Correlative Raman Microscopy for Pharmaceutical Particle Analysis

Raman imaging, used in combination with other laboratory technologies, can analyse particles in greater detail than the techniques could achieve individually
Damon Strom at WITec GmbH
Many pharmaceutical products exist in particulate form during their development or in their final packaging. As new drugs are created at a rapid pace – and each must undergo some of the most rigorous testing and certification processes in any industry – methods for quickly and precisely determining their efficacy and compliance are of critical importance. This has driven the development of laboratory technologies that can evaluate large numbers of particles for statistical insight, visualise particle-like distributions of chemicals on pharmaceutical tablet surfaces, and perform highresolution analyses of individual particles.
Raman microscopy has proven to be one of the most powerful, versatile, and accessible techniques for chemical characterisation in pharma research. It can quickly and non-destructively measure sample components and reveal their physical distribution in great detail without specialised sample preparation. Its outstanding sensitivity enables data acquisition from very small material volumes, which makes it especially well suited to investigations of pharmaceutical particles.
Automated analysis based on Raman spectroscopy can greatly accelerate the process of locating, categorising, characterising, and quantifying large numbers of particles. This yields statistically significant insight into powdered samples and multi-component tablet surfaces in a reasonable amount of time. Other laboratory technologies, such as optical profilometry and scanning electron microscopy (SEM), can be combined with Raman microscopy to link data collected from precisely the same sample position. This integrated approach is known as correlative Raman imaging and it can provide a more comprehensive description of a sample’s chemical and structural properties than the respective techniques in isolation.
This survey will present a brief introduction to Raman microscopy before exploring several applications of correlative Raman microscopy variations in pharmaceutical particle analysis. They include: automated characterisation of an analgesic and antipyretic powder; component evaluation of a pharmaceutical tablet’s curved surface; and a Raman imaging and scanning electron (RISE) microscopy investigation of particles from an anti-asthma inhaler.

Raman Microscopy

The physical phenomenon that underlies Raman microscopy, known as the Raman effect, is the slight wavelength shift in light that has been inelastically scattered by molecules of gaseous, liquid, or solid materials. The incident photons from a monochromatic excitation source cause vibrations of the molecule’s chemical bonds, leading to a specific change in energy that is visible in a Raman spectrum. Every chemical compound produces a unique Raman spectrum when excited and can be quickly identified by it.
Raman imaging microscopy acquires a Raman spectrum at each image pixel over a sample area. This information is then compiled in an image that visualises the distribution of its chemical components. The time required to acquire a Raman spectrum with advanced Raman microscopes is generally a fraction of a second to less than one millisecond, which enables the generation of Raman images in a matter of minutes.
Instruments used to acquire Raman signals should have optimised optical components, including highly sensitive detectors with low noise to allow the detection of Raman signals of even weak Raman scatterers with the lowest excitation energy.

Raman-Based Automated Particle Analysis

For informative evaluations of powder samples, a large number of particles must be analysed. ParticleScout, an automated microparticle analysis tool for WITec’s alpha300 microscope series, accelerates this process by combining Raman spectroscopy with white light microscopy and advanced algorithms to find, classify, and identify microparticles.
Here, an analgesic (pain killing) and antipyretic (antifever) powder sample was investigated with ParticleScout by first acquiring a white light image. Image stitching ensured high resolution and focus stacking sharply defined the edges of each particle, which were used to create a mask of their locations. Criteria were selected that focused the analysis on particles with a Feret diameter of less than 100µm and an area of 1µm² or larger. This highlighted a total of 3,052 particles. A Raman spectrum was then automatically acquired from each particle and identified with the integrated TrueMatch Raman spectral database software (Figure 1A). The measurement revealed that the majority of the particles were the analgesic agents acetaminophen and ethenzamide. Caffeine was present as an effect enhancer, lactose as a carrier, and white pigment was also detected. A quantitative report describing the proportions of the substances found in the sample and their distributions in relation to size was then generated (Figure 1B, page 29).
Figure 1A: Correlative Raman-white light microscopy particle analysis of a powdered pharmaceutical sample
Figure 1B: Particles colour coded corresponding to spectra. Acetaminophen (blue), ethenzamide (cyan), lactose (green), caffeine (orange), and white pigment (light grey), with a quantitative report describing the proportions of the substances found in the sample and their distributions in relation to size

Topographic Raman Imaging

The combination of a Raman microscope with an optical profilometer enables chemical characterisation guided by surface topography. This allows even large samples without a flat surface to remain in constant focus during the measurement. This precise tracing of a sample surface, while acquiring Raman imaging data, combines the convenience of optical profilometry with the sensitivity of Raman microscopy to produce a sharp and detailed topographic Raman image. This technique, pioneered by WITec’s TrueSurface module, makes it possible to investigate the composition of whole, rather than pulverised, tablets.
The following investigation was performed with TrueSurface on a pharmaceutical tablet with a pronounced groove on its curved surface that also contains an array of components that could be evaluated in the manner of the particle analysis described above. Figure 2A shows the tablet in a topographic Raman image. The tablet’s topography is displayed over a large area (7x7mm²) and the simultaneously recorded Raman image (Figure 2B) is overlaid. The groove in the tablet is clearly visible in the topography representation, but the Raman image remained in focus over the whole range. Several different components were identified and could be analysed individually. For example, the pink component represents paracetamol (acetaminophen). It was isolated from the Raman image by Pearson correlation. The Pearson image (Figure 2C) maps the positions at which the Raman spectrum of the ‘pink component’ is present. It was then analysed with ParticleScout, so areas in which the component was present were recognised as a particle and their size distribution was analysed (Figure 2D). This procedure yielded the size distribution of one component at the surface of the tablet without damaging it.

Raman Imaging and Scanning Electron Microscopy

Figure 2: Topographic Raman imaging analysis of a whole tablet’s surface. Scale bar 1,000µm in all pictures.
A: Representation of the tablet’s topography and chemical composition. The surface of the tablet was traced and a Raman image was simultaneously acquired using TrueSurface;
B: Raman image of the tablet’s surface (7x7mm²). Different components are colour-coded;
C: Pearson map for the pink component (paracetamol);
D: Size distribution of the pink component (217 particles), quantified by the circular equivalent diameter of the spots in C
Microparticles are often used as carrier systems for drugs and their morphology and composition can affect the bioavailability of the delivered active pharmaceutical ingredient (API). Raman imaging and scanning electron (RISE) microscopy can explore these properties in great detail within a common vacuum chamber. Shuttling the sample between measurement positions enables the correlation of structural and chemical information.
This study of a particle from an anti-asthma inhaler used a WITec/TESCAN RISE microscope with a 532nm excitation laser to acquire a Raman image (Figure 3A) that visualises the four chemical components identified and colour-coded by their Raman spectra (Figure 3B). The particle consists mainly of lactose in two different hydration states (blue and green). The API was the glucocorticoid fluticasone propionate (red). A fourth component represented milk constituents (pink). The SEM image revealed the particle’s porous surface structures at high resolution (Figure 3C). It was recorded under low vacuum conditions (30Pa) using 15kV accelerating voltage and a BSE detector. The resulting correlative RISE image is shown in Figure 3D.


The applications presented here demonstrate how Raman microscopy can serve as an effective tool for chemically characterising the components present in pharmaceutical particles. Combined with complementary techniques it can analyse pharmaceutical particle samples with greater speed and detail than conventional techniques in isolation. Raman-based automated particle analysis enables thousands of particles to be located, categorised, and chemically identified through rapid, software-driven routines.
Figure 3: Correlative Raman-SEM (RISE) analysis of particles from an anti-asthma inhaler
3A: Raman image of a particle, colour-coded according to the spectra
3B: Raman spectra. API fluticasone propionate (red), lactose in two different hydration states (blue, green) and milk components (pink)
3C: SEM image of the same particle
3D: RISE image: overlay of the corresponding Raman and SEM images
Topographic Raman imaging can keep curved and textured surfaces, such as those often associated with pharmaceutical tablets, in constant focus. Raman integrated with SEM can provide a more comprehensive view of a particle by overlaying chemical and structural properties in a composite image.
The speed and precision that correlative Raman microscopy offers, along with its ease of use and the minimal sample preparation it requires makes it an accessible yet powerful tool for attaining a comprehensive understanding of a specimen’s chemical properties with sub-micron resolution.
Special acknowledgements to: Miriam Boehmler and Keiichi Nakamoto for performing the ParticleScout measurements, and Ute Schmidt for performing the RISE microscopy measurement.

Damon Strom is a Technical Marketing and Editing specialist at WITec GmbH. He received his Bachelor of Arts in History with a focus on the history of science from the University of Illinois Urbana-Champaign, US. During his coursework he was employed at the Laser Facility of the Frederick Seitz Materials Research Laboratory, where he was responsible for fabricating specialised components and helping researchers to set up experiments. He joined WITec GmbH in 2002, working in mechanical and industrial design until 2017 when he moved to the marketing department.