The document discusses using Raman spectroscopy to monitor polymorphic crystallizations and the influence of particle properties on Raman spectra. It shows that Raman intensities change with particle size, shape, and concentration due to light scattering effects. Case studies demonstrate that accounting for changing particle dynamics over time is important for quantitative Raman analysis of polymorph ratios during crystallization processes. Taking particle properties into account through techniques like FBRM-Raman coupling and chemometrics allows Raman to be utilized for dynamic crystallization monitoring.

1. LASENTEC Monitoring and Quantifing Polymorphic Crystallizations The Application of Raman James Ward Paul Barrett MT AutoChem Polymorphism & Crystallization Forum 2003 November 12, 2003 Internet - [email_address] Phone (484) 343-5514

3. PVM Images: Particle shape and crystallization

4. FBRM: Lab to Production Lab to Plant installations

6. FBRM/Raman in 1 probe Source: Laser Illumination Fiber Collection Fiber Probe CCD Detector Echelle Grating Mirror Probe Diameter : 19 mm

8. Raman Spectroscopy Elastically Scattered light Majority of scattered light is elastically scattered light Raman scattering is a low probability event Approx 0.0001% of photons show a shift in frequency – i.e. Raman scattering Anti- Stokes Infrared Raman Electronic levels Vibrational levels Virtual levels Rayleigh Fluorescence Stokes Molecule of interest

9. What does Raman measure in polymorphism? => Shift in Raman spectra Polymorphs => Different intermolecular bonding Slightly different electron distributions in ‘molecular enviroment’ & ‘lattice enviroment’

10. Spectrum Comparison-Polystyrene FT-IR Transmission Spectrum FT-Raman Spectrum 20 40 60 80 %Transmittance 1 2 3 4 Raman Intensity 1000 2000 3000 4000 Wavenumbers (cm -1 )

12. Recent References Solution-mediated phase transformation of anhydrous to dihydrate carbamazepine and the effect of lattice disorder , International Journal Of Pharmaceutics, Volume 246, Issue 1-2, October 10, 2002, Pages 121-134 Murphy, D; Rodríguez-Cintrón, F; Langevin, B; Kelly, R C; Rodríguez-Hornedo, N Solid-state study of polymorphic drugs: carbamazepine , Journal Of Pharmaceutical And Biomedical Analysis, Volume 23, Issue 1, August 1, 2000, Pages 41-54 Rustichelli, C; Gamberini, G; Ferioli, V; Gamberini, M C; Ficarra, R; Tommasini, S

14. The Hydration - Particle Dynamics via FBRM & PVM Anhydrous CBZ Charged PVM Image Trended FBRM particle counts Chords Per Second (not to scale) Time Microns Microns

15. The Hydration - Particle Dynamics via FBRM & PVM Anhydrous CBZ Charged PVM Image Trended FBRM particle counts Chords Per Second (not to scale) Time Microns Microns

16. The Hydration - Particle Dynamics via FBRM & PVM Dry material disperses Increase in coarse particles as material aggregates and dip in fines PVM Image Trended FBRM particle counts Chords Per Second (not to scale) Time Microns Microns

17. The Hydration - Particle Dynamics via FBRM & PVM PVM Image Trended FBRM particle counts Chords Per Second (not to scale) Time Microns Microns

18. The Hydration - Particle Dynamics via FBRM & PVM Platelets disappear (drop in coarse) Needles appear (increase in fine counts) PVM Image Trended FBRM particle counts Chords Per Second (not to scale) Time Microns Microns

19. The Hydration - Particle Dynamics via FBRM & PVM PVM Image Trended FBRM particle counts Chords Per Second (not to scale) Time Microns Microns

20. The Hydration - Particle Dynamics via FBRM & PVM PVM Image Trended FBRM particle counts Chords Per Second (not to scale) Time Microns Microns

21. The Hydration - Particle Dynamics via FBRM & PVM PVM Image Trended FBRM particle counts Chords Per Second (not to scale) Time Microns Microns

22. The Hydration - Particle Dynamics via FBRM & PVM Coarse increase again - Needle lengthening PVM Image Trended FBRM particle counts Chords Per Second (not to scale) Time Microns Microns

23. The Hydration - Particle Dynamics via FBRM & PVM Steady state is achieved No additional significant change in dimension, shape or number of crystals PVM Image Trended FBRM particle counts Chords Per Second (not to scale) Time Microns Microns

24. How can Raman be utilized for the CBZ example?

26. Raman & FBRM – Complementary technologies HOWEVER, We know that particle number, dimension and shape are changing over time. Can this effect the Raman interpretation? Chemometrics utilized to trend ‘concentration of each form over time Form 1 Dihydrate

27. Why is Raman signal influenced by particles? Consider backscatter turbidity: Light striking a particle is scattering in all directions Vast majority of light collected coming back towards the probe has the same wavelength as the outgoing light. This phenomena is termed elastic light scattering How does light interact with a particle system?

28. Turbidity: Time/Intensity The turbidity measurement is a convoluted function of: Solids Concentration Particle Size Particle Shape Particle Size/Shape Distribution If three of these four properties are held constant, the fourth can be quantified.

29. Turbidity: same reading, different particle systems Same Turbidity Measurement Same Turbidity Measurement FBRM can be utilized to detect differences based on dimension, number and shape of particles under investigation = = = Same Projected area, different size and different solids concentration. = = = Same Projected area, different shape and possibly different solids concentration.

30. What about Raman? From a theory perspective, it appears that the backscatter intensity, for a given material at a given wavelength as measured by a bulk measurement instrument like turbidity, is directly proportional to the Raman intensity. It is evident from experimental data that particle concentration, particle dimension and particle shape can effect the Raman intensity.

31. Utilizing Raman for crystallizations Is there a relationship between particle dimension and particle concentration and the intensity of the peaks in the Raman Spectra? Raman intensities change as material dimension (and shape) are changed Some Recent Work: Ensuring Robust Polymorph Isolation Using In-Situ Raman Spectroscopy George Zhou, Ph.D., Jian Wang*, Ph.D., Zhihong Ge, Ph.D., Yongkui Sun, Ph.D Merck Research Laboratories, Merck & Co.,

32. Example 3: The influence of Particle Dimension and Particle Number on Raman Spectra

34. As the solids concentration of the fine sucrose increases, there is a direct linear relationship between the solids concentration and the intensity of the Raman Sucrose peak 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 0 2 4 6 8 10 12 Fine Sucrose concentration (g/100 ml Toluene) Raman Peak Intensities Increasing concentration of fine sucrose

35. Raman is a function of the particle system The Raman measurement is a convoluted function of: A - Solids Concentration B - Particle Size C- Particle Shape D - Particle Size/Shape Distribution Just like turbidity, if 3 of these variables are held constant the other can be quantified directly. In this case, B,C,D are constant => Directly correlate Raman to solids concentration

36. 10 g of large Sucrose, 10 g of milled Sucrose So what if size of material is changed? Chord Length Microns Chords Per Second Reduction in coarse Increase in fines Milled Large Sucrose

37. Raman Spectra Peak height of coarse 5025 Peak height of fines 4315 No change in solids concentration, but a 15% change in signal !!! Particle size changed, solids concentration held constant Intensity Change N.B. Although we have seen behavior in both directions, ( i.e. increase in signal with reduction in size at same concentration, as well as decrease for different materials) Always the same behavior for the same materials.

38. As the solids concentration of the fine sucrose and fine mannitol changes, there is a direct linear relationship between the solids concentration and the intensity of the Raman peaks of each ‘form’ Samples of 2 materials of relatively the same size, but changing their ratios Increasing Mannitol conc Decreasing Sucrose conc

39. The Raman intensities no longer correlate to relationship on slide 56 => Particle Dimension and Particle Concentration have large influence Samples of 2 materials of DIFFERENT sizes, and changing their ratios Fine material obscures the coarse

41. Liquid Phase peaks also change with changing particle size and particle concentration The toluene peak is heavily effected by the dimension and number of particle present. It is therefore difficult to use Raman to track quantitatively the liquid phase concentration in the presence of solids Solids concentration is constant, but dimension varies

42. Building calibration curve without taking into consideration effects of dimension and concentration is not advised Quantitative information from Raman We have to go back and check the reasonable assumptions we have made in the past.