Uv

uv-spectroscopy

1. Uv/visible spectroscopy Submitted by: Savita M Sc. Forensic (Final) Roll No. - 1301 Submitted to: Diksha Mam

2. spectroscopy It is the branch of science that deals with the study of interaction of matter with light. or It is the branch of science that deals with the study of interaction of electromagnetic radiation with the matter.

3. Electromagnetic Radiation

4. Electromagnetic radiation Electromagnetic radiation are different forms of energy which have a dual character; they have the properties of both wave and particle. They are called electromagnetic radiations because they consist of oscillating electric and magnetic field which are perpendicular to each other and perpendicular to the direction of motion of the radiations. Electromagnetic radiation consist of discrete packages of energy which are called photons

5. Some important characteristic The energy of these radiation is inversely proportional to their wavelength and directly proportional to their frequency. These radiation do not require any physical medium and can be transmitted through empty space. All types of electromagnetic radiations travel through space with the same velocity which is nearly same as the speed of light.

6. Wavelength It is the distance between two adjacent crest or trough in a particular wave. It is denoted by the symbol λ Usually expressed in meters , micrometers nanometers and angstrom . Frequency It is the number of waves which passes across a point in one second. Generally expressed as hertz

7. Electromagnetic spectrum The arrangement of entire range of electromagnetic radiations in increasing order of their wavelengths or decreasing order of their frequencies is known as electromagnetic radiation.

8. Electromagnetic spectrum

9. Principles of spectroscopy The principle is based on the measurement of spectrum of a sample containing atoms/ molecules. Spectrum is a graph of intensity of absorbed or emitted radiation by sample versus frequency or wavelength. Spectrometer is a device design to measure the spectrum of a compound.

10. Classification of spectroscopy Absorption spectroscopy- An analytical technique which concerns with the measurement of absorption of electromagnetic radiation. E.g. UV(185-400nmm)/visible(400-800) spectroscopy, IR spectroscopy.

11. Emission spectroscopy Emission spectroscopy is a analytical technique in which emission (of a radiation or a particle ) is dispersed according to some property of the emission and the amount of dispersion is measured. E.g. mass spectroscopy .

12. Principle of absorption spectroscopy When a beam of electromagnetic radiation is passed through a substance, the radiation may be either absorbed or transmitted depending upon the wavelenth of the radiation and molecular structure of the substance. Absorption of radiation would generally increase in the energy of the molecule; the increase in the energy may lead to electronic excitation whereby electrons are raised to higher energy level. Alternatively it may bring about increase in the vibrational and rotational energy of the level.

13. Ultraviolet spectroscopy Ultraviolet spectroscopy involves the measurement of absorption light in the visible and ultraviolet region by a substance. Absorption of light involves transition from one electronic energy level to another within a molecule , so also known as electronic spectroscopy

14. Uv spectrometer A uv spectrometer consist of the following parts: A source of radiation which emits all wavelengths of UV. The common used source of light in the uv region are hydrogen or deuterium discharge lamp while a tungsten filament is used for producing visible light. Monochromator – which separates the radiation into two individual wavelength. The single wavelength of radiation is split into two beams one of which passes through the sample and other passes through the reference sample.

15. Detector- continuously measures the intensity ratio of the beams transmitted through the sample and the solvent respectively. Recorder- Automatically records the absorption of light at each wavelength as a graph

16. Principle of uv spectroscopy Absorption of visible and uv light produces change in electronic states in the molecule associated with excitation of a electron from lower to higher energy level. Each electronic level in a molecule is associated with no of vibrational sub level Each vibrational level in turn are associated with no. of rotational sub-levels.

17. electronic excitations

18. instrumentation Optical system- spectrometers are instrument that provide information about the intensity of light absorbed or transmitted as a function of wavelength. Both single and double beam optical system are used in molecular absorption spectroscopy.

19. Single beam system

20. Double beam system Double beam system is used extensively for spectroscopic studies. The individual component of system have same function as in single beam system, important difference is radiation from source is split into two beams of approximately equal intensity using a beam splitter. The intensity of the two beams after passing through the cells are then compared.

21. Radiation source Radiation source for molecular absorptions must produce light over a continuum of wavelengths. Traditionally the two most common radiation source used for UV spectroscopy were: Tungsten lamp Deuterium lamp

22. Tungsten lamp Tungsten lamp is similar in functioning to the ordinary electric light bulb. It contains a tungsten filament heated electrically to white light, and generates a continuum spectrum. It has two shortcomings: The intensity of radiation at short wavelengths is too low; furthermore to maintain constant intensity, electrical bulb current to the bulb must be carefully controlled

23. Deuterium arc lamp The deuterium lamp consist of deuterium gas in a quartz bulb through which there is electrical discharge. The molecules are excited electrically and the excited molecule dissociates emitting UV radiation.

24. monochromator A monochromator consist of an dispersion element, an entrance slit, and an exit slit plus lenses and mirror for collimating and focusing the beam of radiation. The two most common type of dispersion element are prism and gratings. Entrance slit allows light from the source to fall on the dispersion element. The dispersed light falls on the exit slit of the monochromator. The function of the slit is to permit only a very narrow band of light to pass through to the sample and detector

25. detectors • The purpose of the detector is convert the transmitted radiant energy into an equivalent amount of electrical energy . • Two types of detectors are frequently used :- 1- Photomultiplier tube ( PMT ) : • Incident light strikes the coated cathode , emitting electrons . • The electrons are attracted to a series of anodes , known as dynodes , each having a Successively higher positive voltage . • Theses dynodes are of a material that gives off many secondary electrons when hit by single electrons . • Initial electron emission at the cathodes triggers a multiple cascade of electrons within the PMT itself . • Because of this amplification , PMT are used in instruments designed to be extremely sensitive to very low light levels . • the accumulation of electrons striking the anode produces a current signal that can be fed into a meter or recorder .

26. detectors Photodiode arrays ( PDA ) : Are new detectors being used in modern spectrometers. photodiodes are composed of silicon crystals that are sensitive to light in the wavelength range 170-1100 nm. Upon photon absorption by the diode , a current is generated in the photodiode that is proportional to the number of photons. Although photodiodes are not as sensitive as PMT because of the lack of internal amplification , their excellent linearity , speed , and small size make them useful in applications where light levels are adequate . PDA detectors are available in integrated circuits containing 256 to 2,048 photodiodes in a linear arrangement . Each photodiode responds to a specific wavelength, and as a result, a complete UV/visible spectrum can be obtained in less than 1 second .

27. procedure Are most often used to obtain quantitative information , such as the concentration of a solute in solution or the absorption coefficient of a chromophore . • For fixed-wavelength measurements with a single-beam instrument, a cuvette containing solvent only is placed in the sample beam and the instrument is adjusted to read “Zero” absorbance . • A matched cuvette containing sample plus solvent is then placed in the sample chamber and the absorbance is read directly from the display . • The adjustment to zero absorbance with only solvent in the sample chamber allows the operator to obtain a direct reading of absorbance for the sample

28. Fixed-wavelength measurements using a double-beam spectrophotometer are made by first zeroing the instrument with no cuvette in either the sample or reference holder . Alternatively , the spectrophotometer can be balanced by placing matched cuvettes containing water or solvent in both sample chambers . Then, a cuvette containing pure solvent is placed in the reference position and a matched cuvette containing solvent plus sample is set in the sample position . The absorbance reading given by the instrument is that of the samples; that is, the absorbance due to solvent is subtracted by the instrument

29. For recording the UV spectrum, the given compound is generally dissolved in some suitable solvents which does not absorb light in the region under examination. The commonly employed solvents are 95% ethanol, hexane, and water. The solution of the compound is placed in a suitable transparent container which does not absorb light in the region being studied; a quartz cell of 1cm path length is generally used for this purpose.( glass cannot be used since it absorbs strongly in the ultra violet region)

30. Chromophore and auxochrome CHROMOPHORE : The term chromophore was previously used to denote a functional group of some other structural feature of which gives a color to compound. For example- Nitro group is a chromophore because its presence in a compound gives yellow color to the compound. But these days the term chromophore is used in a much broader sense which may be defined as “any group which exhibit absorption of electromagnetic radiation in a visible or ultra-visible region “It may or may not impart any color to the compound. Some of the important chromophores are: ethylene, acetylene, carbonyls, acids, esters and nitrile groups etc.

31. AUXOCHROMES:  It is a group which itself does not act as a chromophore but when attached to a chromophore, it shifts the adsorption towards longer wavelength along with an increase in the intensity of absorption. Some commonly known auxochromic groups are: -OH, -NH2, -OR, -NHR, and –NR2. For example: When the auxochrome –NH2 group is attached to benzene ring. Its absorption change from λ max 225 (ɛmax 203) to λmax 280 (εmax1430)

32. Change in position and intensity of absorption

33. Bathochromic Shift (Red Shift) When absorption maxima (λmax) of a compound shifts to longer wavelength, it is known as bathochromic shift or red shift. The effect is due to presence of an auxochrome or by the change of solvent. e.g. An auxochrome group like –OH, -OCH3 causes absorption of compound at longer wavelength.

34. Hypsochromic Shift (Blue Shift When absorption maxima (λmax) of a compound shifts to shorter wavelength, it is known as hypsochromic shift or blue shift. The effect is due to presence of an group causes removal of conjugation or by the change of solvent.

35. Hyperchromic Effect • When absorption intensity (ε) of a compound is increased, it is known as hyperchromic shift. • If auxochrome introduces to the compound, the intensity of absorption increases. Pyridine 2-methyl pyridine λ max = 257 nm λ max = 260 nm

36. Hypochromic Effect When absorption intensity (ε) of a compound is decreased, it is known as hypochromic shift. Naphthalene 2-methyl naphthalene ε = 19000 ε = 10250

37. Application of uv Ultraviolet (UV) light technologies are used for multiple purposes in forensic investigations, including authenticating paintings and other fine art, authenticating signatures, analyzing  questioned documents  , illuminating latent fingerprints at crime scenes and  trace evidence  on clothing, analyzing ink stains, and revealing residual stains of body  fluids. Forensic technicians also use UV light technologies, such as ultraviolet monochromators or optical  spectroscopy  , to detect the presence of illegal or controlled substances or their residues in unidentified samples, or to determine how many types of ink or pens were used in a forged document.

38. reference https://www.slideshare.net/422459/uv-visible-spectroscopy- https://en.wikipedia.org/wiki/Ultraviolet https://www.spectroscopyeurope.com/article/shedding-light-evidence-forensic-applications-uvvisible-spectroscopy Undergraduate instrumental analysis-James W. Robinson