Spectroscopic Techniques

Detailed description of spectroscopic techniques.

1. INSTITUTE OF BIOMEDICAL SCIENCES, BUNDELKHAND UNIVERSITY, JHANSI SPECTROSCOPIC TECHNIQUES PRESENTED BY - Ms. PALAK AGRAWAL

2. INTRODUCTION ‘ Spectroscopy’ is a branch of science that deals with the study of interaction of matter with light , using electromagnetic radiation as an investigation to obtain information about atoms and molecules that are too small to see. It is an analytical, structural elucidation technique which helps us to “ see the unseeable ”.

3. ELECTROMAGNETIC RADIATION Electromagnetic radiation consists of particles representing a quantum of light or discrete packages of energy known as ‘ photons ’. A photon carries energy proportional to the radiation frequency but has zero rest mass . It consists of an oscillating electric field (E) and an oscillating magnetic field (B), which are perpendicular to each other.

4. ILLUSTRATION OF ELECTROMAGNETIC WAVES

5.  The relationship between wavelength & frequency can be written as: c = ν λ  As photon is subjected to energy, so E = h ν = h c / λ  The  electromagnetic spectrum  is the range of all possible wavelengths of electromagnetic radiation, ranging from high energy gamma rays through visible light and down to low energy radio waves.  Various astronomical phenomenon can only be observed via specific wavelengths different from visible light.

6. The Electromagnetic Spectrum

7.  On passage through a glass prism , the white light is separated into its component colors - red, orange, yellow, green, blue, indigo and violet. The separation of visible light into its different colors is known as  ’Dispersion of Light’.

8. SPECTROSCOPY ABSORPTION EMISSION ATOMIC MOLECULAR FLOURESCENCE

9. Law of Absorption : THE BEER-LAMBERT LAW SOURCE OF RADIATION BEAM SELECTOR BEAM SPLITTER WAVELENGTH SELECTOR MONOCHROMATIC RADIATION MIRROR REFERENCE CUVETTE SAMPLE CUVETTE ELECTRON PHOTOPLATE ELECTRON CAPTURE CIRCUIT A Io Io I I WATER ABSORBING MEDIA

10. BEER’S LAW  A ∝ c LAMBERT’S LAW  A ∝ l On combining the two laws, we get the BEER-LAMBERT LAW as follows : Log 10 Io/I = Ɛ.l.c or A = Ɛ.l.c where, Io = Intensity of incident light I = Intensity of transmitted light A = Absorbance l = Path length of absorbing media (in cm) c = Concentration (in moles/ litre ) Log 10 Io/I = Absorbance or optical density Ɛ = Molar extinction coefficient or Molar absorptivity constant

11. UV-VIS ABSORPTION SPECTROSCOPY or ELECTRONIC EXCITATION SPECTROSCOPY This spectroscopy is based on the transitions of electron from ground state molecular orbital to excited state due to the absorption of electromagnetic radiation from UV and visible region.

12. ORGANIC ABSORPTION AND ELECTRONIC TRANSITONS GRAPHICAL REPRESENTATION : The Energy Level Diagram These are normally empty C ontain lone pair These contain normal bonding pair of electron

13. TYPES OF ELECTRONIC TRANSITIONS

14. SPECTRAL MODE The absorption spectra λ max

15. PHOTOMETRIC MODE Calliberation curve ( μ l/ml)

16. UV-VIS SPECTROPHOTOMETER ABSORBANCE SCREEN WAVELENGTH SELECTOR METHYLENE BLUE SAMPLES WITH DIFFERENT CONCENTRATIONS CUVETTES CUVETTE HOLDER

17. USES OF UV-VIS SPECTROSCOPY :  Quantitative measurement. Structural elucidation for presence of n electrons (heteroatom). Identify the degree of unsaturation. Determination of λ max. LIMITATIONS OF UV-VIS SPECTROSCOPY : It is measured ideally in micromole( μ mol ) concentration and shows inappropriate results beyond it.

18. INFRARED SPECTROSCOPY / VIBRATIONAL SPECTROSCOPY / FUNCTIONAL GROUP SPECTROSCOPY ‘ IR Spectroscopy ’ is capitalized on the concept that functional groups absorb specific frequencies of energy based on their structure. So basically, it measures the vibrations of atoms which make it possible to determine which functional group is present. The radiations used in IR spectroscopy are low energy infrared radiation and microwaves . Infrared radiation is commonly divided into three sub-regions : (1) Near – IR or NIR (2) Mid – IR or MIR (3) Far – IR or FIR

19. TYPES OF VIBRATIONS : (1) STRETCHING (2) BENDING (3) ROTATIONAL

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21. USES OF IR SPECTROSCOPY :  It is used to identify the presence or absence of a heteroatom in a molecule. Various parameters of a molecule like bond length, bond angle and bond energy are also identified from this technique . LIMITATIONS OF IR SPECTROSCOPY : P oor sensitivity to molecular units with small oscillatory dipoles during a vibrational transition since these modes do not absorb strongly in the infrared . It d oes not provide information about the relative location of the functional groups of a molecule.

22. NUCLEAR MAGNETIC RESONANCE ‘ NMR ’ is a spectroscopic technique that involves change in nuclear spin energy in the presence of an external magnetic field. It is based on the absorption of electromagnetic radiation in the radio-frequency/ radiowave region. The radiowaves flip the nucleus from lower energy state to higher energy state. The nucleus now wants to return to the lower energy state and when it does so, the energy comes out again and this is what we detect.

23. Radiowave flip

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25. USES OF NMR SPECTROSCOPY : It is one of the most advanced spectroscopic techniques used to identify the position of heteroatom in molecular form. A very important diagnostic tool in medicine that is based on the principle of NMR is a technique known as M agnetic R esonance I maging (MRI). It uses strong magnetic fields and radiowaves to form images of the body. It is used for the diagnosis and evaluation of diseases. LIMITATIONS OF NMR SPECTROSCOPY : Long duration of analysis. High cost.

26. ELECTRON SPIN RESONANCE ESR is a method for studying materials with unpaired electrons (radical study). The basic concept of ESR are analogous to those of NMR but it is electron spins that are excited instead of the spins of atomic nuclei. ESR is particularly useful for studying metal complexes or organic radicals.

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28. OPTICAL ROTATORY DISPERSION / CIRCULAR DICHROISM Most of the biological compounds are optically active, and show optical rotation . The technique of ORD measures the ability of optically active compounds to rotate the PPL as a function of wavelength. When PPL is passed through a solution that contains an optically active compound , there is net rotation of the PPL. The light is rotated either clockwise (dextrorotatory) or counterclockwise (laevorotatory) by an angle that depends on the molecular structure and concentration of the compound, the pathlength and wavelength of light.

29. NORMAL UNPOLARISED LIGHT POLARISER PLANE POLARISED LIGHT OPTICALLY ACTIVE COMPOUND IN SAMPLE TUBE ANALYSER ROTATION OF PLANE POLARISED LIGHT RAY DIAGRAM OF A SPECTROPOLARIMETER

30. In CD, circularly polarised light is used, which is obtained by superimposing two PPL’s of same wavelengths and amplitudes which are polarised in two perpendicular planes, but there is a phase difference of  90° between them . POLARISER PLANE POLARISED LIGHT PHASE CHANGER SUPERIMPOSED RADIATION (CIRCULARLY POLARISED)

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32. USES OF ORD/CD SPECTROSCOPY Excellent method for the study of conformations adopted by proteins and nucleic acids due to various interactions in a solution. Comparison of secondary and tertiary structure of wild type and mutant proteins. Used in determining direction and angle of deflection of chiral compounds (D or L). LIMITATIONS OF ORD/CD SPECTROSCOPY It only provides qualitative analysis of data and does not provide atomic level structural analysis. The observed spectrum is not enough for claiming one and only possible structure.

33. X-RAY DIFFRACTION (XRD) “ Every crystalline substance gives a pattern; the same substance always gives the same pattern; and in a mixture of substances each produces its pattern independently of the others ” The X-Ray diffraction pattern of a pure substance is like a fingerprint of the substance. It is based on the scattering of X -Rays by crystals . The atomic planes of a crystal cause an incident beam of X-rays to interfere with one another as they leave the crystal. This phenomenon is called X-Ray diffraction.

34. K Shell L shell M shell X-Rays PRODUCTION OF X-RAYS :

35.  When an incident X-Ray beam hits a scatterer , scattered X-Rays are emitted in all directions. Interference occurs among the waves scattered by the atoms when crystalline solids are exposed to X-Rays. There are 2 types of interference patterns depending on how the waves overlap one another :

36. The constructive interference from a diffracting crystal is observed as a pattern of points on the detector. The relative positions of these points are related mathematically to the crystal’s unit cell dimensions.

37. BRAGG’S LAW (BY W.L. BRAGG & W.H. BRAGG) The X-Ray diffracted from atoms in crystal planes obey the laws of reflection. The two rays reflected by successive planes will be in phase if the extra distance travelled by the second ray is an integral number of wavelengths. BRAGG’S EQUATION : n λ = 2 d sin θ   where, n = Integer d = Lattice spacing θ  = Angle of incidence λ = Wavelength of incident X-Rays

38. INSTRUMENT Picture : INNOVATION CENTRE, BU Model : RIGAKU

39. USES OF X-RAY CRYSTALLOGRAPHY : To find the structure of an unknown material. To determine the atomic arrangement of a crystal. To measure the thickness of thin films and multilayers. The powder XRD pattern may be thought of as finger print of the single crystal structure, and it may be used to conduct qualitative and quantitative analysis. LIMITATIONS OF X-RAY CRYSTALLOGRAPHY : High cost. Time consuming. The technique ionizes the sample, thereby, the sample cannot be used again.

40. MASS SPECTROMETRY Mass S pectrometry  (MS) is an analytical technique that measures the  mass-to-charge ratio of ions. The results are typically presented as a  mass spectrum , a plot of intensity as a function of the mass-to-charge ratio . It is based on the absorption of electromagnetic radiation in the gamma wave region. This technique basically studies the effect of ionizing energy on molecules. In the past few years, ongoing technological developments have contributed substantially to progress in biochemistry, molecular biology, and medicine.

41. COMPONENTS OF MS : General Block Diagram

42. MASS SPECTROMETRY APPARATUS

43. TYPES OF IONISATION TECHNIQUES : VOLATILE SAMPLES  Electron ionisation  Chemical ionisation NON-VOLATILE SAMPLES  Fast atom bombardment  Thermospray  M atrix A ssisted L aser D esorption I onisation (MALDI)  Electrospray ionisation  Atmospheric pressure chemical ionisation .

44. INSTRUMENT Picture : INNOVATION CENTRE, BU Model : CLARUS SQ 8 C

45. USES OF MS : The mass spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical identity or structure of molecules and other chemical compounds. The high sensitivity, detection selectivity, qualitative capability and mass accuracy of the instrument makes its use in drug testing and discovery, food contamination detection, pesticide residue analysis, isotope ratio determination, protein identification and carbon dating. LIMITATION OF MS : If the isomers of a compound have the same m/z ratio, they will not be distinguished by the MS.

46. EMISSION SPECTROSCOPY Atoms or molecules that are excited to high energy levels can decay to lower levels by emitting radiation. The substance first absorbs energy and then emits this energy as light. Emissions can be induced by sources of energy such as flames or EMR. For atoms excited by high temperature, the light emission is commonly called atomic emission (emission spectroscopy) and for atoms excited with EMR, the light emission is called atomic flourescence ( flourometry ) .

47. FLOURESCENCE SPECTROSCOPY Flourescence is an emission phenomenon and is observed when after excitation by the absorption of a photon, an electron returns from the first excited state to the ground state. When photons are incident with original intensity Io, they induce some energy to the sample, thereby, exciting the electrons of the same energy level. These particles then emit radiations in all directions.

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49. USES OF FLOURESCENCE SPECTROSCOPY : Used for heavy metal detection. Used in flourescent solar collector. Diagnostic and research tool in medical field. Quantitative measurement up to femtomoles . Allows real-time labelling of molecules of interest. LIMITATIONS OF FLOURESCENCE SPECTROSCOPY : All molecules are not flourescent hence, only a few can be detected. Loss of recognition capability and photostability . Susceptible to auto- flourescence .

50. ‘SCIENCE’ is simply the word we use to describe a method of organizing our ‘CURIOSITY’ - TIM MINCHIN Thank You!