources of radiant energy (UV and visible) Collimating System (making parallel) Wavelength selector (monochromator) Sample holder Detector Signal processor and readout

Slide 6: 

Collimating system Lenses, mirror, slit, Monochromator Filters Glass filters Gelatin Interference Prism Diffraction grating The beam is split into its component wavelengths by the grating or prism. By moving the dispersing element or the exit slit, radiation of only a particular wavelength leaves the monochromator through the exit slit.

Sample Holder : 

Sample Holder Cuvette : Quartz Sample Holder Quartz

Detectors : : 

Detectors : Barrier layer cell (photovoltaic cell): ♣ consist of a semiconductor, such as a selenium, which is deposited on a strong metal base, such as iron. ♣ Then a very thin layer of silver or gold is sputtered over the surface of the semiconductor to act as a second collector electrode.

iii) The photomultiplier tube : : 

iii) The photomultiplier tube : Consists of Photoemissive cathode (emits electrons when struck by photons) several dynodes (emits several electrons for each electron striking them) anode. A photon of radiation entering the tube strikes the cathode, causing the emission of several electrons. These electrons are accelerated towards the first dynode. The electrons strike the first dynode, causing the emission of several electrons for each incident electron. These electrons are then accelerated towards the second dynode, to produce more electrons which are accelerated towards dynode three and so on. Eventually, the electrons are collected at the anode. The resulting current is amplified and measured. Very sensitive to UV and visible radiation, fast response times. Intense light damages photomultipliers; they are limited to measuring low power radiation.

Cross section of a photomultiplier tube : 

Cross section of a photomultiplier tube

Recording systems : : 

Recording systems : The signal from the photomultiplier tube is finally received by the recording system. The recording is done by recorder pen. The type of arrangement is only done in recording UV spectrophotometers. Power supply : The power supply serves triple function » Decreases the line voltage to the instruments operating level with a transformer. » Converts A.C. to D.C. with a rectifier if direct current is required by the instrument. » smooths out any ripple which may occur in the line voltage in order to deliver a constant voltage to the source lamp & instruments.

Description of a UV spectrophotometer: : 

Description of a UV spectrophotometer: A) single beam system 1.Bausch and Lamb 2.Beckman D.U.

Slide 13: 

All of the light passes through the sample cell. Io must be measured by removing the sample. This was the earliest design, but is still in common use in both teaching and industrial labs. Single Beam instrument

Double beam instrument : : 

Double beam instrument :

Double beam instrument : : 

Double beam instrument : The light splits into two beams before it reaches the sample. One beam is used as the reference; the other beam passes through the sample. Some double-beam instruments have two detectors (photodiodes), and the sample and reference beam are measured at the same time. In other instruments, the two beams pass through a beam chopper, which blocks one beam at a time. The detector alternates between measuring the sample beam and the reference beam.

Slide 16: 

1. Identification 2. Structural Elucidation 3. Quantitative Applications Analysis of Organic compounds Analysis of Inorganic Compounds Mixtures of Absorbing substances Applications

Operational Methodology : 

Operational Methodology Estimation of λmax Study of Beer Lambert law and determination of Linearity range. If the system is multicomponent, Study of Spectra of all components to determine absorbances at different wavelengths, Isobestic point etc. Derivative spectrum Calculations Validation

Ultraviolet-visible spectrophotometer : 

Ultraviolet-visible spectrophotometer It measures the intensity of light passing through a sample (I), and compares it to the intensity of light before it passes through the sample (Io). The ratio I / Io is called the transmittance, and is usually expressed as a percentage (%T). The absorbance, A, is based on the transmittance: A = − log(%T)

Slide 19: 

THANK YOU Shobhit Shrivastava Asst. Prof. Dr. S. Nayak Principal Principal.bcp@gmail.com

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