HSM

Hot-Stage Microscopy

1. HOT-STAGE
MICROSCOPY
Prepared by: Sarah Khallad
MSc Pharmaceutical Sciences
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2. Contents
•Introduction
•Hot-Stage Microscope Instrumentation
•Applications of hot stage microscopy in pharmaceuticals:
1.Morphology studies
2.Amorphous/crystalline form characterization
3.Polymorphism
4.CocrystalScreening
5.Particle size distribution and characterization of an API in a
tablet
6.Solvates/hydrate screening
7.Miscibility
8.Thermal analysis by surface characterization (TASC)
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3. Hot stage microscopy (HSM)
•Coupling of thermal analysis with microscopyfor the
solid-state characterization of materials as a function of
temperature and time.
•In pharmaceuticals HSM is used to supportDSCand
TGAobservations and to detect small changesin the
sample that may be missed by DSC and TGA during a
thermal experiment.
•Like Desolvation, recrystallization, phase transitions
and minor changes in the surface.
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4. Hot-Stage Microscope
•The hot-stage microscope is an optical microscope
equipped with heating and cooling units which allow
observation of a sample under the change of temperature.
•A typical modern hot stage microscope consists of:
1.Computer controlled programmable hot stage
2.Optical microscope for real time observation
3.Polarizing filters
4.Digital camera for recording thermal events
5.Computer and software to control the hot stage and to
carry out the analysis of the thermographs generated
during a thermal event.
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5. Instrumentation
5
Kumar, A., Singh, P. & Nanda, A. Hot stage microscopy and its applications in pharmaceutical characterization.Appl. Microsc.50,12
(2020). https://doi.org/10.1186/s42649-020-00032-9

6. Hot Stage (with glass slide).
Control unit & 2 types of hot stage.
Cont.
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https://en.donho.com.tw/mettler-toledo-hot-stage-microscopy-
systems.html

7. Cont.
•The sample is heated in a sapphire crucible or glass
slide, either in an openor a closed environment and can
be equipped with a liquid nitrogen unit for rapid
cooling, high pressure pumps or purge gas.
•The temperature of a modern computer controlled hot
stage can be varied from − 200 °C to 600 °C.
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8. 8

9. •A part from this basic setup, a hot stage microscope can
be coupledwith various other characterization
techniques such as Fourier Transform Infrared
Spectroscopy (FTIR) or Differential scanning Calorimetry
(DSC), high pressure unit or with other non-optical
imaging tools such as scanning electron microscopy
(SEM), and Raman and mass spectroscopyto avail the
simultaneous benefits of both the techniques.
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10. Applications of Hot Stage Microscopy in
Pharmaceuticals
1-Morphology studies
•Physical features or morphology of a sample under
investigation.
•A wealth of information on how the sample changes when
heatedcan be obtained by HSM studies.
•Avoiding the misinterpretationof the results obtained from
DSC/TGA and prevents flawed conclusions
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11. 2-Amorphous/crystalline form
characterization
•The amorphousform of API is preferredin the
pharmaceutical industry due to their higher solubility and
dissolution rates over crystalline forms.
•Amorphous and crystalline forms can be easily
distinguished using a hot stage microscope equipped with
a polarizing filter;
Crystalline materials are known to show birefringence,
while amorphous compounds lack birefringence, hence
can be easily distinguished from crystalline compounds.
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12. Birefringence
Kumar, A., Singh, P. & Nanda, A. Hot stage microscopy and its applications in pharmaceutical characterization.Appl.
Microsc.50,12 (2020). https://doi.org/10.1186/s42649-020-00032-9
12

13. Cont.
•HSM can be useful to study the conversion of an
amorphous API to crystalline form under the effect of
heating.
•To study the effect of storage, especially exposure to heat
and humidity.
•Other than this HSM allows one to observe the
recrystallization processand measure the crystal
growth rate.
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14. 3-Polymorphism
•Polymorphism is the ability of a substance to exhibit more
than one crystalline structure.
•Different polymorphs of an API may differ in
physicochemical properties and stability.
•It is also possible to study polymorphs which cannot be
grown in a laboratory due to thermodynamic energy
factors, these polymorphs can be grown and studied
thermally on a hot stage.
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15. “At 175.1 °C the melting of form III was observed in
HSM which was missed by DSC.”
M.R. Bakar, Z.K. Nagy, C.D. Rielly, A combined approach of differential scanning calorimetryand hot-stage microscopy with image analysis in the
investigation of sulfathiazole polymorphism. J. Therm. Anal. Calorim. 99(2), 609–619 (2010). https://doi.org/10.1007/s10973-009-0001-z
15

16. M.R. Bakar, Z.K. Nagy, C.D. Rielly, A combined approach of differential scanning calorimetryand hot-stage microscopy with image
analysis in the investigation of sulfathiazole polymorphism. J. Therm. Anal. Calorim. 99(2), 609–619 (2010).
https://doi.org/10.1007/s10973-009-0001-z
16

17. 4-CocrystalScreening
•Cocrystalsare multicomponent solid forms consisting two
or more different molecules non covalently bonded to
each other in same the crystal lattice.
•Cocrystalshave recently gained a lot of attention in
academia due to their ability to modulate
physicochemicalproperties of an API.
•Screening through HSM is rapid, solvent free and
requires small quantities of API and coformer.
•Koflercontact method (also known as Koflermixed
fusion)
17

18. Kavanagh ON, Croker DM, Walker GM, ZaworotkoMJ. Pharmaceutical cocrystals: from serendipity to design to application. Drug DiscovToday. 2019 Mar;24(3):796-804. doi:
10.1016/j.drudis.2018.11.023. Epub2018 Dec 3. PMID: 30521935.
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19. CocrystalScreening of API with GlutaricAcid
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20. CocrystalScreening of API with GlutaricAcid cont.
D.P. McNamara, S.L. Childs, J. Giordano, A. Iarriccio, J. Cassidy, M.S. Shet, R. Mannion, E. O'Donnell, A. Park, Use of a glutaricacid
cocrystalto improve oral bioavailability of a low solubility API. Pharm. Res. 23(8), 1888–1897 (2006). https://doi.org/10.1007/s11095-006-
9032-3
-Glutaricacid was chosen as the potential coformerbased on melt experiments
because of its good aqueous solubility, stability and melting point.
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21. 5-Particle size distribution and characterization
of an API in a tablet
•Particle size distribution has a profound effect on the
processabilityof raw and finished products, which in turn
effects the dissolution, bioavailability, stability profile
and thus ensures the quality, safety and efficacy of the
finished formulation.
•Although there are methods to determine the particle size
of API in a tablet but they have their own limitations such
as the inability to differentiate between agglomerates
of the tablet constituents.
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22. 22

23. Cont.
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24. 24

25. 6-Solvates/hydrate screening
•Solvatesare formed when solvent molecules get
incorporated in the host compound structure. When water
gets incorporated in the host compound, these type of
solvates are called hydrates.
•Hot stage microscopy can also be used for
solvate/hydrate screening since it allows the visual
observation of the gasevolved during the desolvation
of the sample under heat.
25

26. A. Jacobs, F.M. Noa, Hybrid salt–cocrystalsolvate: P-coumaricacid and quinine system. J. Chem. Crystallogr. 44(2), 57–62 (2014). https://doi.org/10.1007/
26

27. 7-Pharmaceutical Incompatibility and Miscibility
•DSC has been successfully used for detection of compatibility
in the binary mixture of the drug and excipient.
•HSM has proven to be advantageous as the interactions can
be visually observed which was limited in DSC studies.
Hence HSM can be used to supplement the data obtained
from DSC to detect possible compatibility or incompatibility.
•Thus, HSM can serve as a rapid, green and cheaper
approach for detectingpharmaceutical incompatibilities and
to observe the miscibility of API and excipients into one another
which can find application in the screening of excipients
compatible with the API and polymersfor preparation of solid
dispersions.
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28. Miscibility Study
•They screened lacidipinewith 11 excipients of polymeric
and non-polymeric nature.
•The miscible components formed an amorphous solid
solutionwhereas the immisciblecomponents resulted in
amorphous drug dispersed in crystalline.
A. Forster, J. Hempenstall, I. Tucker, T. Rades, Selection of excipients for melt extrusion with two poorly water-soluble drugs by solubility parameter calculation and
thermal analysis. Int. J. Pharm. 226(1–2), 147–161 (2001). https://doi.org/10.1016/S0378-5173(01)00801-8
28

29. M. Alhijjaj, M. Reading, P. Belton, S. Qi, Thermal analysis by structural characterization as a method for assessing heterogeneity in complex solid pharmaceutical dosage forms. Anal. Chem.
87(21), 10848–10855 (2015).
8-Thermal analysis by surface characterization (TASC)
29

30. M. Reading, Thermal analysis by structural characterization (TASC): Structural and thermo-rheological information from hot stagemicroscopy. Microscopy
Today 25(5), 18–23 (2017). https://doi.org/10.1017/S1551929517000815
30

31. References:
•Kumar, A., Singh, P. & Nanda, A. Hot stage microscopy and its applications in pharmaceutical characterization.Appl.
Microsc.50,12 (2020). https://doi.org/10.1186/s42649-020-00032-9
•M.R. Bakar, Z.K. Nagy, C.D. Rielly, A combined approach of differential scanning calorimetryand hot-stage microscopy with
image analysis in the investigation of sulfathiazole polymorphism. J. Therm. Anal. Calorim. 99(2), 609–619 (2010).
https://doi.org/10.1007/s10973-009-0001-z
•M.R. Bakar, Z.K. Nagy, C.D. Rielly, A combined approach of differential scanning calorimetryand hot-stage microscopy with
image analysis in the investigation of sulfathiazole polymorphism. J. Therm. Anal. Calorim. 99(2), 609–619 (2010).
https://doi.org/10.1007/s10973-009-0001-z
•Kavanagh ON, Croker DM, Walker GM, ZaworotkoMJ. Pharmaceutical cocrystals: from serendipity to design to application.
Drug DiscovToday. 2019 Mar;24(3):796-804. doi: 10.1016/j.drudis.2018.11.023. Epub2018 Dec 3. PMID: 30521935.
•D.P. McNamara, S.L. Childs, J. Giordano, A. Iarriccio, J. Cassidy, M.S. Shet, R. Mannion, E. O'Donnell, A. Park, Use of a
glutaricacid cocrystalto improve oral bioavailability of a low solubility API. Pharm. Res. 23(8), 1888–1897 (2006).
https://doi.org/10.1007/s11095-006-9032-3
•A. Jacobs, F.M. Noa, Hybrid salt–cocrystalsolvate: P-coumaricacid and quinine system. J. Chem. Crystallogr. 44(2), 57–62
(2014). https://doi.org/10.1007/
•A. Forster, J. Hempenstall, I. Tucker, T. Rades, Selection of excipients for melt extrusion with two poorly water-soluble drugs by
solubility parameter calculation and thermal analysis. Int. J. Pharm. 226(1–2), 147–161 (2001). https://doi.org/10.1016/S0378-
5173(01)00801-8
•M. Alhijjaj, M. Reading, P. Belton, S. Qi, Thermal analysis by structural characterization as a method for assessing
heterogeneity in complex solid pharmaceutical dosage forms. Anal. Chem. 87(21), 10848–10855 (2015).
•M. Reading, Thermal analysis by structural characterization (TASC): Structural and thermo-rheological information from hot
stage microscopy. Microscopy Today 25(5), 18–23 (2017). https://doi.org/10.1017/S1551929517000815
31