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,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Modern methods in techniques of analytical chemistry: Spectroscopic techniques.,Describe the absorption of radiation by,molecules and its relationship to molecular,structure.,Make quantitative calculations, relating,the amount of radiation absorbed to the,concentration of an absorbing analyte.,Describe the instrumentation required for,making measurements.,Scope:,The Electromagnetic Radiation,Light is a form of electromagnetic radiation.,Electromagnetic radiation can be considered as a form of radiant energy that is propagated as a transverse wave.,It vibrates perpendicular to the direction of propagation, imparts a wave motion to the radiation,Wavelength,=,c/ v,Wavenumber, = v/c,=1/,Wavelength,is the distance between the neighboring peaks of two wave.,Frequency,v,(nu) the number of cycles passing a fixed point per unit time.,Frequency,is measure in,Hertz, 1 Hz= 1 s,-1,.,Wavenumber,(nu tilde),the number of complete wavelengths in a,given length.,e.g.: A wave number of 5 cm,-1,indicates there are 5 complete,wavelength in 1 cm.,Where c = velocity of light (3 x 10,10,cm/s),Electromagnetic radiation possesses certain amount of energy,E = h,= hc/,E = energy of a photon,h = Plancks Constant (6.62 x 10-34 joule second (J-s),It is apparent that the shorter the wavelength or the greater frequency,the greater energy.,In spectrometric methods, the sample solution absorbs electromagnetic radiation from appropriate source, and the,amount absorbed is related to the concentration of analyte in the solution.,10,9,10,7,10,5,10,3,10,1,10,-1,10,-3,10,-5,10,-7,10,-9,10,-11,gamma,X-rays,Ultra Violet,Infra red,microwave,Radio waves,500,600,700,Violet, indigo,blue,Green,yellow,Orange,red,Color,Wave length (nm),violet,400-435,indigo,435-480,blue,480-500,green,500-560,yellow,560-595,orange,595-610,red,610-750,Electromagnetic Spectrum,How does matter absorb radiation?,There are 3 basic process:,Rotational transition,molecules rotate, absorb radiation and raised to higher rotational energy level,2) Vibrational transition,Atoms or group of atoms within molecule vibrate relative to each other, absorb radiation and raised to higher vibrational energy level.,3) Electronic transition,-Electrons of molecule raised to higher electron energy.,Wavelenghts and Color,Wavelength of Maximum absorption (nm),Color absorption,Color Observed,380-420,Violet,Green-yellow,420-440,Violet-blue,Yellow,440-470,Blue,Orange,470-500,Blue-green,Red,500-520,Green,Purple,520-550,Yellow-green,Violet,550-580,Yellow,Violet-blue,580-620,Orange,Blue,620-680,Red,Blue-green,680-780,Purple,Green,Irradiance or radiant power, P (Wm,-2,), is the energy per second per unit area of the beam of light.,Schematic of a simple spectrometer,.,Derivation of Beers Law,Irradiance In, P,0,Irradiance Out, P,Pathlenght,Readout,Transmittance is T=P,o,/P =10,-kb,Transmittance, T, is the fraction of original light not absorbed by the sample.,P,o,= power of incident light,P= power of transmitted light,Putting transmittance in logarithmic form:,log T = log P/P,o,= -kb,Similar law holds for the dependence of T on concentration, c,T=P,o,/P =10,-kc,and log T = log P/P,o,= -kc,Combining these two laws,T = P/P,0,=10,-abc,(Beers Law),Where,is the analytes molar absorptivity. = a x molecular weight,log T = log P/P,0,= -abc,A = -log T = log 1/T = log P,0,/P = abc,= log 100 log %T,Continue.,Where,A = absorbance,a = analytes absoptivity,b = pathlenght through the material;,c= concentration,OR,A = bc,Spectroscopy Nomenclature,Recommended Name,Unit,Absorbance (A),-,Absorptivity (a),cm,-1,g,-1,L,Pathlength (b),cm,Transmittance (T),-,Wavelength (),nm,Concentration (c),moles per liter (moles L,-1,),Example 1:,A sample in a,1.0-cm cell,is determine with a spectrometer to,transmit,80%,light at certain wavelength. If the absorptivity of this substance,at this wavelength is,2.0, What is the,concentration,of the substance?,Solution:,The percent of the transmittance is 80%, and so T = 0.80.,log 1/T =abc,log 1/0.80 = 2.0 cm,-1,g,-1,L x 1.0 cm x c,log 1.25 = 2.0 g,-1,L x c,c = 0.10/2.0 = 0.050 gL,-1,Example 2:,A solution containing,1.00 mg ion,(as thiocyanate complex) in,100 mL,was,observed to,transmit 70.0%,of the incident light compared to an appropriate,blank.,What is the,absorbance,of the solution at this wavelength?,What,fraction of light,would be,transmitted,by a solution of iron,four,times as concentrated.,Solution:,T = 0.700,A = log 1/0.700 = log 1.43 = 0. 155,(b) 0.155 = ab (0.0100 g/L),ab = 15.5 L/g,From A= abc,A = 15.5 L/g x (4x0.1000 g/L) = 0.620,log 1/T = 0.620,T = 0.240,The absorbance of the new solution could have been calculated more directly:,Beers Law Assumption/Limitation,The light being shined on the sample must be,monochromatic (one color or wavelenght),The analyte must not be participate in a,concentration dependent equilibrium,This isnt good technique for many weak,acid systems, as dilution increases,dissociation and HA and A- probably dont,have the same absorbance.,Beers Law Assumption/Limitation,Problems:,Calibration curves are found to be nonlinear because occuring of deviation.,Deviation from linearity are divided into three categories:,Fundamental,: Law is valid for low concentration analyte. At higher concentration, there will be interaction between particle of analyte that may change the value of,.,2),Chemical,: when the absorbing species is involved in an equilibrium reaction.,HA H,+,+ A,-,HA will absorbs the wavelength and contribute to,and C value. However, if the equilibrium shifts to right, less HA available for absorption and will result in non linearity of the curve.,3),Instrumentation,: 2 principal limitation:,i) Stricly valid for purely monochromatic instrumentation.,ii) The effect of leakage light from imperfections within wavelength selector. This phenomena is called stray radiation.,Deviation from beers law:continue,From the beers law, the absorbance against the conc.,A straight line passing through origin is obtained (linear graph),However, deviation might occurs. Deviation is due to the following factors:-,A foreign substance having colour particle may affect the absorption & extinction coefficient.,Deviation also occur if colored solute ionized or dissociates in the solution; e.g.- benzyl alcohol in chloroform,Due to the presence of impurities that fluoresce or absorb at the absorption wave length.,If monochromatic light is not used deviation may occurs.,If width of the slit is not proper.,If the solution species undergoes polymerization,Block of Diagram - Spectrometer,Source,Monochromator,Sample,Detector,Readout,(meter or recorder),Sources : Tungsten lamp (visible), hydrogen or deuterium discharge tube (ultraviolet), hot wires, light bulb or glowing seramic (IR), laser,Monochromator: prism, difraction grating, optical filters.,Sampel cell: cuvets, KBr, UV/IR quartz.,Detector: phototube, photomultiplier tube, spectrophotometer (UV),thermocouples, bolometers (IR),Two types of Monochromator,Grating:,A fundamental property of gratings is that the angle of deviation of all but one of the diffracted beams depends on the wavelength of the incident light.,Therefore, a grating separates an incident polychromatic beam into its constituent wavelength components, i.e., it is dispersive. Because of their dispersive properties, gratings are commonly used in monochromators and spectrometers.,Two types of Monochromator,Prism:,Electromagnetic radiation is refracted because index of refraction of prism material is different from air.,Shorter wavelengths are refracted more than longer wavelength,The effects of refraction is to spread the wavelength apart into different wavelength,By rotating the prism, different wavelength can be made to pass through an exit slid and to the sample,To do a Spectroscopic Analysis,You need:,A continuous light source,A wavelength selector,A sample cell,A detector,The sample cell is called,cuvet,and can be made of many substances,Glass (good for visible),Quartz (UV-vis),NaCl/KBr (IR),Spectroscopic Procedure,You may have a single-beam or double beam,Single-beam instrument has one sample holder, you must swap blank and sample,Double-beam instrument splits light output between two holders so you can measure blank and sample,A,baseline,spectrum is a spectrum of a reference solution (solvent or reagent blank),We try to do an analysis at the,max,if we can,Sensitivity is greatest at maximum absorbance,Curve is relatively flat in case the monochromator drifts and is off by a little in wavelength,The Single-Beam Spectrometer,How Do UV spectrometers work?,Two photomultiplier inputs, differential voltage drives amplifier.,Matched,quartz cuvettes,Sample in solution at ca. 10,-5,M,.,System protects PM tube from stray light,D2 lamp-UV,Tungsten lamp-Vis,Double Beam makes it a difference technique,Rotates, to achieve scan,The polychromatic light is separated into monochromatic wavelengths by diffraction. n, = d(sin + sin ),Optics of the Grating Monochromator,In the equation n, = d(sin + sin ) n is the order of the diffraction n = 1, 2, 3 etc, d is the number of lines etched on the grating, is the angle of the incident beam and is the angle of the emerging beam.,Optics of the Grating Monochromator,The photodiode array detector,The photodiode array detector,Key components:,Light Source,Monochromator,Sample/reference holder,Radiation detection,Readout device,UV Instrumentation,Spectroscopic Procedure,You should always try to keep the absorbance reading of your sample below 1.,Because % transmittance is related logarithmically with concentration, it means that from 1-99% transmittance you can detect 2 orders of magnitude in analyte concentration.,Any orders of magnitude greater than that will be detected in the range of 0-1% T.,In order to maximize accuracy, you should dilute the solution if you have to so that the transmittance reading is not maxed out in that region.,An Electronic Spectrum,Absorbance,Wavelength,generally in nanometers (nm),0.0,400,800,1.0,200,UV,Visible,max,with certain extinction,Make solution of concentration low enough that A 1,(Ensures Linear Beers law behavior),Even though a dual beam goes through a solvent blank, choose solvents that are UV transparent.,Can extract the,value if conc. (,M,) and b (cm) are known,UV bands are much broader than the photonic transition event. This is because,vibration levels,are superimposed on UV.,Ultraviolet Spectroscopy,200-400 nm photons excite electrons from a, bonding orbital to a * antibonding orbital.,Conjugated dienes have MOs that are closer in energy.,A compound that has a longer chain of conjugated double bonds absorbs light at a longer wavelength. =,Chromophore,Example,Solvent,l,max,(nm),e,max,Type of transition,Alkene,n,-Heptane,177,13,000,p,p,*,Alkyne,n,-Heptane,178,196,225,10,000,2,000,160,p,p,*,_,_,Carbonyl,n,-Hexane,n,-Hexane,186,280,180,293,1,000,16,Large,12,n,s,*,n,p,*,n,s,*,n,p,*,Carboxyl,Ethanol,204,41,n,p,*,Amido,Water,214,60,n,p,*,Azo,Ethanol,339,5,n,p,*,Nitro,CH,3,NO,2,Isooctane,280,22,n,p,*,Nitroso,C,4,H,9,NO,Ethyl ether,300,665,100,20,_,n,p,*,Nitrate,C,2,H,5,ONO,2,Dioxane,270,12,n,p,*,Absorption Characteristics of Some Common Chromophores,Solvents for UV (showing high energy cut-offs),Water205,CH,3,C,N210,C,6,H,12,210,Ether210,EtOH210,Hexane210,MeOH210,Dioxane220,THF220,CH,2,Cl,2,235,CHCl,3,245,CCl,4,265,benzene280,Acetone300,Various buffers for HPLC, check before using.,Deviation from Beers Law,Beers law is only valid for low concentration, up to 10 mM;,The intermolecular distances in a given solution will decrease, eventually reach a point at which neighboring molecules mutually affect the charge distribution of the other, affect,Chemical processes such as the reversible association-dissociation of analyte molecules, or the ionization of a weak acid in an unbuffered solvent.,Instrumentation limitation-incident beam may be polychromatic .,Processes other than analyte absorption,result in significant decrease in the power,of the incident beam;,Reference cell is used to correct,these processes;,Reference cell is often prepared by,adding distilled water to an absorption,cell;,The reference cell is then placed in the,path of the light beam, and the power,of the radiation exiting the reference cell,is measured and taken as P,0,for the sample cell.,Background Correction,Linear calibration curve;,Nonlinear calibration,Calibration Curves,An Example-Pulegone,Frequently plotted as,log,of molar extinction,So at 240 nm, pulegone has a molar extinction of 7.24 x 10,3,Antilog of 3.86,Infrared Spectroscopy,Introduction,Spectroscopy is an analytical technique which helps determine structure,It destroys little or no sample,The amount of light absorbed by the sample is measured as wavelength is varied,Infrared spectroscopy is very useful for obtaining qualitative information about the molecules. But molecule must possess certain properties in order to undergo absorption.,IR Spectroscopy,The presence and also the environment of functional groups in organic molecule can be identified by infrared (IR) spectroscopy. Infrared spectroscopy is nondestructive. Moreover, the small quantity of sample needed, the speed with which spectrum can be obtained, the relatively low cost of the spectrometer, and I wide applicability of the method combine to make infrared spectroscopy one the most useful tools available to the organic chemist,low,high,Frequency (,n,),Energy,X-RAY,ULTRAVIOLET,INFRARED,MICRO-,WAVE,RADIO,FREQUENCY,Ultraviolet,Visible,Vibrational,infrared,Nuclear,magnetic,resonance,200 nm,400 nm,800 nm,2.5,m,m,15,m,m,1 m,5 m,short,long,Wavelength (,l,),high,low,BLUE,RED,THE ELECTROMAGNETIC SPECTRUM,X-ray,UV/Visible,Infrared,Microwave,Radio Frequency,Bond-breaking,Electronic,Vibrational,Rotational,Nuclear and,Electronic Spin,REGION,ENERGY TRANSITIONS,Types of Energy Transitions in Each Region,of the Electromagnetic Spectrum,(NMR),Principles IR Spectroscopy,Energy: E=h,where: is the frequency in hertz,In IR, frequency is commonly expressed as wave numbers ( , in Reciprocal cm, or cm-1),Where,Absorption of radiation in this region by a typical organic molecule results in the excitation of vibrational, rotational, and bending modes, while the molecule itself remains in its electronic ground state.,Molecular asymmetry is a requirement for excitation by infrared,radiation and fully symmetric molecules do not display absorbance in this region unless asymmetric stretching or bending transitions are possible.,Symmetric stretch,Assymmetric stretch,Symmetric bending,Principles IR Spectroscopy,Principles IR Spectroscopy,For the purpose of routine organic structure determination, the most important absorptions in the infrared region are the simple stretching vibrations. For simple systems, these can be approximated by considering the atoms as point masses, linked by a “spring” having a spring constant k and following Hookes Law.,Principles IR Spectroscopy,Using this simple approximation, the equation shown in below can be utilized to approximate the characteristic stretching frequency (in cm-1) of two atoms of mass m1 and m2, linked by a bond with a spring constant k:,Where,=m1m2/(m1+m2) , also called “reduced mass”,Absorption of Infrared Radiation,Only bonds which have significant dipole moments will absorb infrared radiation.,Dipole is the polar covalent bond in which a pair of electron is shared unequally.,For absorption occur, there must be a charge in the dipole moment (polarity) of the molecule. A diatomic molecule must have a permanent dipole in order to absorb, but larger molecule do not.,DIPOLE MOMENTS,Bonds which do not absorb infrared include:,Symmetrically substituted alkenes and alkynes,Many types of C-C Bonds,Symmetric diatomic molecules,H-H Cl-Cl,Molecular Vibrations,Light is absorbed when radiation frequency = frequency of vibration in molecule,Covalent bonds vibrate at only certain allowable frequencies,Associated with types of bonds and movement of atoms,Vibrations include stretching and bending,Light source,:,Nichrome wire,that glows when an electrical current is passed through;,Interferometer,: no monochrometer,Detector,: thermocouple detector,whose output voltage varies with changes,caused by varying levels of radiation striking the detector.,IR Instrumentation,IR Instrumentation,No two molecules of different structure will have exactly the same natural frequency of vibration, each will have a unique infrared absorption pattern or spectrum.,Two Uses:,IR can be used to,distinguish,one compound from another.,Absorption of IR energy by organic compounds will occur in a manner characteristic,of the types of bonds and atoms in the functional groups present in the compound; thus, infrared spectrum gives,structural information,about a molecule.,The absorptions of each type of bond (N,H, C,H, OH, CX, C=O, CO, CC, C=C, C,C, C,N, etc.) are regularly found only in certain small portions of the vibrational infrared region, greatly enhancing analysis possibilities.,Infrared Spectroscopy (IR),The Infrared Spectrum,A plot of absorption intensity (% Transmittance) on the y-axis vs. frequency (wavenumbers) on the x-axis.,Infrared Spectroscopy (IR),Principal Frequency Bands (from left to right in spectrum),O,H3600 cm,-1,(Acids - Very Broad, Alcohols - Broad),N,H3300 - 3500 cm,-1,(2, 1, 0 peaks 1,o, 2,o, 3,o,),C,N2250,cm,-1,(Nitrile),C,C2150,cm,-1,(Acetylene),C=O1685 - 1725 cm,-1,(1715) (Carbonyl),C=C1650 cm,-1,(Alkene); 4 absorptions 1450-1600 (aromatic),CH,2,1450 cm,-1,(Methylene Group),CH,3,1375 cm,-1,(Methyl Group),C,O900 - 1100 cm,-1,(Alcohol, Acid, Ester, Ether, Anhydride),-C,H(Saturated Alkane absorptions on Right side of 3000 cm,-1,),=C-H(Unsaturated Alkene absorptions on Left side of 3000 cm,-1,),=C-H(Aromatic absorptions) Verify at 1667 2000 cm,-1,C-H(Unsaturated Alkyne absorptions on Left side of 3000 cm,-1,),Infrared Spectroscopy (IR),Suggested approach for analyzing IR Spectra,Step 1. Check for the presence of the,Carbonyl group,(C=O) at 1715 cm,-1,. If molecule is conjugated, the strong (C=O),absorption,will be shifted to the right by 30 cm,-1,i.e., 1685 cm,-1,If the Carbonyl absorption is present, check for:,Carboxylic Acids-Check for OH group (broad absorptionnear 3300-2500 cm,-1,),Amides-Check for NH group (1 or 2 absorptions near 3500 cm,-1,),Esters-Check for 2 C-O group (medium absorptions near 1300-1000 cm,-1,),Anhydrides-Check for 2 C=O absorptions near 1810 and 1760 cm,-1,Aldehydes-Check for Aldehyde CH group (2 weak absorptions near 2850 and 2750 cm,-1,),Ketones-Ketones (The above groups have been eliminated),Infrared Spectroscopy (IR),Step 2. - If the Carbonyl Group is,Absent,Check for Alcohols, Amines, or Ethers.,Alcohols & Phenols-Check for OH group (Broad absorption near 3600-3300 cm,-1,Confirm present of C,O near1300-1000,cm,-1,Amines-Check for NH stretch (Medium absorptions) near 3500 cm,-1,Primary Amine- 2 Peaks,Secondary Amine- 1 Peak,Tertiary Amine- No peaks,N-H Scissoring at 1560 - 1640
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