Raman spectroscopy

Raman spectroscopy

1. Raman spectroscopy Presented by Y.Suvarna M.Pharm, [Ph.D.] Dept. of Pharm. Analysis

2. History When radiation passes through a transparent medium, the species present scatter a fraction of the beam in all directions. In 1928, the Indian physicist C. V. Raman discovered that the visible wavelength of a small fraction of the radiation scattered by certain molecules differs from that of the incident beam and furthermore that the shifts in wavelength depend upon the chemical structure of the molecules responsible for the scattering

3. Light scattering

4. Basic theory Electric field (E) due to electromagn. wave with frequency (ν ) E = E cos 2 π ν t E is Amplitude of wave

5. Energy transfer

6. Energy levels of Diatomic molecule

7. Energy levels

8. Scattering and energy

9. Classical description If a diatomic molecule is irradiated by light there is spatial charge separation under influence of electric field E i.e. induced dipole moment μ: μ = α E ------- (1) α: polarizability Electric field E due to electromagn. wave with frequency ν E = E cos 2 π ν t ------- (2) μ = α E cos 2 π ν t ------- (3) emission of light at same frequency ν I = (2/3c 3 ) μ * μ * = (d 2 µ/dt 2 ) 2 = (16 π 4 α 2 E 2/3c 3 ) ν 4 ----- (4)

10. Cont…. Internal vibrational motion with Eigen frequency v M q = q cos 2 π ν M t ------- (5) Polarizability α → develop in series α = α + ( ∂ α/ ∂ q ) q + higher order terms -------- (6) μ = α E (7) = [ α + ( ∂ α/ ∂ q ) q cos 2 π ν M t] E cos 2 π ν t = α E cos 2 π ν t + ½ ( ∂ α/ ∂ q ) q E [cos 2 π ( ν - ν M ) t ↑ ↑ + cos 2 π ( ν + ν M ) t] Rayleigh Stokes/Anti-Stokes

11. Raman spectra of CCl 4

12. Hook’s law

13. Factors affecting vibrational frequencies Order of frequency H 2 > HD > D 2 --------- mass effect HF > HCl > HBr > HI --------- force constant F 2 >Cl 2 >Br 2 >I 2 ----- Both mass effect & force const. N 2 > CO > NO > O 2 ----- Both

14. Vibrations of polyatomic molecule No. of normal vibrations shown by polyatomic molecule is given as 3N-5 ------ linear molecules 3N-6 ------ Non-linear mol. 3N is no. degrees of freedom Subtracting translational and rotational motion of whole molecule about three principle axes (x,y,z)

15. Vibrations in CO 2 molecule Symmetric Asymmetric 2350 cm -1 1340 cm -1 667 cm -1

16. Vibrations in H 2 o molecule

17. Selection rules for IR & Raman spectra For IR active, dipole moment is changed during vibration For Raman active, polarisability is changed during vibration A vibration is Raman active, if size, shape or orientation of ellipsoid changes during vibration

18. Changes in Dipole moment and polarisability

19. Changes in polarisability ellipsoid during vibration of CO 2 molecule

20. Changes in polarisability ellipsoid during vibration of H 2 O molecule

21. Mutual exclusion rule If a molecule has a centre of symmetry then IR active vibrations are Raman inactive and vice versa. Note that there may be modes inactive in both. If a molecule has no centre of symmetry then some (but not necessarily all) vibrations may be both IR and Raman active.

22. Advantages Symmetric vibrations are Raman active and ‘ Mutual exclusion rule’ holds. Vibrations are strong in Raman if bond is covalent Ex. C≡C, C═C, P═S, C─H, C─S if bond is ionic IR vibrations are strong Ex. O─H, N─H Ratio of relative intensities of C≡C, C═C, C─C stretching vibrations is 3:2:1 Bending vibrations are weaker than stretching vibrations

23. Measurements of depolarization ratio gives reliable information on symmetry of vibrations Selective enhancement of vibrations of particular chromophoric group is possible with R esonance Raman effect which is useful for study of large biological molecules containing chromophoric groups Small sample area is enough in Raman spectra as the diameter of laser beam is 1-2nm Water can be used as solvent without major interferences Raman spectra of hygroscopic and air sensitive compounds can be obtained by placing in sealed glass tubing In a single recording , region from 4000-50cm -1 can be done in Raman

24. Disadvantages Powerful laser source is required to observe weak scattering Some compounds may fluoresce by irradiation Less resolution than IR spectra Costs more than FT-IR instrument

25. Instrumentation

26. Light source As Raman spectra are weak, high intensity sources are required Mercury lamp: 435.8nm and 253.6nm are most commonly used Lasers: Thousand times brighter and far superior to mercury arc. Because the intensity of Raman scattering varies as the fourth power of the frequency, argon and krypton ion sources that emit in the blue and green region of the spectrum have an advantage over the other sources. Optics are arranged so that scattered radiation is observed 90 o to the incident radiation

27. Common LASER sources

28. Sample illumination system Sample handling for Raman spectroscopic measurements is simpler than for IR spectroscopy because glass can be used for windows, lenses, and other optical components instead of the more fragile and atmospherically less stable crystalline halides. In addition, the laser source is easily focused on a small sample area and the emitted radiation efficiently focused on a slit. Consequently, very small samples can be investigated. A common sample holder for non-absorbing liquid samples is an ordinary glass melting-point capillary.

29. Sample handling Liquid Samples : A major advantage of sample handling in Raman spectroscopy, as water is a weak Raman scatterer. Thus, aqueous solutions can be studied by Raman spectroscopy but not by infrared. This advantage is particularly important for biological and inorganic systems and in studies dealing with water pollution problems. Solid Samples : Raman spectra of solid samples are often acquired by filling a small cavity with the sample after it has been ground to a fine powder. Polymers can usually be examined directly with no sample pretreatment. Gas samples : Gas are normally contain in glass tubes, 1-2 cm in diameter and about 1mm thick. Gases can also be sealed in small capillary tubes

30. Filters  It is essential to have monochromatic radiations and to remove high energy radiations that may cause photodecomposion or fluorescence. For getting monochromatic radiations filters are used. They are made of nickel oxide, glass or quartz. Sometimes a suitable colored solution such as an aqueous solution of ferricyanide or iodine in CCl 4 may be used as a monochromator

31. Monochromators Laser-Raman spectrometers incorporate two or more grating type monochromators . i ) to increase resolving power of spectrometer ii) to reduce stray light due to exciting radiation iii) to reduce background caused by Rayleigh scattering by sample

32. Sample holder  The type of sample holder to be used depends upon the intensity of sources ,the nature and availability of the sample. The study of Raman spectra of gases requires samples holders which are generally bigger in size than those for liquids. Solids are dissolved before subjecting to Raman spectrograph. Any solvents which is suitable for the ultraviolet spectra can be used for the study of Raman spectra. Water is regarded as good solvents for the study of inorganic compounds in Raman spectroscopy.

33. Holders for different samples Gases: use gas cell Liquids and solids can be sealed in a glass capillary :

34. Detectors  Researchers traditionally used single points detectors such as photocounting, photomultiplier(PMT), not because of the weakness of a typical Raman signal, longer exposure times were often required to obtains Raman spectrum of a decent quality. Now days multichannel detectors like photodiode arrays(PDA), charged couple devices(CCD) Sensitivity & performance of modern CCD detectors are high

35. CCD detector Most of the current dispersive Raman set-ups are now equipped with multichannel two-dimensional CCD detectors. The main advantages of these detectors are: The high quantum efficiency. The extremely low level of thermal noise (when effectively cooled). Low read noise. The large spectral range available. Many CCD chips exist, but one of the most common spectroscopy sensor formats is the 1024 x 256 pixel array.

36. CCD TE cooled charge-coupled device (CCD) detector or “Camera” that allows simultaneous collection of a wide spectral wavelength range. A water cooling option allows -90º C operation. Thermoelectric (TE ) cooling is efficient, maintenance-free and requires no liquid nitrogen TE cooling provides long-term stability at optimum quantum efficiency Longer wavelengths can be detected more efficiently at higher temperatures than liquid nitrogen cooling . Thermoelectrically (TE) cooled CCD.

37. Calibration CCl 4 and Indene are used as standards for routine calibration Record the emission lines from the Laser source Standard frequencies for emission lines of He, Ar, Kr are available

38. Raman spectrometer

39. FT-Raman spectrometer

40. Variations of Raman spectroscopy Resonance Raman spectroscopy (RRS) Surface-enhanced Raman spectroscopy (SERS) Micro-Raman spectroscopy Nonlinear Raman spectroscopic techniques

41. Resonance Raman spectroscopy Resonance Raman scattering refers to a phenomenon in which Raman line intensities are greatly enhanced by excitation with wavelengths that closely approach that of an electronic absorption peak of an analyte. Under this circumstance, the magnitudes of Raman peaks associated with the most symmetric vibrations are enhanced by a factor of 10 2 to 10 6 . As a consequence, resonance Raman spectra have been obtained at analyte concentrations as low as 10 -8 M .

42. Surface-enhanced Raman spectroscopy Surface enhanced Raman spectroscopy involves obtaining Raman spectra in the usual way on samples that are adsorbed on the surface of colloidal metal particles (usually silver, gold, or copper) or on roughened surfaces of pieces of these metals. For reasons that are not fully understood, the Raman lines of the adsorbed molecule are often enhanced by a factor of 10 3 to 10 6 . When surface enhancement is combined with the resonance enhancement technique discussed in the previous section, the net increase in signal intensity is roughly the product of the intensity produced by each of the techniques. Consequently, detection limits in the 10 -9 to 10 -12 M range have been observed.

43. SERS Can increase the Raman signal by a factor of 10 4 -10 6 regularly, with even 10 8 -10 14 for some systems. Surface selective, highly sensitive: allows for trace analysis. Best when (Au, Ag, Cu) or (Li, Na, K) used a-C:H a-C:H The spectra at right show the regular spectra of a-C:H and a-C (bottom curves), and the SERS enhancement (top curves)

44. Micro-Raman spectroscopy Raman spectra can be collected from a very small volume (< 1 µm in diameter); these spectra allow the identification of species present in that volume. Water does not generally interfere with Raman spectral analysis. Thus, Raman spectroscopy is suitable for the microscopic examination of minerals, materials such as polymers and ceramics, cells, proteins and forensic trace evidence. A Raman microscope begins with a standard optical microscope, and adds an excitation laser, a monochromator, and a sensitive detector (such as a charge-coupled device (CCD), or photomultiplier tube (PMT)).

45. Nonlinear Raman spectroscopic techniques These are based on the contributions of the non-linear part of the induced dipole moment or the induced polarization to the intensity of the frequency - shifted light . Nonlinear spectroscopy is used to refer to cases that fall outside this view, including: (1) Watching the response of matter subjected to interactions with two or more independent incident fields, and (2) the case where linear response theory is inadequate for treating how the material behaves, as in the case of very intense incident radiation.

46. Non linear Raman spectroscopy Hyper Rayleigh and Hyper Raman spectroscopy coherent anti-Stokes Raman Spectroscopy (CARS) Raman Gain Spectroscopy Inverse Raman Spectroscopy Photoacoustic Raman Spectroscopy (PARS) Raman Induced Kerr Effect (RIKE). 

47. Applications Structural chemistry Solid state Analytical chemistry Applied materials analysis Process control Microspectroscopy /imaging Environmental monitoring Biomedical

48. Raman spectrum of Cholesterol

49. Fingerprinting a Molecule Raman spectra are molecule specific Spectra contain information about vibrational modes of the molecule Spectra have sharp features, allowing identification of the molecule by its spectrum Examples of analytes found in blood which are quantifiable with Raman spectroscopy

50. Applications In solid-state physics, spontaneous Raman spectroscopy is used to, characterize materials, measure temperature, and find the crystallographic orientation of a sample. to investigate the chemical composition of historical documents such as and contribute to knowledge of the social and economic conditions at the time the documents were produced as a means to detect explosives for airport security to discriminate between healthy and unhealthy tissues, or to determine the degree of progress of a certain disease

51. Applications as a technique for identification of seafloor hydrothermal and cold seep minerals Used in medicine , aiming to the development of new therapeutic drugs and in the diagnosis of arteriosclerosis and cancer. Pharmaceuticals and Cosmetics:- Compound distribution in tablets Blend uniformity API concentration Powder content and purity Raw material verification Polymorphic forms Crystallinity Contaminant identification