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Vibrational Spectroscopy in Pharmaceutical Development

Particle Sciences - Technical Brief: 2011: Volume 7
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Vibrational spectroscopy (VS) is the analysis of molecular properties based on vibrations at the molecular level and it is highly selective1. VS produces a spectral fingerprint of a material, such as an active pharmaceutical ingredient (API), so is well-suited to both identify and verify the API raw material and its presence in a final product. There are three related analytical techniques that use VS: Fourier-transform infrared (FTIR), near-infrared (NIR), and Raman. The objective of this technical brief is to compare these analytical modalities with an emphasis on the advantages that are inherent to Raman spectroscopy.

Any advantage for a particular spectroscopy modality is rooted in the fundamentals of the measurement itself. A key difference between the Raman and either FTIR or NIR is that Raman is a scattering measurement and FTIR and NIR are absorption based. In absorption methods, the sample is exposed to a range of wavelengths of light and the amount of light absorbed by the sample is compared to a reference and a difference spectrum is produced. With scattering technologies, the sample is exposed to a single wavelength of light usually in the form of a laser and a range of wavelengths are monitored for light emitted. The light emitted is as a result of the interaction (Raman) of the material with that single wavelength light. The requirement of the FTIR/NIR technique to have a reference light path will limit the sampling and instrument configurations since the reference and sample light path lengths must remain similar and short.

Absorption techniques are sensitive to the dipole vibrations in O-H, C-H, and N-H bonds whereas Raman is sensitive to polarizable vibrations like those present in C=C, C=N, C≡N, and aromatics. The dispersive technique is sensitive to the molecular backbone and analysis can be performed in aqueous preparations. The sensitivity of absorption techniques (i.e., FTIR and NIR) to these particular molecular vibrations limits this modality to samples that are non-aqueous and the spectra are dominated by signals from functional groups. Raman sensitivity to a molecular backbone suffers from the possibility that similar spectra are seen for materials which have small structural differences (i.e., potassium laurate vs. potassium palmitate; Figure 1).

Fourier-Transform Infrared Spectroscopy

FTIR (also known as mid-IR) is the most commonly used vibrational technique for material identification and authentication2. It owes its name to a popular instrument configuration for data input. In this technique, light absorption by the material of interest is measured across a range of wavelengths (~ 400 - 4,000 cm-1) corresponding to fundamental molecular vibrational modes. FTIR has exquisite molecular selectivity and the absorptions are strong. However, due to a requirement for similar and short sample and reference light paths, the instrument configurations are limited. Often, sample presentation requires specialized sample preparation procedures (i.e., KBr pellets or Nujol mulls for solids) or through use of attenuated total reflectance (ATR) crystals. Also, due to significant water interference, deuterated solvents are generally employed in order to examine samples in aqueous solution. With few exceptions, the sample of interest must be in direct contact with the instrument and when utilizing ATR a few microns of material depth are interrogated. Despite these limitations, FTIR remains a common method of API characterization including purity, identification, and solid-state properties such as polymorphism. FTIR microscopy is useful on a micro-scale with a spatial resolution on the order of 50 μ.

Near-Infrared Spectroscopy

NIR has gained favor over the last 20 years because of convenience and relatively large volume sampling3. NIR characterizes the material based on its absorption in the ~ 4,000 - 12,500 cm-1 wavelength region, corresponding to the vibrational overtone (harmonic) and combination modes of those fundamental vibrations seen in mid-IR. The bands observed in NIR predominantly arise from stretching of O-H, C-H, and N-H bonds and are present in almost any substance containing covalently linked hydrogen. These harmonic overtones are greatly broadened relative to their FTIR counterparts and continue to decrease and broaden with increasing order (third order weaker and broader than second order, etc.). Due to this spectral broadening and the commonality of these sub-structures across organic molecules, the differences between NIR spectra of different compounds are often very subtle and results in a lower molecular selectivity than FTIR. Like FTIR, the sample must be in direct contact with the instrument and the sampling/instrument configurations are thus limited. Also, the sensitivity to the O-H vibrational mode makes the measurement of aqueous samples impossible but NIR has been used in the detection of moisture in drying processes. The amenability of NIR to chemometrics makes it well suited to such processes. Other pharmaceutical NIR applications include detection and quantitation of API in solid dosage forms, polymorph identification, and crystallinity determination.

Raman Spectroscopy

In contrast to FTIR and NIR, Raman is a scattering/dispersive technique. Raman is effective in the 50 - 4,000 cm-1 range and involves a shifting of incident light (Raman shift). Raman, like FTIR, interrogates fundamental vibrational modes and has outstanding molecular selectivity. Advances in Raman instrumentation have provided a technique that incorporates the selectivity of FTIR and the sampling convenience of NIR with independence from some common sources of interference, such as water.

The focus here will remain on various advantages that Raman spectroscopy has in testing and characterizing pharmaceutical formulations. There are various configurations of Raman instrumentation; the two most common are dispersive and FT-Raman. The latter utilizes a lower wavelength excitation laser, minimizing fluorescence and sample heating that can occur in the dispersive mode.


Raman can be easily used in a non-contact fashion through many common container materials such as glass and plastic4. The need for sample preparation is nearly eliminated and in most cases a formulation can be examined directly. There is no need to grind the material or press into KBr pellets as with x-ray diffraction or FTIR. This advantage is particularly useful for the examination of materials in situ. Since the technique is dispersive / scattering, with no need for a reference light path, it is amenable to fiber optics and allows for remote sampling.

Extremely small samples can be interrogated since laser illuminators can focus on very small cross sections. This fact combined with the use of confocal mechanisms limiting the detection of out of focus light makes the adaptation of the Raman spectrometer to microscope systems a natural development. Note that in contrast to FTIR microscopy, a spatial resolution on the order of 1 – 2 μ can be achieved in confocal Raman microscopy (CRM). CRM also allows a change in the beam focus along the z-axis, so that depth profiling of samples is possible.

Crystal Forms

Many pharmaceutical compounds exhibit polymorphism, or different molecular arrangements within a crystal lattice. In general, polymorphs have differing lattice energies, which in turn give rise to differences in many other material properties, such as solubility and melting point. This further differentiates the polymorphs in terms of dissolution rate and bioavailability5. The structural backbone of polymorphic crystalline forms can be examined by Raman since the lattice vibrational mode spectral features (shifts typically near 200 cm-1) are available with the use of modern notch filters. These filters allow the collection of spectral data to within 50 cm-1 of the excitation laser, which isn’t possible with FTIR. This has made Raman particularly useful in the pharmaceutical industry for the correlation of dissolution kinetics with formulation-dependent crystal morphology in situ since in most cases the API's have very defined crystal structures but excipients generally do not. This offers an advantage of chemical specificity over NIR, wherein the signal mainly corresponds to the excipients. The ability of Raman to distinguish different crystalline forms has been integrated into high throughput screening of API’s during preformulation.

Raman has been used very successfully to identify the amorphous (non-crystalline) content in a drug formulation, i.e., solid dispersions. It can discriminate between crystalline and amorphous forms in dispersions, while mapping reveals the distribution of the API within the matrix. CRM is used to track changes in dispersions during storage, such as API homogeneity or in API crystal size and morphology.

Aqueous Samples– A MajorAdvantage

Raman is free from interference because of water bands, which dominate and influence NIR/FTIR spectra. Although the detection of API in solution is limited by the Raman signal strength and spectral bandwidths that are significantly wider in solutions, it has been used successfully for detection of small molecules and proteins in aqueous solutions (Figure 2). Also, Raman has found utility in the detection and analysis of a variety of ion-ion interactions in aqueous solutions albeit at high or saturation concentrations. Furthermore, Raman has been shown to be superior to NIR in probing API hydration during wet granulation.


The three vibrational spectroscopic techniques are useful in pharmaceutical applications. However, as with most analytical methods, a “one size fits all” VS instrument does not exist and so the choice between FTIR, NIR and Raman will depend upon the specific application. FTIR and Raman share the characteristic of being highly selective and therefore quite useful for compound identification testing. Since the techniques are sensitive to different structural features, the techniques are complementary. Raman’s ease of sampling, owing to its independence from packaging materials as well as water, will benefit users who have struggled with applying FTIR and NIR techniques to these applications.


  1. M Diem, Introduction to Modern Vibrational Spectroscopy, Wiley, New York (1993).
  2. JA Ryan, SV Compton, MA Brooks, and DAC Compton, J. Pharm. Biomed. Anal., 9, 303 (1991).
  3. M Blanco, J Coello, H Iturriaga, S Maspoch, and C de la Pezuela, Analyst, 123, 135 (1998).
  4. RL McCreery, AJ Horn, J Spencer, and E Jefferson, J. Pharm. Sci., 87, 1 (1998).
  5. IR Lewis, HGM Edwards, Handbook of Raman Spectroscopy, CRC Press, New York (2001).


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