Spectropotometer.08.20 - Copy.ppt
- 1. Presented by Dr. A. N. M. Al-Razee M. Sc., Ph. D. Email: al_razee8@yahoo.co.uk Deputy Chief Chemist, ACESD, TICI (08 years) • Previous experience: 12 years in ammonia plant of Urea Fertilizer Factory 2 years in Quality Control officer in Incepta Pharmaceuticals Ltd.
- 2. Spectroscopy • Light is a type of energy made of electromagnetic waves, a blend of magnetism and electricity. Visible light is only one kind of light, or electromagnetic radiation. Spectroscopy = interactions between light & matter E = hn = hc/l
- 3. Contents • Electromagnetic radiation • Photoelectric effect • Atomic spectroscopy • Beer lamberts law • Instrumentation of UV-Vis Spectrophotometer. • Application
- 4. Electromagnetic radiation Electromagnetic radiation are conveniently described as wave model which employs such as parameters • Wavelength • frequency • Velocity • Amplitude
- 6. Different Wavelengths and Frequencies
- 7. Sky blue 410 490 520 570 640 Red 620~780 650 Orange 585~620 602 Yellow 570~585 577 Green 490~570 530 Blue 440~490 465 Indigo 420~440 430 Violet 400~420 410 ELE Paqualab Photometer Wavelength selection with filter colours VIBGYOR
- 8. • Humans can only see a small section of electromagnetic radiation, and this band is called the visible light spectrum. • Spectrum
- 10. UV-Visible Spectrophotometer • The wavelength range of UV radiation is 200 nm- 400 nm. There are mainly two types of UV region. 1. 200 nm- 400 nm that is called near ultraviolet region. 2. Below 200 nm that is called far ultraviolet region. • The wavelength of visible radiation is 400 nm- 800 nm. • ΔE= E1- E0 = hv = hc/λ • ΔE depends upon how tightly the electrons are bound in the bonds and accordingly, absorption will occur in UV or visible range.
- 12. Theory of Atomic Spectroscopy. Atomic spectroscopy is based upon • absorption, • fluorescence or • emission of electromagnetic radiation by atoms or ions
- 13. Absorption, emission, fluorescence Schematic representation of absorption, emission, and fluorescence.
- 14. Fundamentals • Absorption and emission of light by compounds is generally associated with transitions of electrons between different energy levels 14 E2 E1 E0 DE = hn = hc/l Emission: Sample (in an excited state) produces light/looses energy Absorption: sample takes up energy Consumes light of appropriate wavelength http://physics.nist.gov/PhysRefData/ASD/lines_form.html Atomic spectra: line spectra provide specificity: each element has its own pattern, as each element has its own electronic configuration ground state excited states DE2 DE1 E2 E1 E0 DE2 DE1
- 15. Light and the perception of color Light is a form of electromagnetic radiation. When it falls on a substance, three things can happen: • the light can be reflected by the substance • it can be absorbed by the substance • certain wavelengths can be absorbed and the remainder transmitted or reflected Since reflection of light is of minimal interest in spectrophotometry, we will ignore it and turn to the absorbance and transmittance of light
- 16. Beer-Lambert's law of absorption • According to Beer’s law, when a beam of monochromatic radiation passes through a solution of an absorbing substance, absorption of radiation by the solution is proportional to the concentration of the solution. i.e., A C • According to Lambert’s law, when a beam of monochromatic light passes through a homogeneous absorbing medium, the absorption of radiation by the medium is proportional to the length of the absorbing medium. i.e., A l • Mathematically A C×l • i.e. A = . ×C×l Where, = molar absorptivity ( liter mol-1,cm-1) • This is the fundamental equation of spectrometry, and is often spoken of as the ‘Beer-Lambert’ law.
- 17. • Again if Io is the intensity of the incident light and It is the intensity of the transmitted light, then the ratio It / Io is the fraction of the incident light transmitted by the medium and is termed the transmittance T. • i.e., T= It / Io • Its reciprocal is Io / It , and the absorbance A of the medium is given by • A = log Io / It • There is a relationship between the absorbance A, the transmittance T, and molar absorption coefficient, • A = .C.l = log Io / It = - log It / Io = - logT
- 18. Limitations of Beer-Lamber's law • The electromagnetic radiation should be monochromatic • The light beam should not be scattered • No association or dissociation reaction occurs • The solution should be diluted.
- 19. Components of instrument Technique Source Cell/Material Monochromator Detector Ultraviolet Hydrogenor Deuteriumlamp Quartz Diffractiongratingor quartzprism Phototube, Photomultiplier Visible Tungstenhalogen lamp Glass Diffractiongratingor glassprism Phototube, Photomultiplier
- 20. 20 1- Sources of light Continuous Sources Visible and near IR radiation Ultraviolet radiation Deuterium Lamp 200-400 nm Tungsten Lamp 320-2500 nm 2- Filters Filters permit certain bands of wavelength (bandwidth of ~ 50 nm) to pass through. The simplest kind of filter is absorption filters , the most common of this type of filters is colored glass filters. They are used in the visible region.
- 21. 21 • Filters permit certain bands of wavelength (bandwidth of ~ 50 nm) to pass through. • The simplest kind of filter is absorption filters , the most common of this type of filters is colored glass filters. • They are used in the visible region. • The colored glass absorbs a broad portion of the spectrum (complementary color) and transmits other portions (its color). Disadvantage • They are not very good wavelength selectors and can’t be used in instruments utilized in research. • This is because they allow the passage of a broad bandwidth which gives a chance for deviations from Beer’s law. • They absorb a significant fraction of the desired radiation. i- Filters
- 22. 22 3- Sample compartment (cells) For Visible and UV spectroscopy, a liquid sample is usually contained in a cell called a cuvette. Glass is suitable for visible but not for UV spectroscopy because it absorbs UV radiation. Quartz can be used in UV as well as in visible spectroscopy 1 cm 1 cm Opaque Face Transparent Face Long pathlength Short pathlength (b) 1 cm pathlength cuvet
- 23. Diffraction gratings • Early diffraction gratings were made of glass through which the radiation passed and became diffracted these known as transmission gratings. To achieve the diffraction of ultraviolet radiation however modern grating spectrophtometers employ metal reflection gratings with the radiation is reflected from the surfaces of a series of parallel grooves. These are often known as echelette.
- 24. 24 1. The reflection grating is ruled with a series of closely spaced, parallel grooves with repeated distance d. 2. The grating is covered with Al to make it reflective. 3. When polychromatic light is reflected from the grating, each groove behaves as a new point source of radiation. 4. When adjacent light rays are in phase, they reinforce one another (constructive interference). 5. When adjacent light rays are not in phase, they partially or completely canceled one another (destructive interference). Reflection followed by either constructive or destructive interferences Echellette Reflection Grating 1 2
- 25. Photomultiplier tube • The photomultiplier tube is a commonly used detector in UV-Vis spectroscopy. • There are three main electrodes within a Photomultiplier. o 1. Photocathode. o 2. Dynodes. o 3. Anode.
- 26. 26 Photomultiplier tube It is a very sensitive device in which electrons emitted from the photosensitive cathode strike a second surface called dynode which is positive with respect to the original cathode. Electrons are thus accelerated and can knock out more than one electrons from the dynode. If the above process is repeated several times, so more than 106 electrons are finally collected for each photon striking the first cathode. photochathode anode high voltage voltage divider network dynodes light electrons e-
- 27. Single Beam Spectrometer Mirror Deuterium lamp Grating Movable mirror Detector Cuvette Slit Mirror Tungsten lamp
- 29. Atomic spectra vs molecular spectra: • Lines Bands 29 (nm) Typical atomic spectrum Two typical molecular spectra Y axes: intensity of absorbed light. Under ideal conditions proportional to analyte concentration (I c; Beer’s law). e.g. acquired by AAS Acquired by UV-Vis spectroscopy
- 30. 30 Applications of Ultraviolet/Visible Molecular Absorption Spectrophotometry Molecular spectroscopy based upon UV-Vis radiation is used for identification and estimation of inorganic, organic and biomedical species. Molecular UV-Vis absorption spectrophotometry is employed primarily for quantitative analysis. UV/Vis spectrophotometry is probably more widely used in chemical and clinical laboratories throughout the world than any other single method.
- 32. 32 Selection of wavelength Absorbance measurements are always carried out at fixed wavelength (using monochromatic light). When a wavelength is chosen for quantitative analysis, three factors should be considered 1. Wavelength should be chosen to give the highest possible sensitivity. This can be achieved by selecting lmax or in general the wavelengths at which the absorptivity is relatively high. λmax λmax - wavelength where maximum absorbance occurs
- 33. 33 By performing the analysis at such wavelengths, it will be sure that the lowest sample concentration can be measured with fair accuracy. For example, the lowest sample concentration (10-5 M) can be measured with good accuracy at lmax, while at other wavelength (l1), it may not be detected at all. Absorbance lmax l1 wavelength 10-2 M 10-3 M 10-4 M 10-5 M 5x10-5 M
- 35. Question 1. Use Beer’s Law to determine molar absorptivity and specific absorbtivity. 2. Function of grating 3. Parts of monocromator 4. Basic steps for determination of unknown conc.