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UV-Vis Spectroscopy Ultraviolet and visible spectroscopy Dr. Ahmad Najjar Philadelphia University Faculty of Pharmacy Department of Pharmaceutical Sciences 1st semester, 2020/2021 Spectrophotometry Spectroscopy is a general term referring to the interactions (mainly absorption and emission) of various types of electromagnetic radiation with matter. Spectrophotometry is a method to measure how much a chemical substance absorbs or emits light by measuring the intensity of light (electromagnetic radiation). It refers to the use of light (electromagnetic radiation) to measure chemical concentrations. Electromagnetic spectrum refers to the full range of all frequencies of electromagnetic radiation, which is refers to the waves of the electromagnetic field, propagating through space carrying electromagnetic energy. Electromagnetic radiation Electromagnetic radiation (EMR) has been described in terms of a stream of photons that travel in a wave-like pattern. Each photon contains a certain amount of energy, and all electromagnetic radiations consists of these photons. All electromagnetic radiations travels in a straight line at the Crest Crest speed of light (3 x 108 m/s). The only difference between the various types of electromagnetic radiations is the amount of energy found in the photons. Electromagnetic radiations are described by several terms Trough such as wavelength, frequency, wave number and energy. λ wavelength (units : m,cm, μm, nm) ν 1/λ ν frequency (units of cycles/sec, sec1, Hertz c ν λ ν /ν c ν wavenumber (number of waves per cm; unit : cm1) Energy (E) h ν h h c ν λ 8 1 velocityof light in vacuum c ν λ 3.00 10 m.s h is planck's constant 6.62x10-34 J.s Electromagnetic radiation Crest Crest Electromagnetic wave is also characterized by several fundamental properties, including its velocity, amplitude, phase angle, polarization, and direction of propagation. Trough Wave amplitude measures the magnitude of oscillation of a particular wave. Larger amplitude means higher energy and lower amplitude means lower energy. Amplitude is important because it tells you the intensity or brightness of a wave in comparison with other waves. Power, P, and Intensity, I, of light give the flux of energy from a source of EMR. • P is the flux of energy per unit time • I is the flux of energy per unit time per area Electromagnetic radiation spectrum Electromagnetic radiation in the domain ranging between 180 and 780 nm, has been studied extensively. This portion of the electromagnetic spectrum, designated as the ‘UV/Visible’. Generally provide little structural information but is very useful for quantitative measurements. Legend: γ = Gamma rays HX = Hard X-rays SX = Soft X-Rays EUV = Extreme-ultraviolet NUV = Near-ultraviolet Visible light (colored bands) NIR = Near-infrared MIR = Mid-infrared FIR = Far-infrared EHF = Extremely high frequency (microwaves) SHF = Super-high frequency (microwaves) UHF = Ultrahigh frequency (radio waves) VHF = Very high frequency (radio) HF = High frequency (radio) MF = Medium frequency (radio) LF = Low frequency (radio) VLF = Very low frequency (radio) VF = Voice frequency ULF = Ultra-low frequency (radio) SLF = Super-low frequency (radio) ELF = Extremely low frequency(radio) Problem 1: Calculate the wavenumber of a beam of IR radiation with a wavelength of 3μm. Problem 2: The frequency of a radiation is 3x1012 s-1. Calculate the wavelength of the radiation. Problem 3: Calculate the energy of 530-nm photon of visible radiation J 10 x 3.75 = / h = h = E Answer: 흀 풄 19 19 - m 10 = / = Answer: 풄 흀 4 4 - = 3,333 cm 3,333 = 1/ = wavenumber Answer: 흀 1 - Spectrophotometric methods A group of techniques that relies on the quantitative interaction of EMR and matter, these are mainly: . Absorption (excitation process) . Emission (deactivation (or de-excitation) process): Incandescence (luminescence after thermal heating) Chemiluminescence (luminescence after chemical reaction) Photoluminescence (luminescence after light absorption): Fluorescence: Resonance fluorescence Non-resonance fluorescence Phosphorescence Note: deactivation of absorbed energy could also be done through non-radiative (radiationless) process, such as relaxation, internal conversion (quenching) and intersystem crossing. Spectrophotometric methods: Absorption When a photon is absorbed by an analyte, it is "destroyed," and its energy is acquired by the analyte. This energy promoting the analyte electron from a lower-energy state (ground state) to a higher-energy, (or excited) state. Na atomic HCOH molecular energy levels energy levels Spectrophotometric methods: Absorption . The energy levels have well-defined values (i.e., they are quantized). Absorption only occurs when the photon's energy matches the difference in energy, E, between two energy levels. A plot of absorbance as a function of the photon's energy (expressed as wavelength, ) is called an absorbance spectrum. Absorption spectrum of Hg gas Absorption spectrum of chlorophyll a Why atomic spectrum has sharper peaks!!! Molecular orbital Electrons in atoms exist in atomic orbitals (consist of electronic levels only) while electrons in molecules exist in molecular orbitals (consist of electronic, vibrational and rotational levels). Each molecular orbital has energy level represent electronic state S. Between each electronic states there lies several vibrational levels V, themselves also sub-divided into a collection of rotational levels R. Molecular orbital Spectrophotometric methods: Absorption A molecule absorbs a photon by undergoing an energy transition exactly equal to the energy of the photon. The energy captured during the photon absorption can be expressed as Etot = Erot + Evib + Eelec In atoms (no sub-levels present), the energy captured during the photon absorption can be expressed as Etot = Eelec The reason why electronic absorption bands are usually very broad is that many different vibrational and rotational levels are available at slightly different energies. Therefore, a molecule could absorb photons with a fairly wide range of energies and still be promoted from the ground electronic state to one particular excited electronic state. Spectrophotometric methods: Absorption Absorbing a photon of visible light causes a valence electron in the analyte to move to a higher-energy level. When an analyte absorbs infrared radiation one of its chemical bonds experiences a change in vibrational energy level. Spectrophotometric methods: Emission Emission of a photon occurs when an analyte in a higher-energy state returns to a lower-energy state. The higher-energy state can be achieved in several ways including thermal energy, radiant energy from a photon, or by a chemical reaction. Emission following the absorption of a photon is also called photoluminescence, and that following a chemical reaction is called chemiluminescence. Spectrophotometric methods: Emission In many cases molecules in the excited states loss some energy before emission process occurs. This will give emitted radiations with lower energies than those generated the absorption in first place. The loss of energy could be happened through several radiative or non- radiative deactivation processes such as: vibrational relaxation, internal or external conversions and through intersystem crossing process. In general Eemission = Eabsorption - Erelaxation (and/or -Einternal conversion and/or -Eintersystem crossing) Note that, as the E decrease, the will increase. Spectrophotometric methods: Jablonski diagram Spectrophotometric methods: Jablonski diagram . Absorption Radiative transition from ground state to higher states with the same spin quantum number (S0 S0+n). This may include changing vibrational levels also. Relaxation (R) Radiationless transition between vibrational levels in the same electronic state ( Vn Vn-1). The definition also cover the transition to Vn-2, Vn-3….until V0. Internal Conversion (IC) Radiationless transition between states with same spin quantum numbers ( Sn Sn-1). The transition includes changing the vibrational levels. Intersystem Crossing (ISC) Radiationless transition between states with different spin quantum numbers ( S1 T1). Changing vibrational levels is expected also. Fluorescence Radiation transition between states with the same spin quantum number (S1 S0). The transition may include changing in the vibrational levels. Phosphorescence Radiation transition between states with different spin quantum number (T1 S0) including changing vibrational levels. The UV/Vis spectrum UV/Vis spectrometers collect the data (transmittance or absorbance) over the required range of wavelengths and generate the spectrum of the compound under analysis as a graph. The spectrum exhibit peaks over the investigated wavelengths range. The wavelength at which the top of the peak occurs is called max (lambda max). Some compounds show more than one max. Spectrum profile is affected by several conditions like : sample state, pH, solvent nature, presented metal ions, temperature and concentration. The UV/Vis spectrum . Other examples: . The recorded spectra of compounds in the condensed phase, whether pure or in solution, generally present absorption bands that are both few and broad, while those spectra obtained from samples in the gas state yield spectra of detailed ‘fine structure’. Electronic transitions of organic compounds Organic compounds represent the majority of the studies made in the UV/Vis. The observed transitions involve electrons engaged in or or non-bonding n electron
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