Raman Spectroscopy: Multiple Techniques Can Analyze Pharmaceutical Tablets and Capsules

 Raman Spectroscopy: Multiple Techniques Can Analyze Pharmaceutical Tablets and Capsules

 

Hesamodin Hosseini Ghahi
and Johannes Kiefer
University of Bremen, Engineering
Thermodynamics and MAPEX
Center for Materials and Processes

 

Raman spectroscopy is emerging as a powerful analytical tool for the pharmaceutical industry.

Like the more common FTIR spectroscopy, Raman spectroscopy is part of the vibrational spectroscopy family of analytic methods. Both methods provide a molecular fingerprint of the sample by probing the vibrations of the covalent bonds. Nevertheless, Raman spectra look different from FTIR spectra because the underlying physical principles differ: absorption in FTIR and scattering in Raman. In absorption, light (in this case, infrared light) is sent to the sample, which then absorbs some wavelengths and transmits others. Spectral analysis of the transmitted light reveals the fingerprint signatures of the sample. In Raman scattering, a laser is pointed at the sample and the scattered light is analyzed in a spectrometer. This scattered light carries the same information as FTIR, but through a frequency difference between the laser wavelength and the molecular vibrations of the sample. Figure 1 illustrates both effects in an energy level diagram. It becomes clear that both methods probe the same molecular transitions, but through different paths. As a consequence, the spectra appear similar, but exhibit distinct differences, as there are transitions that are exclusively IR-active or Raman-active, depending on the dipole and polarizability properties, respectively. (For detailed descriptions of the methods, see common text books.1-4

Figure 1
Energy level diagram illustrating the underlying physics of FTIR and Raman spectroscopy.

Raman spectroscopy provides a number of great features for pharmaceutical analysis. Depending on the specific Raman technique applied and instrument employed, it offers a rapid and nondestructive full chemical analysis, as the Raman spectrum contains molecular signatures of all the species in the sample (e.g. the pharmaceutically active ingredient as well as all the excipients). The general appearance of the spectrum enables a qualitative analysis. Taking a closer look at the relative peak intensities allows the concentrations to be extracted. Utilizing appropriate training data facilitates evaluation of individual spectra or even entire data sets in a fast and reproducible fashion. 

Many different individual Raman techniques have been developed for different purposes. For analyzing capsules and tablets, three of them—namely Spatially Offset Raman Spectroscopy (SORS),5 Transmission Raman Spectroscopy (TRS),6 and Confocal Raman Microscopy7—have proven useful. These variants distinguish themselves through the ways in which the sample is irradiated by the laser and how the scattered signal is collected. In the simplest cases, the sample is homogeneous and transparent, like a solution in a glass vial or cuvette. Then the laser beam can simply be guided through the center of the sample and the scattered signal is commonly collected in a direction perpendicular to the laser path or in a backscattering arrangement, i.e. in the opposite direction of the laser propagation. In inhomogeneous samples such as capsules and tablets, the impact of the interfaces on the propagation of the laser and the signal photons needs to be taken into account. On the one hand, the inhomogeneity makes life more difficult, but on the other hand, it also offers new possibilities for excitation-detection schemes. Each Raman technique has its own features, strengths, and weaknesses for analyzing capsules and tablets: 

Confocal Raman Microscopy 

Raman microscopy takes advantage of the backscattered signal. The strength of the confocal Raman microscopy for product analysis lies in its imaging capability, combined with providing chemical contrast of a surface. It enables the discrimination of different visible layers of a sample surface, e.g. resulting from polymorphic forms of a blend of multiple active pharmaceutical ingredients such as in a tablet mixed with different ingredients. 

In a confocal microscope, the entire sample is not illuminated completely at once. Instead, the exciting radiation (i.e. the laser beam) is focused to a small spot by the objective lens and the backscattered signal is collected by the same lens, see Figure 2. The signal is then sent through a spatial filter to block out-of-focus light and to achieve a well-controlled and highly limited depth of field. A dichroic mirror separates the laser beam from the signal. Rapid scanning of the sample position results in a hyperspectral image, which means that a map is created and a full Raman spectrum is available for each point. Confocal Raman spectroscopy provides fantastic spatial resolution, which is on the order of the wavelength of the light (typically sub-micron). 

Figure 2
Laser focusing and signal collection in confocal Raman
spectroscopy in tablets.

The confocal method comes with a number of advantages and disadvantages. The high spatial resolution allows for collecting detailed information about the spatial distribution of ingredients, e.g. at the surface of the tablet. It enables particle size determination of e.g. active pharmaceutical ingredients, since Raman images afford necessary resolution to distinguish discrete domains of a mixed species. Furthermore, a comparison between polymorphic forms of pharmaceuticals can be performed, e.g. when it comes to investigations or inspections between the polymorphic form and hydration state of active pharmaceutical ingredients. However, the need for scanning the sample and recording Raman spectra point by point can be a significant limitation, as it may result in long measurement times. On the other hand, in cases where only a small number of spots are probed, it may bear prediction biases, especially when it comes to quantifications for pharmaceutical formulations. Therefore, this technique is best suited for the image-resolved analysis of a sample surface, when measurement time is not crucial. Pharmaceutical applications may include the discrimination of different top layers on a tablet surface, e.g. discrimination of a coating region and the core of a reference tablet. It can also verify the homogeneity of the surface and identify potential imperfections of a coating. 

Spatially Offset Raman Spectroscopy 

Spatially offset Raman spectroscopy takes advantage of the inhomogeneous nature of a sample. In an inhomogeneous sample—like tissue, a suspension, or a pharmaceutical tablet or capsule—both the laser light and the signal photons will scatter at particles or phase boundaries, as illustrated in Figure 3. Consequently, the collection point can be moved away from the point where the laser irradiates the sample. Essentially, the farther away the laser spot is from the signal collection point, the more likely signal photons from deeper layers will contribute to the overall signal. Hence, a systematic variation of the distance between the two spots can provide a depth profile with chemical information. SORS is therefore a useful tool for analyzing the inside or subsurface, such as deeper lying features in multi-layered systems in materials of interest. This can be achieved with different illumination-detection approaches. The most common SORS variants include the point-like SORS, ring-collection SORS, ring-illumination SORS (inverse SORS), and defocusing SORS, see Figure 4. 

Figure 3
Laser propagation and signal collection in spatially offset Raman spectroscopy.

The point-like SORS belongs to the simplest SORS variants and is similar to the conventional Raman setup, because it uses a point-like illumination and collection geometry, which is separated by the spatial offset. Through a sequential move of the laser or collection area across the sample surface, multiple collection points can be read between acquisitions, or alternatively, data collection from multiple points can be performed simultaneously by collecting signals from each spatial offset to a separate channel of the detector. This technique is known as hyperspectral SORS, and works well in situations where a sample is evolving in time, e.g. undergoing a chemical reaction or as a moving sample. However, slightly different Raman spectral profiles and distortions can appear due to different detection channels, caused by the spectrograph’s imaging imperfection. This can lead to artefacts in the processed data and requires a numerical correction, which marks the limitation of this method. Furthermore, high illumination intensities at all spatial offsets can destroy samples. 

Figure 4
Selected illumination-detection schemes (top view) of spatially offset Raman spectroscopy.

In order to optimize signal levels, the collection area can be increased, giving rise to the ring-collection SORS geometry. Alternatively, ring-illumination—aka inverse SORS—can be employed. The latter has the advantage of a decreased irradiance on the sample. This can be important when the sample is sensitive to radiation and may be damaged during the measurement. Another option to increase signal levels and decrease irradiation is to defocus SORS. Because the defocusing SORS uses overlapping illumination and collection areas, it can be regarded as less effective with respect to contrast between the surface and subsurface than the above introduced modalities. The key advantage of its application, however, relies on its simple implementation on conventional Raman instruments. By moving the sample away from the imaging position, both the laser illumination and Raman collection areas can be increased as a result of the defocusing SORS performance. A SORS effect is given while the laser irradiation and the detection of the Raman signal can be spatially offset on the sample, although no actual separation is achieved between two areas. This leads to the simultaneous collection of spectra from a range of SORS offsets, and thus enhances depth sampling without suppressing the surface signal to the same degree as other SORS modalities. 

Figure 5
Illustration of transmission Raman spectroscopy in a tablet.

Transmission Raman Spectroscopy

The concept of transmission Raman spectroscopy (TRS) can be considered a special application of SORS, where the illumination and collection areas are on opposite sides of the sample, see Figure 5. In contrast to other SORS modalities, the transmission geometry does not enable for an individual layer investigation. Furthermore, TRS is only applicable to samples where both surfaces are accessible for analysis. This enables a volumetric signal by approximating the average composition of the probe sample volume. Therefore, the transmission geometry represents a sophisticated variant of Raman spectroscopy for the noninvasive and nondestructive optical analysis of tablets and capsules, especially when quantifying pharmaceutical formulations, where the average volumetric sample composition including information about the total amounts of active pharmaceutical ingredients is desirable. This achievement results from the long migration times of Raman photons in non-absorbing powder or in general turbid media without any thinning of an analyzed sample, e.g. tablets. Furthermore, the transmission geometry exhibits gross insensitivity to the depth of impurities within a sample, such as given in a blend turbid medium. 

Conclusion 

Table 1. Summary of discussed Raman spectroscopic methods.

Three variants of Raman spectroscopy are capable of analyzing tablets and capsules. Table 1 provides a summary of the main strengths and weaknesses with respect to pharmaceutical analysis. Table 2 gives an overview of commercially available instruments needed for the three Raman spectroscopy techniques. SORS can, in principle, be performed with any high-performance benchtop Raman instrument, in particular the defocused illumination-detection scheme. Raman spectroscopy offers great features for qualitative and quantitative analyses in the pharmaceutical industry. It is a nondestructive and fast method, and it can provide a comprehensive chemical characterization of a sample. As instruments and sophisticated data evaluation approaches become more user friendly, Raman has emerged as a competitor to conventional analytical methods. 

Table 2. Overview of manufacturers offering SORS, TRS, CRM devices.

References 

1. P. Vandenabeele, Practical Raman Spectroscopy, Wiley, Chichester, UK, 2013. 

2. P.R. Griffiths, J.A. De Haseth, Fourier Transform Infrared Spectrometry, 2nd ed., Wiley, Chichester, UK, 2007. 

3. P.J. Larkin, Infrared and Raman Spectroscopy: Principles and Spectral Interpretation, Elsevier, Amsterdam, NL, 2011. 

4. K. A. Bakeev, Process analytical technology: spectroscopic tools and implementation strategies for the chemical and pharmaceutical industries. John Wiley & Sons: 2010. 

5. S. Mosca, C. Conti, N. Stone, P. Matousek, Spatially offset Raman spectroscopy. Nature Reviews Methods Primers 2021, 1 (1), 1-16. P. Matousek, A.W. Parker, Bulk Raman analysis of pharmaceutical tablets. Applied spectroscopy 2006, 60 (12), 1353-1357. 

6. J. Johansson, A. Sparén, O. Svensson, S. Folestad, M. Claybourn, Quantitative transmission Raman spectroscopy of pharmaceutical tablets and capsules. Applied spectroscopy 2007, 61 (11), 1211-1218. 

7. J. Toporski, T. Dieing, O. Hollricher, Confocal Raman Microscopy, Springer Series in Surface Sciences, Springer, 2018. 

Author Biographies

Hesamodin Hosseini Ghahi is a PhD student at the University of Bremen and develops enantioselective Raman techniques for pharmaceutical analysis.

Johannes Kiefer is professor of engineering thermodynamics. His research interests include the development of optical spectroscopic methods for engineering and life science applications. 

 

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