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1 SPECTROSCOPY Downloaded from

2 دیپارتمنت کیمیای فارمسی وکنترول ادویه2 Definition : Spectroscopy - The study of the interaction of electromagnetic radiation with matter.

3 دیپارتمنت کیمیای فارمسی وکنترول ادویه3 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!

4 دیپارتمنت کیمیای فارمسی وکنترول ادویه4 Properties of Light 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)

5 دیپارتمنت کیمیای فارمسی وکنترول ادویه5 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.

6 دیپارتمنت کیمیای فارمسی وکنترول ادویه6 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.

7 دیپارتمنت کیمیای فارمسی وکنترول ادویه7 Regions of the electromagnetic spectrum :

8 دیپارتمنت کیمیای فارمسی وکنترول ادویه8 Interaction of e.m.r. with Matter Interaction of electromagnetic radiation with matter –The wave-length,, 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

9 دیپارتمنت کیمیای فارمسی وکنترول ادویه9 Absorption and Emission of Photons

10 دیپارتمنت کیمیای فارمسی وکنترول ادویه10 Wave Number (cycles/cm) X-RayUV VisibleIR Microwave 200nm 400nm800nm Wavelength (nm) Spectral Distribution of Radiant Energy

11 دیپارتمنت کیمیای فارمسی وکنترول ادویه11 V = Wave Number (cm -1 ) Wave Length C = Velocity of Radiation (constant) = 3 x cm/sec. = Frequency of Radiation (cycles/sec) The energy of photon: h (Planck's constant) = 6.62 x (Erg sec) C = Electromagnetic Radiation

12 دیپارتمنت کیمیای فارمسی وکنترول ادویه12 Visible Ultra violet Radio Gamma ray Hz cmcm -1 Kcal/moleV Type Quantum Transition Type spectroscopy Type Radiation Frequency υ Wavelength λ Wave Number VEnergy 9.4 x x x x x x x x x x x x x x x x x x x x x x x x X-ray Infrared Micro- wave Gamma ray emission X-ray absorption, emission UV absorption IR absorption Microwave absorption Nuclear magnetic resonance Nuclear Electronic (inner shell) Molecular vibration Electronic (outer shell) Molecular rotation Magnetically induced spin states Spectral Properties, Application and Interactions of Electromagnetic Radiation

13 دیپارتمنت کیمیای فارمسی وکنترول ادویه13 Atomic Spectra 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. n = 1 n = 2 n = 3, etc. energy E ground state Excited state Energy n=1 n=2 n=3 n=4 1s 2s 2p 3s 3p 4s 3d 4p 4d 4f

14 دیپارتمنت کیمیای فارمسی وکنترول ادویه14 Atomic Spectra 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 … n = 1 n = 2 n = 3, etc. energy E Energy n=1 n=2 n=3 n=4 1s 2s 2p 3s 3p 4s 3d 4p 4d 4f

15 دیپارتمنت کیمیای فارمسی وکنترول ادویه15 Atomic Spectra 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 n = 1 n = 2 n = 3, etc. energy E Energy n=1 n=2 n=3 n=4 1s 2s 2p 3s 3p 4s 3d 4p 4d 4f

16 دیپارتمنت کیمیای فارمسی وکنترول ادویه16 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 amplitude 2 Energy required of photon to give this transition: h E = E 1 - E o (excited state) (ground state)

17 دیپارتمنت کیمیای فارمسی وکنترول ادویه17 B.)Terms: 1.) Beers Law: A = bc 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 ( – molar absorptivity ) Amount of light absorbed is dependent on frequency ( ) c Absorbance is directly proportional to concentration Fe +2 Increasing Fe +2 concentration

18 دیپارتمنت کیمیای فارمسی وکنترول ادویه18 B.)Terms: 1.) Beers Law: A = bc Transmittance (T) = P/P o %Transmittance = %T = 100T Absorbance (A) = log 10 P o /P No light absorbed- % transmittance is 100% absorbance is 0 All light absorbed- % transmittance is 0% absorbance is infinite

19 دیپارتمنت کیمیای فارمسی وکنترول ادویه19 Relationship Described in Terms of Beers Law A = Absorbance = bc = -log(%T/100) = molar absorptivity : constant for a compound at a given frequency ( ) 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 and. Note: law does not hold at high concentrations, when A > 1

20 دیپارتمنت کیمیای فارمسی وکنترول ادویه20 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. Molar Absorptivity A = lc A concentration

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48 دیپارتمنت کیمیای فارمسی وکنترول ادویه48 Cuvettes (sample holder) Polystyrene – nm Methacrylate – nm Glass – nm Suprasil Quartz – nm

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55 UV-Visible Spectrophotometry سپکتروفوتومتری ماورای بنفش – قابل دید

56 دیپارتمنت کیمیای فارمسی وکنترول ادویه56 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.

57 دیپارتمنت کیمیای فارمسی وکنترول ادویه57 What does the absorbed light (electromagnetic radiation) do to the molecule? 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) 700 nm400 nm IR UV visible Energy increasing

58 دیپارتمنت کیمیای فارمسی وکنترول ادویه58 UV/visible light absorption In organic molecules, electronic transitions to higher energy molecular orbitals – double bonds: * 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(NH 3 ) 4 ++ – strong absorption Valence electrons

59 دیپارتمنت کیمیای فارمسی وکنترول ادویه59 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.

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61 دیپارتمنت کیمیای فارمسی وکنترول ادویه61 Electronic Molecular Energy Levels The higher energy transitions ( *) occur a shorter wavelength and the low energy transitions ( *, n *) occur at longer wavelength.

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63 دیپارتمنت کیمیای فارمسی وکنترول ادویه63 Electronic Transitions in Organic Molecules

64 دیپارتمنت کیمیای فارمسی وکنترول ادویه64 and * orbitals

65 دیپارتمنت کیمیای فارمسی وکنترول ادویه65 and * orbitals

66 دیپارتمنت کیمیای فارمسی وکنترول ادویه66 Electronic Transitions: * The * transition involves orbitals that have significant overlap, and the probability is near 1.0 as they are symmetry allowed.

67 دیپارتمنت کیمیای فارمسی وکنترول ادویه67 * transitions - Triple bonds Organic compounds with -CC- or -CN groups, or transition metals complexed by CN - or CO ligands, usually have low- lying * orbitals

68 دیپارتمنت کیمیای فارمسی وکنترول ادویه68 Electronic Transitions: n * The n-orbitals do not overlap at all well with the * orbital, so the probability of this excitation is small. The of the n * transition is about 10 3 times smaller than for the * transition as it is symmetry forbidden.

69 دیپارتمنت کیمیای فارمسی وکنترول ادویه69 UV Activity h

70 دیپارتمنت کیمیای فارمسی وکنترول ادویه70 Excited States

71 دیپارتمنت کیمیای فارمسی وکنترول ادویه71 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.

72 دیپارتمنت کیمیای فارمسی وکنترول ادویه72 Chromophore absorptions ChromophoreExampleExcitation max, nm Solvent C=CEthene 17115,000hexane C 1-Hexyne 18010,000hexane C=OEthanal n ,000 hexane N=ONitromethane n ,000 ethanol C-X X=Br X=I Methyl bromide Methyl Iodide n hexane

73 دیپارتمنت کیمیای فارمسی وکنترول ادویه73 Organic Chromophores Chromophore Transition max (nm) log( ) Nitrile (-CN) to 160<1.0 Alkyne (-CC-) to Alkene (-C=C-) to Alcohol (ROH) to Ether (ROR) to Ketone (-C(R)=O) to to Aldehyde (–C(H)=O) to to Amine (-NR 2 ) to Acid (-COOH) to Ester (-COOR) to Amide (-C(=O)NH 2 ) to Thiol (-SH) to Nitro (-NO 2 ) to 271<1.0 Azo (-N=N-) to 340<1.0

74 دیپارتمنت کیمیای فارمسی وکنترول ادویه74 Single Beam Spectrophotometer

75 دیپارتمنت کیمیای فارمسی وکنترول ادویه75 Dual Beam Spectrophotometer

76 دیپارتمنت کیمیای فارمسی وکنترول ادویه76 Sample Cells UV Spectrophotometer Quartz (crystalline silica) Visible Spectrophotometer Glass

77 دیپارتمنت کیمیای فارمسی وکنترول ادویه77 Cuvettes (sample holder) Polystyrene – nm Methacrylate – nm Glass – nm Suprasil Quartz – nm

78 دیپارتمنت کیمیای فارمسی وکنترول ادویه78 Components of an Instrument for UV/Vis Absorbance Measurements: 1.) Basic Design: Hitachi Instruments U-3010 Light Source, selector, Sample cell holder, Detector (amplifier, recorder)

79 دیپارتمنت کیمیای فارمسی وکنترول ادویه79 a) Desired Properties of Components of UV/Vis: Light Source Selector Creates Proper Narrow Bandpass: Stable: Selects Desired Constant PLarge Light Throughput: Good PrecisionIncrease P Intense: Increase P Easier to See Absorbance Sample Cell Holder Detector Fixed Geometry:Stable Constant bSensitive to of Interest Transmits of Interest: Increase P

80 دیپارتمنت کیمیای فارمسی وکنترول ادویه80 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 H 2 or D 2 at Low pressure In presence of arc, some of the electrical energy is absorbed by D 2 (or H 2 ) which results in the disassociation of the gas and release of light D 2 + E elect D * 2 D + D + h (light produced) Excited state

81 دیپارتمنت کیمیای فارمسی وکنترول ادویه81 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 Temperature Dependence of

82 دیپارتمنت کیمیای فارمسی وکنترول ادویه82 b) Wavelength Selectors: 1. Monochromator - separates frequencies ( ) from polychromatic light in time or space. - allows only certain s to be selected and used. i.) Dispersing Monochromator: a) Prism: based on refraction of light and fact that different s have different values of refraction index ( i ) in a medium.

83 دیپارتمنت کیمیای فارمسی وکنترول ادویه83 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

84 دیپارتمنت کیمیای فارمسی وکنترول ادویه84 IoIo I Cell with Pathlength, b, containing solution light source detector blank where I o = I concentration 2 concentration 1 b with sample I < I o The process of light being absorbed by a solution As concentration increased, less light was transmitted (more light absorbed).

85 دیپارتمنت کیمیای فارمسی وکنترول ادویه85 Some terminology I – intensity where I o is initial intensity T – transmission or %T = 100 x T (absorption: Abs = 1 – T or %Abs = %T) T = I/ I o A – absorbance A = - log T = -log I/ I o

86 دیپارتمنت کیمیای فارمسی وکنترول ادویه86 Beers Law A = abc where a – molar absorptivity, b – pathlength, and c – molar concentration See the Beers Law Simulator

87 دیپارتمنت کیمیای فارمسی وکنترول ادویه87 Analyze at what wavelength? Scan visible wavelengths from 400 – 650 nm (detector range) to produce an absorption spectrum (A vs. ) max max - wavelength where maximum absorbance occurs phototube detector range

88 دیپارتمنت کیمیای فارمسی وکنترول ادویه88 The BLANK The blank contains all substances except the analyte. Is used to set the absorbance to zero: A blank = 0 This removes any absorption of light due to these substances and the cell. All measured absorbance is due to analyte.

89 دیپارتمنت کیمیای فارمسی وکنترول ادویه89 Light source Grating Rotating the grating changes the wavelength going through the sample slits Sample filter Phototube The components of a Spec-20D occluder When blank is the sample I o is determined otherwise I is measured Separates white light into various colors detects light & measures intensity - white light of constant intensity

90 دیپارتمنت کیمیای فارمسی وکنترول ادویه90 Uses of visible spectrophotometry Analysis of unknowns using Beers 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!)

91 دیپارتمنت کیمیای فارمسی وکنترول ادویه91 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

92 دیپارتمنت کیمیای فارمسی وکنترول ادویه92 Quantitative Analysis (Beers Law): 1) Widely used for Quantitative Analysis Characterization - wide range of applications (organic & inorganic) - limit of detection to M (10 -6 to M; 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) ChromophoreExampleExcitation max, nm Solvent C=CEthene __ > * 17115,000hexane C 1-Hexyne __ > * 18010,000hexane C=OEthanal n __ > * __ > * ,000hexane N=ONitromethane n __ > * __ > * ,000ethanol C-X X=Br X=I Methyl bromide Methyl Iodide n __ > * hexane

93 دیپارتمنت کیمیای فارمسی وکنترول ادویه93 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:, 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, V x, of the unknown sample. (2) Add a variable volume, V s, 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.


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