Pharmaceutical Particle Analysis with Correlative Raman Microscopy

Damon Strom explores comprehensive particle analysis with correlative Raman microscopy

Raman microscopy provides exceptional chemical sensitivity that allows data acquisition from very small material volumes, such as those encountered in microparticle analysis. The combination of Raman with other technologies, including confocal white light microscopy and scanning electron microscopy (SEM), to acquire data from the same measurement position is known as correlative Raman microscopy.

Investigations of particles are especially important in pharmaceutical research as a precise understanding of properties and composition is necessary to refine production processes and determine efficacy. Analyses of many particles and the identification of different substances within an individual particle are routine challenges. The following measurement examples demonstrate how correlative Raman microscopy can expedite these tasks.

Raman and Confocal White Light Microscopy

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 confocal white light microscopy and advanced algorithms to find, classify and identify microparticles.

Here, an analgesic (pain-killing) and antipyretic (anti-fever) powder sample was investigated with ParticleScout by first acquiring a confocal white light image (Fig. 1A). 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 100 µm and an area of 1 µm² or larger. This highlighted a total of 3,052 particles (Fig. 1B). A Raman spectrum was then automatically acquired from each particle and identified with the integrated TrueMatch Raman spectral database software (Fig. 1C). 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.

Raman Imaging and Scanning Electron Microscopy

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 (Fig. 2A) that visualises the four chemical components identified and color-coded by their Raman spectra (Fig. 2B). 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 (Fig. 2C). It was recorded under low vauum conditions (30Pa) using 15kV accelerating voltage and a BSE detector. The resulting correlative RISE image is shown in Fig. 2D.


Raman microscopy combined with complementary techniques can analyse pharmaceutical particle samples with greater speed and detail than conventional techniques in isolation. Raman together with confocal white light microscopy and an advanced particle analysis software enables thousands of particles to be located, categorised and chemically identified through rapid, automated routines. Raman integrated with SEM can provide a more comprehensive view of a particle by overlaying chemical and structural properties in a composite image.

Damon Strom is with WITec

Recent Issues