Abstract
When drug crystals are heated they undergo changes that can be observed under a microscope. Characteristics that can be analyzed under a microscope include melting point, melting range, crystal nucleation, crystal growth, crystal transformations and more. For example, under a microscope it is easy to see when a solid melts or how crystals nucleate and grow when cooled. This data can then be used to obtain important information about the solid such as kinetic information about crystal growth and transformation. This review will highlight some examples and practical suggestions for using a hot-stage microscope to learn more about pharmaceutical solids.
Introduction
Thermal microscopy (TM), also known as thermomicroscopy or hot-stage microscopy (HSM), is the combination of microscopy and thermal analysis to enable the study and physical characterization of materials as a function of temperature and time. Development of the techniue is often credited to Ludwig and Adelheid Kofler, but its origins can be traced back to the work of Otto Lehman in the 1880s [1]. Maria Kuhnert-Brandstätter was the first researcher to focus on the use of TM in the characterization of pharmaceutical compounds and this has since become a well-established practice.
There are several reasons why this thermo-optical technique is quite extensively used to characterize the solid-state properties of active pharmaceutical ingredients (APIs). Among these reasons, HSM allows the observation of the most obvious property of a material, namely physical appearance. It also allows characterization of small quantities of API to rapidly obtain valuable data about its solid-state properties. This technique is also a simple and relatively inexpensive method since the instrument setup basically comprises of a heating stage with a sample holder and gaseous atmosphere control coupled with a suitable polarized-light microscope and a system that allows the capturing and measurements of observations and temperatures. Figure 1 is a schematic representation of an HSM and a summary of its applications in characterizing solids and some of the results that can be obtained by this technique.
Figure 1 - A schematic representation of an HSM and a summary of its application in characterizing pharmaceutical solids.
Basic Configuration of Thermal Microscopes
The early thermomicroscopists had to develop and build their own equipment, but today all the necessary components, as well as some integrated systems, are available commercially.
Many manufacturers have developed accessories that can be added to expand analytical capabilities. This means that a typical simple optical thermal microscopy setup for the analysis of pharmaceuticals can now include an optical stereo microscope; polarizer; hot stage; digital programmable temperature controller; digital camera; computer and software for the capture and analysis of micrographs.
Such a system allows controlled heating of samples that are usually placed on glass discs or slides to allow for viewing on the optical microscope. Heating of the sample is typically achieved by heat transfer from a metal block that is heated thermoelectrically. If passively cooled, a controlled rate of cooling can only be achieved if the desired rate is relatively low. Some modern hot-stages off er active controlled cooling by utilizing chiller units or liquid nitrogen. Some also feature ports for connecting vacuum pumps, high pressure pumps or purge gas.
Many variations and customizations of this basic theme have been described to date. In particular an HSM may be combined with other equipment, like Fourier transform infrared spectroscopy (FTIR) or differential scanning calorimetry (DSC), or heating and cooling may be achieved by regulating the flow of hot and cold gasses. The use of hot- stages in combination with non-optical imaging, including scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman and confocal microscopy, is also available.
Figure 2 - A: Multiple crystal habits of indomethacin; B: Birefringence of nevirapine Form I crystals grown from a melt; C: Gas evolution during desolvation of nevirapine 1-hexanol solvate (sample under mineral oil); D: Solid-state transformation, with associated changes in crystal habit, during the desolvation of nevirapine 1-octanol solvate; E: The growth of salicylic acid and nicotinamide co-crystals from a melt; F: Charring of streptomycin, which has no defi nite melting point, above 250°C.
Pharmaceutical Applications
In the study of solid-state active pharmaceutical ingredients (APIs), excipients and pharmaceutically relevant polymers and lipids, various observations (Figure 2) can be made with the help of TM:
- Compound morphology (liquid/solid/semi-solid, crystalline/ amorphous, crystal habit)
- Birefringence (polarizing filter required)
- Gas evolution (solvates under mineral oil)
- Solid-solid transformations
- Interaction between different compounds
- Dissolution of one compound in another
- Sublimation and/or evaporation
- Vapor deposition
- Melting or liquefaction upon heating (solid-liquid transformations)
- Boiling
- Solidifi cation upon cooling (liquid-solid transformations)
- Crystal growth and rate thereof
- Charring or decomposition
These observations can be applied in the study and characterization of single- and multi-component systems. The range of variables that may be altered is determined by the design and capabilities of the hot-stage used and include: temperature, heating/cooling rate, time, pressure, gas atmosphere (inert or reactive, solvent vapors, humidity, etc.).
Single-component Pharmaceutical Systems
Important basic information about a compound can be obtained by visual observation if analyzed on an optical TM. It should be immediately obvious whether a substance is a liquid, solid or semi- solid. If it is a solid, one can distinguish between crystalline and amorphous forms – it might even be possible to identify a specifi c polymorph of a crystalline substance if the crystal habit is distinct. Through heating the melting/liquefaction temperature, and any possible associated charring, can be directly observed. If the melting temperatures of different polymorphs of a solid are known, this can serve as a means of identifi cation. The temperature of liquefaction can also be useful in distinguishing between lipids or lipid groups within a small subset of possible candidates.
Polymorphism
TM is especially useful for screening and characterizing API polymorphs and amorphous or glassy forms. Polymorphism is the ability of a substance to exhibit different crystal structures. It aff ects the physicochemical properties, stability and performance of APIs. TM is perhaps most widely used for identifying different polymorphs of a given API through visualization of their diverse crystal habits and by determining their unique melting points [2]. This is often done complementary to DSC (differential scanning calorimetry) and PXRD (powder X-ray diffraction) analyses [3]. The technique is however also a powerful tool for polymorph screening and for studying relative polymorph stability at a given temperature. During heating, a metastable polymorph can often be seen converting to the more stable form prior to melting (see Figure 2A). By repeated heating and cooling, it is possible to determine whether such transformations are reversible enantiotropic phenomena or irreversible monotropic events. Information gathered in this way sheds light on the energetics of polymorphic systems and on the relative stability of the different forms involved.
In cases where solid-state transformations cannot so easily be seen, the relative stability of different polymorphs can still be determined at a given temperature: by placing a small amount of each form in a suitable solvent, at opposite sides of the field, one can observe how the most stable form grows at the expense of the metastable species. It is important to keep in mind that relative stability of polymorphs in enantiotropic systems is a function of temperature. Some polymorphs that cannot be prepared by recrystallization from solvents, because their formation is not energetically favored, may be prepared thermally on a hot-stage. The yield will be small, but these crystals can be utilized for seeding the desired form in conventional solvent-based crystallization processes.
Amorphism
Amorphous forms of APIs play an important role in the pharmaceutical industry due to the advantageous properties of improved solubility and higher rates of dissolution. Due to these improved properties there tends to be a keen interest in amorphous forms of APIs. Hot-stage microscopy (HSM) is a very useful tool for rapidly distinguishing between the crystalline and amorphous forms of pharmaceutical materials. Birefringence is an indication for crystallinity, therefore upon utilizing a cross polarizer, amorphous forms will usually show a lack of interference colors (see Figure 2B). The USP test for amorphous forms or presence of amorphous forms involves the exclusion of birefringence.
Several techniques exist through which amorphous forms of an API can be prepared. One of these methods is by heating a crystalline API beyond its melting point followed by cooling it to its original temperature. This process will produce a glassy solid which is usually brittle and transparent. The glassy form of an API can easily be prepared on a microscope slide by heating the crystalline form on the hot-stage until melting is achieved. To render a glassy form the melt is then allowed to cool, either slowly or through quenching.
Amorphous forms tend to be physically and chemically unstable compared to their crystalline counterparts. This instability leads to recrystallization to the more stable crystalline form of the API. HSM is useful to determine whether an amorphous form will recrystallize to a more stable form upon heating. Furthermore the eff ect of storage, especially exposure to heat and humidity, on an amorphous form can be observed. HSM not only allows one to observe the recrystallization process but also to measure the crystal growth rate and further investigation of the subsequent recrystallization product in terms of morphology, melting point and degradation [4].
Seeding is also a factor that might influence the recrystallization of amorphous forms. Different recrystallization products can be obtained if the amorphous form is seeded with different crystalline forms of an API. During the recrystallization process facilitated by seeding, interesting observations can sometimes be made. One polymorphic form might crystallize faster than another or the crystallization and crystal growth of the one form may dominate to such an extent that it causes form transformation. Since the crystallization of amorphous forms due to seeding greatly depends on temperature, the hot-stage microscope is a great tool for monitoring sample temperature while observing and measuring the growth of crystals.
Sublimation and Physical Vapor Deposition
Sublimation occurs when a solid substance transforms to the gaseous phase without passing through the intermediate liquid phase. The ability of an API to sublime can easily be observed by means of hot-stage microscopy. Hot-stage microscopy is also useful in the preparation of different polymorphic forms by means of sublimation. The technique involves that the API (crystalline or amorphous), which is covered with a microscope cover slip, is heated on a microscope slide. The microscope cover slip will act as a condenser. The sample is usually heated up to the temperature where the sublimation occurs. After enough sublimate is formed the microscope cover slip can be removed and the resulting product can be investigated further by means of hot-stage microscopy and other supplementary thermal methods.
Physical vapor deposition is a high-vacuum deposition process, using thermal energy to remove a material from a source and deposit it on a substrate. With hot-stage microscopy this process will involve the heating of the material under vacuum until a sufficient vapor pressure is reached to allow evaporation or sublimation of the material. Subsequently, the liberated particles condense on a substrate and will usually form a thin amorphous film, although crystalline products have also been reported. The product can then be investigated further by means of HSM or other thermal techniques.
Multi-component Pharmaceutical Systems
Although many of the above observations, as well as certain phenomena such as polymorphism and amorphism, may apply to both single- and multi-component systems, certain TM applications are unique to mixtures, dispersions and inclusion compounds:
Solvatomorphism
A solvate of a crystalline solid is an inclusion compound containing both the molecular solid and one or more types of solvent which may be entrapped in isolated sites, layers or channels within the crystal structure [5]. A hydrate is a solvate in which the included solvent is water. If a solvate/hydrate sample is placed under mineral oil and heated, a TM allows one to observe gas evolution when the crystals desolvate. This is often associated with internal changes in structure that render the desolvated crystals opaque, making them appear darker, due to decreased light transmittance if the sample is illuminated from below. These observations are useful for substantiating thermo-gravimetric analysis (TGA) and DSC results and can aid in identifying the nature of a thermal event and distinguishing between true desolvation and the loss of adsorbed solvent.
Co-crystals
HSM is widely used in the discovery of new pharmaceutical co-crystals [6]. In the context of co-crystal screening, the new phase generated from the mixed fusion of contacted components is likely to be a co-crystal. HSM allows the melting and eutectic melting profile of a binary system to be observed, mapped and screened for co-crystals (see Figure 2E). In practice, the component with higher melting point (A) is melted first then allowed to solidify, and then the other component (B) is melted and brought into contact with A. In the contact zone solidified A is dissolved in the liquid of B, producing a mixing zone when the sample is quenched and recrystallized. This mixing zone is flanked with pure component A at one side and pure B at the other side. This process is shown in Figure 3 (see also Figure 2E).
Figure 3 - A schematic representation of the method used to prepare co-crystals under an HSM is shown on the left and the formation of salicylic acid: nicotinamide co-crystals is shown on the right.
When the sample is heated again until melting, under an HSM equipped with a polarizer, it is possible to observe the newly formed co-crystal, flanked by two eutectic mixtures, in the mixing zone. This co-crystal phase will retain birefringence and is usually clearly distinguishable from the eutectic and components A and B. This means that HSM has the advantage of determining the thermodynamic landscape of the binary system while screening for new co-crystals. The new co-crystal phase formed in the contact region can be confirmed in situ if for example a Raman microscope is equipped with a hot-stage. This new co-crystal formed on the hot-stage can then be used as seeds for growing co-crystal from solution, which may provide single crystals good enough for further structure determination.
Compatibility Studies
The challenges associated with incompatibilities between APIs and excipients are well recognized within the pharmaceutical industry [7]. Several techniques are routinely used for compatibility studies. Usually compatibility studies are very costly and time-consuming due to the long term and accelerated storage studies, sample preparation and sometimes the lack of sufficient quantity of materials. Currently, there is a growing interest in the use of rapid methods that use small amounts of sample to test for incompatibilities. DSC or isothermal microcalorimetry is currently the methods of choice for compatibility studies. Although these are very effective methods, several studies have proved that the concurrent use of HSM assists in the proper identification of incompatibilities. Since HSM is a visual thermal analysis technique it allows the monitoring of solid-state interactions. For example, DSC does not allow one to see possible dissolution of one component into another, therefore what might have been interpreted as an incompatibility is in fact not. Conversely, the degradation of one component might be masked by a thermal event caused by another. The fact that HSM allows visual observation on small quantities of sample can be a great advantage when performing compatibility studies.
Conclusion
HSM is a well-established technique within the pharmaceutical development industry. The greatest advantages of HSM are the fact that it is a fairly easy technique to master and that data is obtained rapidly. Furthermore, there is often no real substitute for the convenience of physically observing the attributes of a sample as a function of temperature and time. For these reasons, TM is both a complementary thermal analysis technique, useful for visualizing thermal events recorded by DSC and TGA, and a versatile tool for solid- state screening. Since the earliest reports of hot-stage microscopy, much progress has been made in the refi nement and diversifi cation of the equipment used for such studies. It is only reasonable to expect progress along these lines to continue into the future. Further advances are likely to include: a greater degree of automation; improvements in image capturing with greater resolution and rate of capture; more sophisticated software for image analysis with integrated kinetics and statistical calculations; and novel means of achieving heating and cooling of samples – these being more effi cient, more precise and more rapid than current commercially available hardware allows.
Acknowledgements
We are grateful to the University of Wisconsin-Madison, North-West University and the National Research Foundation of South Africa for research support.
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Author Biographies
Dr. Nicole Stieger, is a Senior Researcher at the Unit for Drug Research and Development of the North-West University of South Africa currently on sabbatical leave at the School of Pharmacy at the University of Wisconsin. She earned a M.Sc. (2005) and a Ph.D. (2010) in Pharmaceutics from North-West University. Her research focuses on the solid-state characterization and optimization of active pharmaceutical ingredients. She has several publications, patents and book chapters to her credit.
Dr. Marique Aucamp is a Post-Doctoral researcher from the Unit for Drug Research and Development at the North-West University, Potchefstroom, South Africa. She is currently working as an Honorary Fellow, at the School of Pharmacy, University of Wisconsin. She obtained her M.Sc in 2005 and her Ph.D in 2010, from North-West University. Her research interests are solid-state properties of active pharmaceutical ingredients, especially amorphous forms and analytical methods. She is the inventor of two patents, author of several manuscripts and serves as reviewer for peer-rieviewed journals.
Si-Wei Zhang, is a PhD student at the School of Pharmacy at the University of Wisconsin. He received a BS in pharmaceutical sciences and MS in medicinal chemistry from Peking University Health Sciences Center. His research focuses on the characterization of the thermodynamics of co- crystals and the stability of amorphous drugs in the presence of excipients.
Dr. Melgardt M. de Villiers serves as Associate Professor in the School of Pharmacy at the University of Wisconsin. His research program focuses on developing innovative nano and micro particulate drug delivery systems for treating and preventing communicable and chronic diseases. He is the author or co-author of more than a hundred peer-reviewed publications and has presented his research either orally or as posters more than 150 times at meetings all over the world.