Spectroscopy * Spectroscopy is the study of the interaction between matter and electromagnetic radiation. * Historically, spectroscopy originated through the study of visible light dispersed according to its wavelength by a prism + The electromagnetic spectrum is the range of frequencies of electromagnetic radiation and their respective wavelengths and photon energies* The history of spectroscopy began with Isaac Newton's optics experiments (1666-1672). * Newton applied the word "spectrum" to describe the rainbow of colors that combine to form white light and that are revealed when the white light is passed through a prism. * During the early 1800s, Joseph von Fraunhofer made experimental advances with dispersive spectrometers that enabled spectroscopy to become a more precise and quantitative scientific technique.Principle of spectroscopy * The term "spectroscopy" defines a large number of techniques that use radiation to obtain information on the structure and properties of matter. * The basic principle shared by all spectroscopic techniques is to shine a beam of electromagnetic radiation onto a sample, and observe how it responds to such a stimulus.WHAT IS SPECTROSCOPY? SPECTROSCOPY 2 = —_ Ry ~~ @. oe — ny ae oS e S+ Applications + Spectroscopy is used as a tool for studying the structures of atoms and molecules. The large number of wavelengths emitted by these systems makes it possible to investigate their structures in detail.+ Spectroscopy also provides a precise analytical method for finding the constituents in material having unknown chemical composition. + Ina typical spectroscopic analysis, a concentration of a few parts per million of a trace element in a material can be detected through its emission spectrumDes igel tae) hd By stole deel tad It is the science of “ The spectrometry is the studying the method used to acquire a interaction between quantitative matter and radiated measurement of the | __ energy spectrum. “It does not generate * It is the practical any results. It is the application where the theoretical approach results are generated of science.How to classify spectroscopy * Spectroscopy can be defined by the type of radiative energy involved. The intensity and frequency of the radiation allow for a measurable spectrum. * Electromagnetic radiation is a common radiation type and was the first used in spectroscopic studies. * Both infrared (IR) and near IR use electromagnetic radiation, as well asmicrowave techniques.¢ Another way of classifying spectroscopy is by the nature of the interaction between the energy and the material. These interactions include absorption, emission.Long radio wave| Broadcasting, aircraft, and marine ete. Gamma rays Radioactive. Ultraviolet {Short radio wave MicrowaveIR Spectroscopy * Range of electromagnetic spectrum that is used is Infrared radiation. * Infrared is makeup of different radiations. * The measurement of the interaction of infrared radiation with matter by absorption, reflection. * Used to find functional group in molecules can liquid gaseous forms.Principle of IR spectroscopy * Molecules are made up of atoms linked by chemical bonds. The movement of atoms and the chemical bonds like like spring and balls (vibration) * This characteristic vibration are called Natural frequency of vibration.Infrared Spectroscopy Features of infrared spectrophotometer: Recorder “a source of infrared rediation Sepmerted rotating [ | *sample/and Sacee Detector reference cell shemstely Sipteistabencth Seuice of radiation selector ‘an infrared ‘Semele detector =+ After absorbing energy molecules vibrate. Vibration of two typ * Streching 1. Symetrical 2. Assymetrical * Bending 1. Scissoring 2. Wagging 3. Rocking 4. Twisting* Bonds show different vibrations at different wavelengths. * Different functional groups absorbs different wavelength of light so show different peaks.+ Formula to find vibrations in linear molecules. Question: How many normal modes of vibration does the CO2 molecule possess? Answer: CO2 is Linear molecule. For linear molecules formula is 3N -5 In Case of CO2, N is 3 where N = No. Of atoms, So 3N-5=(3"3-5) = (9-5) = 4 Vibrations‘Typical infrared Absorption Values For Various Types of Bonds 1. 32003400 em" 2850-3100 em 2100-2260 em‘! 1650-1800 em* 00-1000 em* % (0-4 strotch CH stretch =N CSC C00 stretch T = = as ' 4 a n 8 m i t t a n € e 0 4000 3500 3000 2500 2000 1500 1000 500Spectrum * Spectrum have two main regions. * Absorbtion region:- * Individual peaks we can identify easily. * Fingerprinting region:- * Multiple peaks * We can’t identify easily * But by matching with spectrum library. * Represents bands of bending and stretching.Applications * To establish Identity of two compounds. * To determine the structure of new compound from its functional group. * To determine nature of contaiminants in a sample. * Some advanced physical properties of material.* Vibrational energy depends upon following:- *Mass of atom * Strength of bonds and bond distance * The arrangement of atom within molecule.UV visible spectroscopy * Why we need UV visible spectroscopy. * We need to find concentration of different substances compounds mixtures. * It can also tell us about chemical groups but data is not reliable. + Also use in kinetic study in enzymatic activity. * Also known as colour emmitery. * Determining molar concenteration * Determining ppmDESCRIPTION OF UV- SPECTROPHOTOMETER Advantage of double beam spectrophotometer:- It is not necessary to continually replace the blank with the sample or to adjust the auto zero. The ratio of the powers of the sample & reference is constantly obtained. It has rapid scanning over the wide wavelength region because of the above two factors. log(l/l) =A UV-VIS sources 4 fei} ; Spf : monochromator! beam splitter optics A,* Detector can be * Photodiode * PMT * Photomultiplier * Monochromator consists of * 2slits * 1 prism * First slit fall light to prism * Second slit allow one wavelength of light to pass.* Cuvette with organic compounds ai or proteins. * Proteins absorb wavelength if 260 NM. * More protein more absorbtion. * Less intensity of reflected light. * | decrease. * Transmitance ~ 1/absorbance * Transmitance depends upon concentration of molecule.* Detector only detect transmitance than how we find absorbance. * So we get graph. I\ .+ To convert a value from percent transmittance (%T) to absorbance, use the following equation: + Absorbance = 2 - log(%T) + Example: convert 56%T to absorbance: + 2—log(56) = 0.252 absorbance units. + Absorbance=10-1(concenterationx path length of cuvette) + Concenteration less =transmitance more + A=E xCx!* If we know absorbance than can calculate concenteration. * C=A/€x! Because absorbance and concenteration does not have linear relationship. In order to know unknown sample we need a linear graph. Absorbance Beer's Law ‘Standard 1 P standard 2 Standard 4 Concentration of Concentration (mol/L)