2. 10/11/2005 1 ENGINEERING RESEARCH CENTER FOR S TRUCTURED O RGANIC P ARTICULATE S YSTEMS RUTGERS UNIVERSITY PURDUE UNIVERSITY NEW JERSEY INSTITUTE OF TECHNOLOGY UNIVERSITY OF PUERTO RICO AT MAYAGÜEZ Vibrational Spectroscopy for Pharmaceutical Analysis Part VII. Introduction to Raman Spectroscopy Rodolfo J. Romañach, Ph.D.

3. 2 Scattering Mid-IR and NIR require absorption of radiation from a ground level to an excited state, requires matching of radiation from source with difference in energy states. Raman spectroscopy involves scattering of radiation (matching of radiation is not required). E. Smith and G. Dent, “Modern Raman Spectroscopy. A Practical Approach.”, Wiley 2005, pages 3 – 5.

4. 3 Raman Spectroscopy A single frequency of radiation irradiates the molecule and the radiation distorts (polarizes) the cloud of electrons surrounding the nuclei to form a short-lived state called a “virtual state”. This state is not stable and the photon is quickly re-radiated. E. Smith and G. Dent, “Modern Raman Spectroscopy. A Practical Approach.”, Wiley 2005, pages 3 – 5.

5. 4 What is Raman Spectroscopy? Rayleigh scattering: Elastic scatter 200 400 600 800 1000 1200 1400 Raman Intensity -400-200 0 200 400 Raman Shift (cm-1) Raman : Stokes Anti-Stokes Inelastic scatter LASER Raman is a scattering technique Slide courtesy Kaiser Optical Systems.

6. 5 Raman Scattering from Molecular Vibrations 2c2c 1 = ( k  ) ½ = Vibrational frequency k = Spring force constant  = Reduced mass of atoms, m 1 m 2 /(m 1 +m 2 ) Higher vibrational frequency with stronger chemical bond and lighter atoms. Rayleigh – Elastic Strongest Component Anti-Stokes – Photon Gains Energy Stokes – Photon has less energy Adapted from Kaiser Optical Systems slide Only one in 10 6 or 10 8 photons is Raman scattered.

7. 6 Quantum Mechanical Model of Raman Scattering StokesRayleighAnti-Stokes E0E0 v=3 v=2 v= 1 v=0 Virtual state hv ex h ( v ex- v v ) h ( v ex+ v v ) E1E1 Courtesy Kaiser Optical Systems.

8. 7 Raman Scattering The difference in wavelength between the incident and scattered visible radiation corresponds to wavelengths in the mid-infrared region. An Indian physicist C.V. Raman discovered this effect in 1928. This has been considered an experimentally difficult technique for many years; but in recent years a number of advances in instrumentation has made it more available to non-specialized labs. Courtesy Kaiser Optical Systems.

9. 8 Raman Scattering Sample is irradiated with intense monochromatic radiation usually in the visible or NIR region of the spectrum. The wavelength is well away from any absorption peaks of the analyte. The abscissa in the spectra are in terms of wavenumber shift Δυ between the observe radiation and that of the source, and we speak of Raman shift instead of frequency of absorption. Skoog Holler Niemann, p. 429-433, 435 – 441.

10. 9 Raman Scattering and Polarizability

11. 10 Stokes Scattering Stokes scattering is, by convention, positive-shifted Raman scatter. Most Analytical work is done in this region. Represents inelastic scattering to a region of lower energy. This means that the energy of the detected radiation is higher in wavelength relative to the laser. The scattered spectrum appears similar to an IR spectrum and is interpreted similar IR spectrum. Adapted from Kaiser Optical Systems slide

12. 11 Raman Scattering C=C, and C≡C, C≡N bonds are strong scatterers, bonds undergo polarization. Symmetric stretches undergo greater changes in polarization, and are stronger in Raman than asymmetric stretches. E. Smith and G. Dent, Wiley 2005, page 6.

13. 12 Advantages of Raman Spectroscopy – Chemical Information Raman bands can provide structural information (presence of functional groups). Raman spectroscopy can be used to measure bands of symmetric linkages which are weak in an infrared spectrum (e.g. -S-S-, -C-S-, -C=C-). The standard spectral range reaches well below 400 cm - 1, making the technique ideal for both organic and inorganic species.

14. 13 Advantages of Raman Spectroscopy – Ease of Use for Process Measurements Fiber optics (up to 100's of meters in length) can be used for remote analyses. Purging of sample chamber is unnecessary since Water and CO 2 vapors are very weak scatterers. Little or no sample preparation is required Water is a weak scatterer - no special accessories are needed for measuring aqueous solutions Inexpensive glass sample holders, non-invasive probes and immersion probes are ideal in most cases

15. 14 Disadvantages of Raman Spectroscopy Inherently not sensitive (need ~ 1 million incident photons to generate 1 Raman scattered photon) Fluorescence is a common background issue Typical detection limits in the parts per thousand range Fluorescence Probability versus Probability of Raman Scatter ( 1 in 10 3 -10 5 vs 1 in 10 7 -10 10 ) Requires expensive lasers, detectors and filters. Small sample volume can make it difficult to obtain a representative sample.

16. 15 Complementary Nature of IR and Raman Spectroscopy IR absorption intensities are proportional to the change in dipole moment as the molecule vibrates. Raman scattering intensities are proportional to the change in molecular polarizabilities upon vibrational excitation. For molecules with a center of inversion IR and Raman and mutually exclusive.

17. 16 Need to emphasize complementarity with more specific examples.

18. 17 Placzek's Equation for Raman Scattering Intensity I R proportional to I L I R proportional to N I R stronger at shorter wavelength Statistical factor: (1-e - h/kT ) (45)(3 2 )c 4 243243  (1-e -h /kT ) hI L N( 0 - ) 4 [45(  a ') 2 +7(  a ') 2 ] = IRIR Where c = speed of light h = Planck's constant I L = laser intensity N = number of scattering molecules = molecular vibrational frequency in Hz L = laser excitation frequency, in Hz  = reduced mass of the vibrating atoms k = Boltzmann's constant T = absolute temperature  a ' = mean value invariant of the polarizability tensor  a ' = anisotropy invariant of the polarizability tensor courtesy Kaiser Optical Systems

19. 18 I  LCI I  Raman intensity  = Raman cross section L = Pathlength C = Concentration I = Instrument parameters Analytical Raman Spectroscopy Sample courtesy Kaiser Optical Systems

20. 19 Raman Scattering is Stronger from Some Vibrations than from Others 3N-6 vibrations possible, many have no Raman bands Change in polarizability during a molecular vibration leads to Raman scattering. –Covalent bonds more polarizable than ionic bonds –Intensity from stretching vibration increases with bond order –Intensity tends to increase with increasing atomic number –Symmetry-forbidden vibrations Adapted from Kaiser Optical Systems slide

21. 20 Raman Scattering is Stronger from Some Vibrations than from Others Stretching bands often stronger than bending ones Symmetric bands often stronger than anti-symmetric ones Crystalline materials often have stronger Raman bands than non- crystalline materials Adapted from Kaiser Optical Systems slide

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24. 23 Kaiser Optical Systems Rxn-1-785 nm Raman Spectrometer.

25. 24 Fluorescence Properties – Very efficient conversion of laser photons into unwanted light – Emission spectrum usually changes little, if at all, with changing laser wavelength – Fluorescence lifetime typically 1 to 10 nanoseconds Sources – impurities – additives Elimination – Near-infrared wavelength excitation – Far-UV wavelength excitation – Photobleaching – Spectral subtraction methods – Time-resolved detection Energy Fluorescence Energy Level Diagram Stokes Anti- Stokes Two photon Slide Courtesy of Kaiser Optical Systems

26. 25 Example of a Raman Spectrum 0 2 4 6 400 600 800 1000 1200 1400 1600 1800 4 component mixture: omp-xylene and ethylbenzene Raman shift in wavenumbers from the laser line Intensity in detected photons x10 -5 p m o e p o m e p p m, e o p, e o m omp ompe Slide Courtesy of Kaiser Optical Systems

27. 26 Glass/Amorphous Materials Stress on molecular groups from local environment changes vibrational energy. Discrete peaks become broad bands. Slide Courtesy of Kaiser Optical Systems

28. 27 Raman Scattering from Crystals Periodicity of a crystalline lattice reduces the number of vibrations that Raman observes. Spectrum consists of narrow peaks. Spectrum effected by orientation XX X X X XXX X X XX X X X XXX X X Polarizability changes add together Polarizability changes cancel out Slide Courtesy of Kaiser Optical Systems

29. 28 Theophylline Anhydrous vs. Monohydrate 1941.9180017001600150014001300120011001000900793.5 Raman Shift / cm-1 anhydrous monohydrate Slide Courtesy of Kaiser Optical Systems, work by Lynne Taylor’s group,. Industrial & Physical Pharmacy, Purdue University

30. 29 Theophylline Phase Stability as a Function of Temperature Literature Transition temperature for hydrate to anhydate is around 60°C 69°C 64°C 58°C 54°C 48°C 1816.117001600150014001300120011001000877.4 Raman Shift / cm -1 Intensity Slide Courtesy of Kaiser Optical Systems

31. 30 Components of a Raman Spectrograph Laser Fiber optic sampling device Notch filter Grating CCD Detector Slide Courtesy of Kaiser Optical Systems

32. 31 RamanRxn1 Schematic Overview Imaging Spectrograph HoloPlex TE Cooled CCD Detector Slit Notch Control Electronics ProbeHead Invictus NIR Laser ProbeHead Filtering Universal ProbeHead Immersion and Non-Contact Sampling Optics Axial Transmissive Spectrograph HoloPlex Grating TE Cooled CCD Detector Invictus NIR Laser Slide Courtesy of Kaiser Optical Systems

33. 32 Innovative all refractive design! HoloPlex Advantages: Quantitative Raman!  Full Simultaneous Spectral Coverage  High Throughput  High Spectral Resolution  No Moving Parts Low 1.8 f/# means Higher Optical Throughput (~4X) Improved Thermal Stability (5X) Rugged Compact Design Holographic Transmission Grating Entrance Slit Multi-element Lenses Output Plane Axial Transmissive Design Slide Courtesy of Kaiser Optical Systems

34. 33 Lasers Commonly used for Raman 1064 nm – Nd:YAG laser – FT Instrumentation – Out of range of CCD, must use InGaAs or Ge 830 nm – Not common but could help avoid fluorescence 785 nm – Diode laser – Most common laser used for Raman work – Good compromise between fluorescence and Raman efficiency…makes it somewhat universal – Stable to environment – Electronically efficient Slide Courtesy of Kaiser Optical Systems

35. 34 Lasers Commonly used for Raman 633 nm – He-Ne laser – Longer lifetime 532 nm – Frequency doubled Nd:YAG laser – Good efficiency, low power – Watch out for fluorescence! – Sensitive to temperature 514 nm – Ar-ion laser 488 nm – Ar-ion laser UV lasers – Resonance Raman – EXPENSIVE Slide Courtesy of Kaiser Optical Systems

36. 35 Sampling Options Pilot Plant TrialProduction Installation Immersion ProbeNon-Contact Optic Stream 532 nm excitation Slide Courtesy of Kaiser Optical Systems

37. 36 Purpose of Notch Filters Filter out non-informative radiation In the case of Raman instrumentation, this means filtering the Rayleigh scattered energy Holographic notch filters are the most common…in nearly every Raman instrument you will find a Kaiser notch filter Slides by courtesy of: Mark Kemper, kemper@kosi.com

38. 37 Properties of Holographic Notch Filters High attenuation Narrow bandwidth Sharp spectral edges Good transmission High damage threshold Environmentally stable Center = 785 nm FWHM at 50%T = 12 nm 0.3 to 4.0 OD edge = 7.1 nm 0 40 80 700 720 740 760 780 800 820 840 Wavelength (nm) % Transmission Slide Courtesy of Kaiser Optical Systems

39. 38 CCD Detector Multi element silicon detector (1024 x 128) Maintained at low temperature (-40ºC) Key reason for lack of moving parts High sensitivity Detection range 400 – 1050 nm Slides by courtesy of: Mark Kemper, kemper@kosi.com

40. 39 To learn more about Raman Spectroscopy: E. Smith and G. Dent, “Modern Raman Spectroscopy A Practical Approach”, John Wiley & Sons Ltd; (Chichester, United Kingdom), 2005.

41. 40 Comparing FT-IR and Raman  FT-IR  Absorption  Fundamental information  Sample preparation  Process measurements difficult  High spectral density  Organics  Dipoles  O-H, C=O, N-H  Water a problem  Raman  Emission  Fundamental information  No sample preparation  Process measurements  High spectral density  Sampling challenges  Organics and inorganics  Polarizability  Aromatics, C=C  Water no problem Slide courtesy Kaiser Optical Systems.