UV-Visible Spectroscopy Instrumentation PDF

INSTRUMENTATION OF UV- VISIBLE SPECTROSCOPY DR. AFSHAN ABDUL SHAKOOR LECTURER LIST  Instrumentation Spectrophotometer can be divided into I. Single beam spectrophotometer II. Double beam spectrophotometer Single beam spectrophotometer: Practically all UV vis...

INSTRUMENTATION OF UV- VISIBLE SPECTROSCOPY DR. AFSHAN ABDUL SHAKOOR LECTURER LIST  Instrumentation Spectrophotometer can be divided into I. Single beam spectrophotometer II. Double beam spectrophotometer Single beam spectrophotometer: Practically all UV visible spectrophotometer can possess five essential components. i. Source ii. Monochromator iii. Cell iv. Detector v. Recorder Instrumentation  SOURCE In UV spectrophotometer, the source of radiant energy used is the hydrogen discharge lamp. It is composed of a tube fitted with two electrodes and hydrogen gas at low pressure. when high voltage is applied across the electrode, the electrons of hydrogen gas molecules are excited to higher energy state. The return of excited molecule to the ground state result in the emission of continuous radiation in the region between 180-350nm. For visible spectrophotometer, source is usually a tungsten filament lamp and the radiation lies in the range of 350-2500µm.  Monochromator Devices that are employed to isolate the wavelength are called as the monochromator. A typical monochromator usually consist of i. Entrance slit ii. Prism or diffraction grating iii. Mirror iv. Exit slit v. Polychromatic light from the source enter the monochromator system through entrance slit and collimated by either a lens or a mirror. This collimated light is then dispersed by the prism or grating. Depending upon the type of dispersion element, there are two types of monochromators. Prism type Grating type  Monochromator….. Prism type: In this type, a prism is used to get monochromatic light. The prism breaks light into the narrow bands, when it is passed through the prism. For visible region: glass prism is used. For UV region: quartz prism is used. Grating type: Diffraction grating is of two types a) Transmission grating b) Reflection grating Reflection grating is usually used for UV region which consists of large number of equispaced lines ruled on a glass coated with thin film of aluminium. The desired portion of dispersed spectrum is then focussed by a lens or mirror on the exit slit on the test sample.  Filter Filters used mostly in simple analysis while monochromators are used for ultraviolet, visible and infrared radiation. Filters used mostly commonly are colored glass, gelatin filter and interference filter. Filter may be used to filter out the light of undesirable wavelength. Filters are of following types; Glass filter (pieces of colored glasses that transmit limited wavelength) Gelatin filter (consist of thin gelatin sheet between a pair of glass plates) Interferometer filter (consist of two parallel glass plates silvered internally and separated by a thin film of cryolite or some other dielectric material.)  Sample cell Gas or liquid samples for analysis for the UV and visible region are examined in cuvette (absorption cell). Shapes of the cell may be cylindrical or rectangular. For visible region, glass cells are used. For UV region, quartz cells are used.  Detectors Several types of the detectors are used but most commonly phototubes and photomultiplier tubes are used. Phototubes: It consists of semi cylindrical cathode and metal wire anode sealed inside an evacuated glass envelop. A potential difference is applied between the anode and the cathode. When the photons are absorbed by the detector, they transfer their energy to the photo-emissive material on the cathode surface. The loosely bound electrons leave the cathode surface and are collected at the anode, causing current to flow in the phototube circuit.  Detectors Photomultiplier tubes: A photomultiplier tube is a type of phototube that contains a number of electrodes called dynodes in addition to photosensitive cathode and anode. When an electron leaves the photo-emissive cathode and strikes a dynode, it transfer some of its energy and cause the ejection of several no of electron. These electrons in turn are accelerated toward the next dynode where the same process takes place. After several stages of amplification each photoelectron emitted by the cathode will have been amplified to a great level.  Detectors Other photodetectors include: Barrier layer cells (photovoltaic cells) are also used.  Recorders The output from detector is amplified and is observed on a recorder in term of %age transmittance or absorbance. Working: UV-visible spectrometer records the spectra of the compound in the range of 200-800nm. This range consists of two parts, one is uv range and other is visible range. For recording the spectrum, the given sample is dissolved in a suitable solvent which itself does not absorb light. The commonly used solvents are 95% ethanol, methanol, hexane etc. A quartz cell of path length 1cm is used as a container for the sample solution. The solution is exposed to UV-visible light by the prism selector. The prism selector is rotating continuously to emit lights of varying wavelengths. This light pass through the detector and amplified at recorder and graph is draw between wavelength of radiation absorb and intensity of absorption.  Double beam spectrophotometer In research purposes, double beam spectrophotometer is mostly used. In this case, monochromatic radiations is split into two beams of equal intensity. One beam is passed through the sample cell. And the other beam is passed through the reference cell. The two cells should be matched. Then its focused on the detector that in turn is connected to an amplifier which transmits the signal to the recorder. Double beam spectrophotometer is more advantageous as compare to single beam spectrophotometer because in single beam the blank and the sample have to be replaced continuously according to wavelength and the ratio of the absorption power. Because we cant use the sample and the reference cell together. Single beam Spectrophotometer: Double-beam Spectrophotometer: 14  Applications of UV-visible spectroscopy There are two main application of UV-visible spectroscopy. Detection of conjugation: It helps to establish the relationship with special reference to conjugation in a compound i.e. Carbon-carbon double or triple bond And also the carbon-oxygen double bond and also in aromatic ring. So , by finding the λmax , we can determine the location of the substituents.  Applications (continue) Detection of functional group: It is possible to determine the certain functional group with the help of UV-visible spectrum specially alpha, beta unsaturated carbonyl system or any other functional group.  Applications (continue) Detection of impurities: UV-visible spectroscopy is one of the best methods for detection of impurities in organic compounds. The bands due to impurities are very intense. Additional peaks can be observed due to impurities in the sample and it can be compared with that of standard raw material.  Applications (continue) Qualitative analysis: In case of qualitative analysis with the help of uv- visible spectrometer, we can identify the given organic molecule. Identification can be done by comparing the absorption spectrum with the spectrum of known compound. Molecular weight determination: o Molecular weights of compounds can be measured spectrophotometrically by preparing the suitable derivatives of these compounds.  Applications (continue). Quantitative analysis o Prepare samples o Make series of standard solutions of known concentrations o Set spectrophotometer to the λ of maximum light absorption o Measure the absorption of the unknown, and from the standard plot, read the related concentration. 19 Elucidation of the structure of Organic Compounds: From the location of peaks and combination of peaks UV spectroscopy elucidate structure of organic molecules: o the presence or absence of unsaturation, o the presence of hetero atoms Chemical Kinetics: Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is passed through the reaction cell and the absorbance changes can be observed. 20  Reference Books  Introduction to Spectroscopy  Donald A. Pavia  Elementary Organic Spectroscopy  Y. R. Sharma  Practical Pharmaceutical Chemistry  A.H. Beckett, J.B. Stenlake o Pharmaceutical Analysis by Anees A. Siddiqui