Downloaded from www.pharmacy123.blogfa.com SPECTROSCOPY سپکتروسکوپی Downloaded from www.pharmacy123.blogfa.com.

Downloaded from www.pharmacy123.blogfa.com
SPECTROSCOPY سپکتروسکوپی Downloaded from

دیپارتمنت کیمیای فارمسی وکنترول ادویه
Definition : Spectroscopy - The study of the interaction of electromagnetic radiation with matter . دیپارتمنت کیمیای فارمسی وکنترول ادویه

Electromagnetic radiation :
An oscillating electric and magnetic field which travels through space A discrete series of “particles” that possess a specific energy but have no Mass BOTH! دیپارتمنت کیمیای فارمسی وکنترول ادویه

Properties of Light Downloaded from Light can be thought of as a wave or particle. The wavelength, , is the distance between crests of a wave (m) The frequency, , is the number of oscillations per second (Hz) دیپارتمنت کیمیای فارمسی وکنترول ادویه

Introduction of Spectrometric Analyses
Downloaded from Introduction of Spectrometric Analyses The study how the chemical compound interacts with different wavelengths in a given region of electromagnetic radiation is called spectroscopy or spectrochemical analysis. The collection of measurements signals (absorbance) of the compound as a function of electromagnetic radiation is called a spectrum. دیپارتمنت کیمیای فارمسی وکنترول ادویه

Energy Absorption The mechanism of absorption energy is different in the Ultraviolet, Infrared, and Nuclear magnetic resonance regions. However, the fundamental process is the absorption of certain amount of energy. The energy required for the transition from a state of lower energy to a state of higher energy is directly related to the frequency of electromagnetic radiation that causes the transition. دیپارتمنت کیمیای فارمسی وکنترول ادویه

Regions of the electromagnetic spectrum :
دیپارتمنت کیمیای فارمسی وکنترول ادویه

Interaction of e.m.r. with Matter
Interaction of electromagnetic radiation with matter The wave-length, l, and the wave number, v’, of e.m.r. changes with the medium it travels through, because of the refractive index of the medium; the frequency, v, however, remains unchanged Types of interactions Absorption Reflection Transmission Scattering Refraction Each interaction can disclose certain properties of the matter When applying e.m.r. of different frequency (thus the energy e.m.r. carried) different type information can be obtained . refraction transmission absorption reflection scattering دیپارتمنت کیمیای فارمسی وکنترول ادویه

Absorption and Emission of Photons

Spectral Distribution of Radiant Energy Wave Number (cycles/cm) X-Ray UV Visible IR Microwave 200nm 400nm 800nm Wavelength (nm) دیپارتمنت کیمیای فارمسی وکنترول ادویه

Electromagnetic Radiation V = Wave Number (cm-1) l = Wave Length C = Velocity of Radiation (constant) = 3 x 1010 cm/sec. u = Frequency of Radiation (cycles/sec) The energy of photon: h (Planck's constant) = 6.62 x (Ergsec) C = u دیپارتمنت کیمیای فارمسی وکنترول ادویه

Spectral Properties, Application and Interactions of Electromagnetic Radiation Type Radiation Type spectroscopy Type Quantum Transition Energy Wave Number V Wavelength λ Frequency υ Kcal/mol eV cm-1 cm Hz 9.4 x 107 4.9 x 106 3.3 x 1010 3 x 10-11 1021 9.4 x 103 4.9 x 102 3.3 x 106 3 x 10-7 1017 9.4 x 101 4.9 x 100 3.3 x 104 3 x 10-5 1015 9.4 x 10-1 4.9 x 10-2 3.3 x 102 3 x 10-3 1013 9.4 x 10-3 4.9 x 10-4 3.3 x 100 3 x 10-1 1011 9.4 x 10-7 4.9 x 10-8 3.3 x 10-4 3 x 103 107 Gamma ray Gamma ray emission Nuclear X-ray absorption, emission Electronic (inner shell) X-ray Ultra violet UV absorption Electronic (outer shell) Visible Infrared IR absorption Molecular vibration Molecular rotation Micro-wave Microwave absorption Magnetically induced spin states Nuclear magnetic resonance Radio دیپارتمنت کیمیای فارمسی وکنترول ادویه

Atomic Spectra n = 1 n = 2 n = 3, etc. energy DE ground state Excited Shell structure & energy level of atoms In an atom there are a number of shells and of subshells where e-’s can be found The energy level of each shell & subshell are different and quantised The e-’s in the shell closest to the nuclei has the lowest energy. The higher shell number is, the higher energy it is The exact energy level of each shell and subshell varies with substance Ground state and excited state of e-’s Under normal situation an e- stays at the lowest possible shell - the e- is said to be at its ground state Upon absorbing energy (excited), an e- can change its orbital to a higher one - we say the e- is at an excited state. Energy n=1 n=2 n=3 n=4 1s 2s 2p 3s 3p 4s 3d 4p 4d 4f دیپارتمنت کیمیای فارمسی وکنترول ادویه

Atomic Spectra n = 1 n = 2 n = 3, etc. energy DE Electron excitation The excitation can occur at different degrees low E tends to excite the outmost e-’s first when excited with a high E (photon of high v) an e- can jump more than one levels even higher E can tear inner e-’s away from nuclei An e- at its excited state is not stable and tends to return its ground state If an e- jumped more than one energy levels because of absorption of a high E, the process of the e- returning to its ground state may take several steps, - i.e. to the nearest low energy level first then down to next … Energy n=1 n=2 n=3 n=4 1s 2s 2p 3s 3p 4s 3d 4p 4d 4f دیپارتمنت کیمیای فارمسی وکنترول ادویه

Atomic Spectra n = 1 n = 2 n = 3, etc. energy DE Atomic spectra The level and quantities of energy supplied to excite e-’s can be measured & studied in terms of the frequency and the intensity of an e.m.r. - the absorption spectroscopy The level and quantities of energy emitted by excited e-’s, as they return to their ground state, can be measured & studied by means of the emission spectroscopy The level & quantities of energy absorbed or emitted (v & intensity of e.m.r.) are specific for a substance Atomic spectra are mostly in UV (sometime in visible) regions Energy n=1 n=2 n=3 n=4 1s 2s 2p 3s 3p 4s 3d 4p 4d 4f دیپارتمنت کیمیای فارمسی وکنترول ادویه

Absorption Spectroscopy Introduction A.) Absorption: electromagnetic (light) energy is transferred to atoms, ions, or molecules in the sample. Results in a transition to a higher energy state. Transition can be change in electronic levels, vibrations, rotations, translation, etc. Concentrate on Molecular Spectrum in UV/Vis (electronic transition) Power (P): energy of a beam that reaches a given area per second Intensity (I): power per unit solid angle P and I related to amplitude2 (excited state) Energy required of photon to give this transition: hn = DE = E1 - Eo (ground state) دیپارتمنت کیمیای فارمسی وکنترول ادویه

Amount of light absorbed is dependent on frequency (l)
B.) Terms: 1.) Beer’s Law: A = ebc The amount of light absorbed (A) by a sample is dependent on the path length (b), concentration of the sample (c) and a proportionality constant (e – molar absorptivity) Amount of light absorbed is dependent on frequency (l) c Increasing Fe+2 concentration  دیپارتمنت کیمیای فارمسی وکنترول ادویه Absorbance is directly proportional to concentration Fe+2

Transmittance (T) = P/Po %Transmittance = %T = 100T
B.) Terms: 1.) Beer’s Law: A = ebc Transmittance (T) = P/Po %Transmittance = %T = 100T Absorbance (A) = log10 Po/P No light absorbed- % transmittance is 100%  absorbance is 0 All light absorbed- % transmittance is 0%  absorbance is infinite دیپارتمنت کیمیای فارمسی وکنترول ادویه

Relationship Described in Terms of Beer’s Law A = Absorbance = ebc = -log(%T/100) e = molar absorptivity: constant for a compound at a given frequency (l) units of L mol-1 cm-1 b = path length: cell distance in cm c = concentration: sample concentration in moles per liter. Therefore, by measuring absorbance or percent transmittance at a given frequency can get information related to the amount of sample (c) present with an identified e and l. Note: law does not hold at high concentrations, when A > 1 دیپارتمنت کیمیای فارمسی وکنترول ادویه

Molar Absorptivity A = lc  is a measure of the amount of light absorbed per unit concentration at a particular . Molar absorptivity is a constant for a particular substance, so if the concentration of the solution is halved, so is the absorbance at sufficiently dilute concentrations.  (epsilon) is commonly referred to as the absorption coefficient. A concentration دیپارتمنت کیمیای فارمسی وکنترول ادویه

Cuvettes (sample holder)
Polystyrene nm Methacrylate nm Glass nm Suprasil Quartz nm دیپارتمنت کیمیای فارمسی وکنترول ادویه

UV-Visible Spectrophotometry
سپکتروفوتومتری ماورای بنفش – قابل دید

UV-Visible Spectrophotometry
Downloaded from UV-Visible Spectrophotometry The absorption of ultraviolet and visible radiation by molecules are dependent upon the electronic structure of the molecule. So the ultraviolet and visible spectrum are called electronic spectrum. دیپارتمنت کیمیای فارمسی وکنترول ادویه

What does the absorbed light (electromagnetic radiation) do to the molecule? IR UV 700 nm visible 400 nm Energy increasing high energy UV – ionizes electrons low energy UV and visible – promotes electrons to higher energy orbitals (absorption of visible light leads to a colored solution) IR – causes molecules to vibrate (more later) دیپارتمنت کیمیای فارمسی وکنترول ادویه

UV/visible light absorption
Valence electrons In organic molecules, electronic transitions to higher energy molecular orbitals – double bonds: p  p* In transition metals, hydrated ions as Cu++ have splitting of d orbital energies and electronic transitions – weak absorption In complexed transition metals, charge transfer of electrons from metal to ligand as Cu(NH3)4++ – strong absorption دیپارتمنت کیمیای فارمسی وکنترول ادویه

Electronic Excitation
Downloaded from Electronic Excitation The absorption of light energy by organic compounds in the visible and ultraviolet region involves the promotion of electrons in , , and n-orbitals from the ground state to higher energy states. This is also called energy transition. These higher energy states are molecular orbitals called antibonding. دیپارتمنت کیمیای فارمسی وکنترول ادویه

Electronic Molecular Energy Levels
Downloaded from Electronic Molecular Energy Levels The higher energy transitions ( *) occur a shorter wavelength and the low energy transitions (*, n *) occur at longer wavelength. دیپارتمنت کیمیای فارمسی وکنترول ادویه

Electronic Transitions in Organic Molecules

s and s* orbitals دیپارتمنت کیمیای فارمسی وکنترول ادویه

p and p* orbitals دیپارتمنت کیمیای فارمسی وکنترول ادویه

Electronic Transitions: p  p*
The p  p* transition involves orbitals that have significant overlap, and the probability is near 1.0 as they are “symmetry allowed”. دیپارتمنت کیمیای فارمسی وکنترول ادویه

p  p* transitions - Triple bonds
Organic compounds with -C≡C- or -C≡N groups, or transition metals complexed by C≡N- or C≡O ligands, usually have “low-lying” p* orbitals دیپارتمنت کیمیای فارمسی وکنترول ادویه

Electronic Transitions: n  p*
The n-orbitals do not overlap at all well with the p* orbital, so the probability of this excitation is small. The e of the np* transition is about 103 times smaller than e for the pp* transition as it is “symmetry forbidden”. دیپارتمنت کیمیای فارمسی وکنترول ادویه

UV Activity hn p p* دیپارتمنت کیمیای فارمسی وکنترول ادویه

Excited States دیپارتمنت کیمیای فارمسی وکنترول ادویه

Chemical Structure & UV Absorption What is chromophore ? Chromophore is a functional group which absorbs a characteristic ultraviolet or visible region. Chromophoric Group ---- The groupings of the molecules which contain the electronic system which is giving rise to absorption in the ultra-violet region. دیپارتمنت کیمیای فارمسی وکنترول ادویه

Chromophore absorptions
Example Excitation lmax, nm e Solvent C=C Ethene p __> p* 171 15,000 hexane CC 1-Hexyne 180 10,000 C=O Ethanal n __> p* p __> p* 290 15 N=O Nitromethane 275 200 17 5,000 ethanol C-X X=Br X=I Methyl bromide Methyl Iodide n __> s* 205 255 360 دیپارتمنت کیمیای فارمسی وکنترول ادویه

Organic Chromophores Chromophore Transition Lmax(nm) log(e) Nitrile (-C≡N) h to p* 160 <1.0 Alkyne (-C≡C-) p to p* 170 3.0 Alkene (-C=C-) 175 Alcohol (ROH) h to s* 180 2.5 Ether (ROR) 3.5 Ketone (-C(R)=O) 280 1.5 Aldehyde (–C(H)=O) 190 2.0 290 1.0 Amine (-NR2) Acid (-COOH) 205 Ester (-COOR) Amide (-C(=O)NH2) 210 Thiol (-SH) Nitro (-NO2) 271 Azo (-N=N-) 340 دیپارتمنت کیمیای فارمسی وکنترول ادویه

Single Beam Spectrophotometer

Dual Beam Spectrophotometer

Sample Cells UV Spectrophotometer Quartz (crystalline silica) Visible Spectrophotometer Glass دیپارتمنت کیمیای فارمسی وکنترول ادویه

Cuvettes (sample holder)
Polystyrene nm Methacrylate nm Glass nm Suprasil Quartz nm دیپارتمنت کیمیای فارمسی وکنترول ادویه

Components of an Instrument for UV/Vis Absorbance Measurements: 1.) Basic Design: Hitachi Instruments U-3010 Light Source, l selector, Sample cell holder, Detector (amplifier, recorder) دیپارتمنت کیمیای فارمسی وکنترول ادویه

a) Desired Properties of Components of UV/Vis: Light Source l Selector Creates Proper l Narrow Bandpass: Stable: Selects Desired l Constant P Large Light Throughput: Good Precision Increase P Intense: Increase P Easier to See Absorbance Sample Cell Holder Detector Fixed Geometry: Stable Constant b Sensitive to l of Interest Transmits l of Interest: دیپارتمنت کیمیای فارمسی وکنترول ادویه

b) Light Sources UV/Vis (~ 200 – 800 nm): 1. Deuterium & Hydrogen Lamps (UV range) - continuous source, broad range of frequencies - based on electric excitation of H2 or D2 at Low pressure In presence of arc, some of the electrical energy is absorbed by D2 (or H2) which results in the disassociation of the gas and release of light D2 + Eelect  D*2  D’ + D’’ + hn (light produced) Excited state دیپارتمنت کیمیای فارمسی وکنترول ادویه

2. Tungsten Filament Lamp (Vis – Near IR) - continuous source, broad range of frequencies - based on black body radiation: heat solid filament to glowing, light emitted will be characteristic of temperature more than nature of solid filament Low pressure (vacuum) Tungsten Filament دیپارتمنت کیمیای فارمسی وکنترول ادویه Temperature Dependence of l

b) Wavelength Selectors: 1. Monochromator - separates frequencies (l) from polychromatic light in time or space. - allows only certain l’s to be selected and used. i.) Dispersing Monochromator: a) Prism: based on refraction of light and fact that different l’s have different values of refraction index (hi) in a medium. دیپارتمنت کیمیای فارمسی وکنترول ادویه

UV vs. IR vs. NMR UV has broad peaks relative to IR & NMR UV has less information than IR & NMR UV spectra are easier to collect UV spectra are faster to collect UV spectrometers are cheaper UV spectra require only nanograms of material or chemicals دیپارتمنت کیمیای فارمسی وکنترول ادویه

The process of light being absorbed by a solution concentration 2 with sample I < Io concentration 1 blank where Io = I light source detector Io I As concentration increased, less light was transmitted (more light absorbed). b Cell with Pathlength, b, containing solution دیپارتمنت کیمیای فارمسی وکنترول ادویه

Some terminology I – intensity where Io is initial intensity T – transmission or %T = 100 x T (absorption: Abs = 1 – T or %Abs = %T) T = I/ Io A – absorbance A = - log T = -log I/ Io دیپارتمنت کیمیای فارمسی وکنترول ادویه

Beer’s Law A = abc where a – molar absorptivity, b – pathlength, and c – molar concentration See the Beer’s Law Simulator دیپارتمنت کیمیای فارمسی وکنترول ادویه

Analyze at what wavelength?
Scan visible wavelengths from 400 – 650 nm (detector range) to produce an absorption spectrum (A vs. l) lmax phototube detector range lmax - wavelength where maximum absorbance occurs دیپارتمنت کیمیای فارمسی وکنترول ادویه

The BLANK The blank contains all substances except the analyte. Is used to set the absorbance to zero: Ablank = 0 This removes any absorption of light due to these substances and the cell. All measured absorbance is due to analyte. دیپارتمنت کیمیای فارمسی وکنترول ادویه

The components of a Spec-20D
Light source - white light of constant intensity slits filter occluder Grating slits Separates white light into various colors Phototube detects light & measures intensity Rotating the grating changes the wavelength going through the sample Sample When blank is the sample Io is determined otherwise I is measured دیپارتمنت کیمیای فارمسی وکنترول ادویه

Uses of visible spectrophotometry
Analysis of unknowns using Beer’s Law calibration curve Absorbance vs. time graphs for kinetics Single-point calibration for an equilibrium constant determination Spectrophotometric titrations – a way to follow a reaction if at least one substance is colored – sudden or sharp change in absorbance at equivalence point, a piece-wise function (Been there, done that!) دیپارتمنت کیمیای فارمسی وکنترول ادویه

Practical Applications
Medicinal Chemistry compound ID (steroids, nucleosides) monitoring isomerization, chirality Pharmaceutical Biotechnology concentration/purity measurements monitoring conformation of protein drugs Pharmacokinetics/Med. Chem. HPLC monitoring and purification دیپارتمنت کیمیای فارمسی وکنترول ادویه

Quantitative Analysis (Beer’s Law):
1) Widely used for Quantitative Analysis Characterization - wide range of applications (organic & inorganic) - limit of detection  10-4 to 10-5 M (10-6 to 10-7M; current) - moderate to high selectivity - typical accuracy of 1-3% ( can be ~0.1%) - easy to perform, cheap 2) Strategies a) absorbing species - detect both organic and inorganic compounds containing any of these species (all the previous examples) Chromophore Example Excitation lmax, nm e Solvent C=C Ethene p __> p* 171 15,000 hexane 1-Hexyne 180 10,000 C=O Ethanal n __> p* p __> p* 15 10,000 hexane hexane N=O Nitromethane 17 5,000 ethanol ethanol C-X X=Br X=I Methyl bromide Methyl Iodide n __> s* n __> s* دیپارتمنت کیمیای فارمسی وکنترول ادویه

b) non- absorbing species - react with reagent that forms colored product - can also use for absorbing species to lower limit of detection - items to consider: l, pH, temperature, ionic strength - prepare standard curve (match standards and samples as much as possible) Standard Addition Method (spiking the sample) - used for analytes in a complex matrix where interferences in the UV/Vis for the analyte will occur: i.e. blood, sediment, human serum, etc.. - Method: (1) Prepare several identical aliquots, Vx, of the unknown sample. (2) Add a variable volume, Vs, of a standard solution of known concentration, cs, to each unknown aliquot. (3) Dilute each solution to an equal volume, Vt. (4) Make instrumental measurements of each sample to get an instrument response, IR. (5) Calculate unknown concentration, cx, from the following equation. Note: This method assumes a linear relationship between instrument response and sample concentration. دیپارتمنت کیمیای فارمسی وکنترول ادویه

Downloaded from www.pharmacy123.blogfa.com SPECTROSCOPY سپکتروسکوپی Downloaded from www.pharmacy123.blogfa.com.

Similar presentations

Presentation on theme: "Downloaded from www.pharmacy123.blogfa.com SPECTROSCOPY سپکتروسکوپی Downloaded from www.pharmacy123.blogfa.com."— Presentation transcript:

Similar presentations

About project

Feedback

Log in

Auth with social network:

Downloaded from www.pharmacy123.blogfa.com SPECTROSCOPY سپکتروسکوپی Downloaded from www.pharmacy123.blogfa.com.

Similar presentations

Presentation on theme: "Downloaded from www.pharmacy123.blogfa.com SPECTROSCOPY سپکتروسکوپی Downloaded from www.pharmacy123.blogfa.com."— Presentation transcript:

Similar presentations

About project

Feedback