**4. Solid state characterization techniques**

As previously mentioned, changes in the properties of solids can occur during pharmaceutical drug manufacturing and storage. Normally, drugs are manufactured in a stable crystalline form because the risk of solid state transformations during storage is minimized. However, when developing a solid crystalline form, a rigorous control must be made to determine if the crystalline form is maintained or if there were changes during its production [31].

Currently, there are a variety of techniques to characterize a crystal. Characterization techniques are valuable tools that make it possible to determine the structure, chemical composition, and different properties of a pharmaceutical sample. However, simply one technique will not be able to offer complete information for a solid substance. It is vital to utilize them in a complementary manner in order to acquire acceptable outcomes (**Figure 2**). The most important techniques used in the pharmaceutical field for crystal characterization are those described below.

#### **4.1 Thermal analysis techniques**

Thermal analytical methods, which offer information on the thermal behavior of materials, are widely utilized in the physical characterization of pharmaceutical solids. When a sample is exposed to a temperature increase, the observed changes in characteristics can be measured [32, 33]. The thermal methods most commonly applied in the analysis of pharmaceutical solids are differential scanning calorimetry and thermogravimetric analysis.

#### *4.1.1 Differential scanning calorimetry (DSC)*

This technique provides information about the physical and energetic properties of a substance subjected to temperature variations. To obtain a DSC thermogram, the

**Figure 2.**

*A) Powder X-ray diffraction patterns and B) Fourier-transform infrared spectra of a) furosemide form I, b) furosemide form II, c) oxytetracycline hydrochloride form I, d) oxytetracycline hydrochloride form II, and e) oxytetracycline hydrochloride form III.*

#### **Figure 3.**

*A) Differential scanning calorimetry curves of a) albendazole form I, b) albendazole form II, c) clarithromycin form II, d) clarithromycin form 0 and B) thermogravimetric curves of a) oxytetracycline hydrochloride form I, b) oxytetracycline hydrochloride form II, c) clarithromycin form 0, d) clarithromycin form II.*

difference in heat flux of a sample as a function of temperature or time is measured. Typically, this study is performed by comparing the thermogram of a sample of interest, such as a crystal, to that of a reference sample. The deviation in the thermogram below the reference corresponds to an endothermic transition, while the deviation above the reference relates to an exothermic transition [34]. The curve obtained can be used to determine enthalpies of crystal fusion, phase transition temperatures, purity, degree of crystallinity, type of interaction between molecules, and thermal stability [34, 35].

Numerous studies reported the most popular application of DSC in the field of pharmaceuticals. Their application in the characterization of polymorphic drugs such as albendazole and clarithromycin can be seen in **Figure 3A**. The representative DSC curve for albendazole form I exhibits a fusion endotherm, whereas the profile for form II shows a preceding endo-exothermic event that indicates its polymorphic transformation to form I, followed by an endotherm attributed to form I melting, and lastly an exotherm of decomposition. Similarly, the profile for clarithromycin form 0 exhibits an exotherm corresponding to a solid phase transition, followed by an endotherm assigned to the form II melting event due to its coincidence with the melting endotherm evidenced in the clarithromycin form II DSC curve.

#### *4.1.2 Thermal gravimetric analysis (TGA)*

TGA measures the mass change of a sample as a function of temperature or heating time. It is a simple technique that requires a smaller sample size [36]. A thermogravimetric curve shows the mass change due to physical and chemical phenomena such as absorption, melting, sublimation, vaporization, oxidation, reduction, and decomposition events [32].

It is a useful tool to quantify different processes such as crystalline melting, sublimation, or decomposition of a sample, and to elucidate the degree of purity of the API [37]. On the other hand, it is possible to elucidate on the curves, whether the crystals

*Pharmaceutical Crystals: Development, Optimization, Characterization and Biopharmaceutical… DOI: http://dx.doi.org/10.5772/intechopen.105386*

under study contain water or a solvent [36]. Moreover, it allows the detection of solvent loss in a crystal. For instance, information on dehydration/desolvation events for clarithromycin and oxytetracycline hydrochloride polymorphs was obtained (**Figure 3B**), as demonstrated by a mass loss at low temperatures in the TGA profiles. When the TGA profiles of Clarithromycin form 0 and form II are compared, it is clear that form 0 is a solvate, as demonstrated by the weight loss caused by the ethanol evaporation process. On the other hand, TGA profiles of oxytetracycline hydrochloride form I showed a larger mass loss until 100°C than those of form II, indicating variations in solid dehydration. These TGA curves also revealed that form II had higher thermal stability.

#### **4.2 X-ray diffraction**

The technique most commonly utilized for identifying and characterizing crystalline materials is X-ray diffraction. Differentiating between crystalline and amorphous forms, identifying distinct solid forms of crystals, defining the crystalline structure of the API, and analyzing the differences between different crystal forms are some of its applications. As a result, it is commonly used in the pharmaceutical field [36]. For example, X-ray diffraction experiments have provided an unequivocal identification of furosemide and oxytetracycline hydrochloride polymorphs (**Figure 2A**), which exhibited clear differences in terms of reflection positions and relative intensity.

Single crystal X-ray diffraction is employed to determine the molecular structure of pharmaceutical materials that exist as single crystals [7, 38, 39]. A three-dimensional picture of the molecule and geometrical properties data in the solid state can be produced by studying a perfectly crystalline sample [40]. Powder X-ray diffraction is applied when the crystalline material is found as a fine-grained powder, rather than a single crystal [7, 38, 39, 41].

#### **4.3 Vibrational spectroscopic techniques**

Vibrational spectroscopic techniques are widely used in the pharmaceutical field to identify crystalline solids due are fast, non-destructive, and can characterize solid samples with minimal or no preparation. The most commonly used methods for analyzing crystalline samples are Fourier-transform infrared (FT-IR) and Raman spectroscopy [7, 31].

These techniques are extensively utilized in the study of pharmaceutical solids to characterize amorphous and crystalline phases, identify the structure and composition of different pharmaceutical solid forms, determine the compatibility of mixtures, and establish molecular interactions [42].

For instance, FT-IR spectroscopy has been used in several studies to identify the individual polymorphic forms of a drug confirming that they are structurally distinct. Differences in characteristic FT-IR bands assigned to sulphonamide NH and secondary amine NH stretches were identified between furosemide polymorphs (**Figure 2B**). In the same way, significant differences between oxytetracycline hydrochloride polymorphs were observed in the bands attributed to the OH and amide NH stretching vibrations (**Figure 2B**).

#### **4.4 Solid state nuclear magnetic resonance (ssNMR)**

ssNMR is a non-destructive and multinuclear technique that exploits the magnetic properties of certain nuclei, for example, 1 H, 13C, 15N, 17O, and 19F. Although it is a

non-routinary expensive methodology that has extensive experimental times and robust expertise users are required, it is widely used in pharmaceutical applications [32].

This technique is used to analyze crystalline and amorphous pharmaceutical samples qualitatively and quantitatively, as well as to characterize both APIs and formulations. Structural or dynamic information is obtained from mono and bidimensional experiments based on different nuclear interactions. Their pharmaceutical applications included identification, characterization, and quantitation of different solid forms of an API in bulk samples; determination of conformational and crystalline packing behavior, intra- and intermolecular interactions, internuclear distances; study of amorphous phase properties, stability of API forms, the effects of drug processing, molecular motions, chemical and physical interactions between APIexcipient and excipient-excipient, solid state chemical reactivity; and identification of contaminants or degradation products, among others [7, 43, 44].

#### **4.5 Microscopy**

Microscopy is considered a tool of great interest in the pharmaceutical field, which is mainly used to examine shape and size and to identify the solid state form in the sample. Different types of microscopes are currently used for the characterization of pharmaceutical crystals [32, 45]. The most relevant are described below.

#### *4.5.1 Scanning electron microscopy (SEM)*

SEM is a very useful and versatile tool in the pharmaceutical field. It provides quantitative as well as qualitative information such as morphology, size, size distribution, crystal shape, and consistency of powders or compressed dosage forms by analyzing the images obtained by microscopy. In addition, it allows studying the effects of any interaction with its environment [7, 45].

Microscopic analysis of pharmaceutical crystals using SEM microphotographs reveals significant morphological differences between solids produced using distinct crystallization techniques, allowing each polymorphic form of the drug to be identified. In **Figure 4**, for example, significant differences in particle size and shape can be observed. Albendazole form I appear as small and irregular particles with a predisposition to aggregate while in contrast albendazole form II exhibits self-agglomerate lamellar particles with a smooth surface. Furosemide form I presented hexagonal and tubular compact crystals with a defined surface while furosemide form II shows fine and elongated prism particles. On the other hand, a compact structure with small particles adhered to the surface is observed for norfloxacin form BI, while the norfloxacin form C crystals are typical hexagonal-like faceted, compact, and with well-defined smooth structures. Finally, oxytetracycline hydrochloride form I have particles with a smooth surface and well-defined edges, form II crystals show compact particles with an irregular surface, form III presents rod-shaped crystals with a smooth surface with defined edges, while the form IV appeared as thin agglomerated needles.

#### *4.5.2 Optical microscopy and polarized light microscopy*

An optical microscope is used to observe crystals directly providing information on particle size and shape. In addition, the nucleation events can be visualized by monitoring within situ cameras [32].

*Pharmaceutical Crystals: Development, Optimization, Characterization and Biopharmaceutical… DOI: http://dx.doi.org/10.5772/intechopen.105386*

#### **Figure 4.**

*SEM images of the morphology of a) abendazole form I, b) albendazole form II, c) furosemide form I, d) furosemide form II, e) norfloxacin form BI, f) norfloxacin form C, g) oxytetracycline hydrochloride form I, h) oxytetracycline hydrochloride form III, i) oxytetracycline hydrochloride form II, and j) oxytetracycline hydrochloride form IV.*

The utilization of polarized light, on the other hand, optimizes the utility of optical microscopes. Using polarized light microscopy, the interior structure of crystals can be analyzed and determined if the sample is amorphous or crystalline. Due to birefringence, several colors can be seen in a crystalline particle when viewed through crossed polarizers [7, 32, 45].

For example, significant differences in particle size and shape of sulphathiazole precipitated from a variety of solvents and techniques between them and compared

#### **Figure 5.**

*a), c), e), g), i) and k) images obtained using an optical microscope. b), d), f), h), j) and l) images obtained using a polarized light microscope.*

additionally with commercial sulphathiazole can be observed by an optical microscope. In addition, these different sulphathiazole crystal forms exhibit different birefringence under a polarized light microscope. **Figure 5** shows images of commercial sulphathiazole (**Figure 5a** and **b**) and samples obtained by the crystallization of commercial sulphathiazole from methanol heating the solution below the boiling point to the total solution (**Figure 5c** and **d**), from aqueous solution heating below the boiling point to total solution and immediately cooled at freezer temperature (**Figure 5e** and **f**), and from the saturated aqueous solution obtained below 80°C that was exposed to a temperature ramp of 90 to 25°C for one hour and then kept at 25°C for 24 hours (**Figure 5g** and **h**), 1hour (**Figure 5i** and **j**) and 30 minutes (**Figure 5k** and **l**).
