UVVIS Spectrophotometry Fluorometry Polarometry Atomic Absorption Spectrometry Flame
UV-VIS Spectrophotometry Fluorometry Polarometry Atomic Absorption Spectrometry Flame Photometry Infra-Red Spectrometry (IR) Mass Spectrometry (MS) Nuclear Magnetic Resonance Spectrometry (1 H-NMR/13 C-NMR) 10/31/2021 1
Light and radiation Electromagnetic spectrum Interaction of Photons with matter Absorption spectrum, Characteristics and shifts Chromophores, and auxochromes Factors affecting absorption spectrum (p. H, solvent) Beer’s law and quantitative analysis Instrumentation (Basic component of spectrophotometer) Analysis of some pharmaceutical drugs 10/31/2021 2
Definition of Spectrometry: The underlying principle of spectrophotometry is to shine light on a sample and to analyze how the sample affects the light. Principle of Spectrophotometry: Absorption spectrophotometry: is the measurement of an interaction between electromagnetic radiation and the molecules, or atoms, of a chemical substance Colorimetry: Spectrophotometric measurement in the visible region was formerly referred to as colorimetry; however, it is more precise to use the term “colorimetry” only when considering human perception of color. Fluorescence spectrophotometry is the measurement of the emission of light from a chemical substance while it is being exposed to ultraviolet, visible, or other electromagnetic radiation. In general, the light emitted by a fluorescent solution is of maximum intensity at a wavelength longer than that of the exciting radiation, usually by some 20 to 30 nm. 10/31/2021 3
Spectroscopy is basically an experimental subject and is concerned with the absorption, emission, or scattering of electromagnetic radiation by atoms and molecules. Spectroscopy is the study of the interaction of electromagnetic radiation with matter. Analyst spectrum molecules, or atoms, of a chemical substance light prism spectrometer 10/31/2021 4
E A Electric field Light can be described as a wave. This wave has an electric component and a magnetic component which are perpendicular to each other Velocity = c Direction of propagation H Wavelength ( ) 10/31/2021 5
Electric magnetic spectrum 10 -11 10 -9 x-ray 1021 1019 Wavelength ( , cm) 10 -6 10 -5 10 -4 UV visible 1016 1015 10 -2 102 IR Rf 1014 1012 108 Frequency ( , Hz) Wavelength (nm) 390 UV 10/31/2021 450 520 590 Increasing Energy Increasing Wavelength 620 780 IR 6
When a light wave encounters a particle, or molecule, it can be either scattered (i. e. , direction changed) or absorbed (energy transferred). Molecules can only absorb discreet packets of energy, or quanta. Absorption occurs when the energy of the photon corresponds to differences between energy levels in that particular molecule. Absorption of the energy from the photon elevates the molecule to an excited state. A molecule or substance that absorbs light is known as a chromophore. Chromophores exhibit unique absorption spectra and can be defined by a wavelength of maximum absorption, or λmax. Reflection losses at interfaces I 0 (radiant intensity) 10/31/2021 Scattering losses in solution I (transmitted intensity) Reflection and scattering losses 7
A molecule may absorb light energy in three ways: [1] by raising an electron to a higher energy level ( transitional energy) when molecule absorb visible and UV light. [2] by increasing the vibration of constituent nuclei (vibrational), when molecule absorb IR irradiation. [3] by increasing the rotation of molecule about its axis ( rotational) when molecule absorb FT-IR irradiation. When a molecule interact with photons in the UV-VIS region the absorption of energy results in displacing an outer electron, (valence electron) in the molecule. The molecule is said to undergo transition from the ground state of energy level to an excited state of energy level. 10/31/2021 8
Absorbance absorption spectrum, UV Scan, UV-VIS scan spectrum 10/31/2021 max Absorption spectrum is: Characteristic to substance, and the wavelength at which the maximum absorption is recorded is used to trace the substance strength to enhance the sensitivity. 9
Chromophores: (Chrom = color, phore = carrier) They are functional groups which confer color on substances capable of absorbing UV and/or visible light. They have unsaturated bonds such as C=C, -C=O, -N=N, and -C≡N (etc π electrons). Auxochromes: They are functional groups which can not confer colors on substances but have the ability to increase the coloring power of chromophores, they does not absorb radiations longer than 200 nm, but when attached to a given chromophore, cause a shift to a longer wavelength with increase in absorption intensity. Ex: -OH, -NH 2. Bathochromic shift (red shift): it is the shift of max to a longer wavelength due to substitution or solvent effects. Hypsochromic shift (blue shift): it is the shift of max to a shorter wavelength due substitution or solvent effects. Hyperchromic effect: enhancement of molecule absorptivity (or absorption intensity). Hypochromic effect: decrease of molecule absorptivity (or absorption intensity). 10/31/2021 10
Hyperchromic Absorbance, m. AU APEX Bathochromic Hyporchromic Hypsochromic Wavelength, nm 10/31/2021 11
Affect what? Intensity Maximum wavelength Effect of p. H on absorption spectra Effect of solvent on absorption spectra Effect of redox reaction on the absorption spectrum 10/31/2021 12
Ex 1: the UV spectrum of phenol in acidic medium is completely different from its spectrum in alkaline medium (using same concentration). The spectrum in alkaline medium exhibits bathochromic shift with hyperchromic effect. The red shift is due to the participation of the pai electrons in resonance with the π-electrons of the benzene ring, thus increasing the delocalization of the π-electrons. Phenol in acid medium max = 270 nm Phenol in alkaline medium max = 290 nm Aniline in alkaline medium max = 280 nm Anilinium ion in acid medium max = 254 nm Ex 2: On the other hand, UV spectrum of Aniline in acid medium shows hypsochromic shift with hypochromic effect. This blue shift is due to the protonation of the amino group, hence the pair of electrons is no longer available and the spectrum in this case is similar to that of benzene (thus called benzenoid spectrum). 10/31/2021 13
detector monochromator lens lamp 10/31/2021 Sample cuvette amplifier readout 14
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