Developing methods to analyze drug substances and finished drug products is crucial for ensuring the quality of pharmceutical
products. Several industry experts discuss applications in pharmaceutical analysis. Jerry Sellors, IR business manager at
PerkinElmer, examines attenuated total internal reflectance sampling in Fourier tranform–infrared spectroscopy. Bo Wang, research
scientist, and Laurence A. Nafie, chief technology officer, both with BioTools, and Elena Eksteen, senior business manager,
business development and planning at Chiral Technologies, discuss vibrational circular dichroism technology in determining
the absolute configuration of enantiomers. Robert H. Clifford, PhD, industrial business unit manager at Shimadzu Scientific
Instruments, discusses ultraviolet–visible spectrophotometry in determining the quantitation limit of residual samples in
cleaning validation. Richard Godec, new product development manager, Jon Yourkin, pharmaceutical market manager, and Kevin
Aumiller, product manager, at GE Analytical Instruments, examine on-line total organic carbon analysis for pharmaceutical
water. St. John Skilton, PhD, senior marketing manager, business operations, LSD, pharmaceutical life sciences at Waters,
explains the role of hydrogen/deterium exchange with mass spectrometry in biopharmaceutical analysis.
Attenuated internal reflectance sampling in FT–IR
Jerry Sellors, IR business manager at PerkinElmer
Most pharmaceutical laboratories use a Fourier transform–infrared (FT–IR) spectrometer for testing materials. Mid-IR spectroscopy
provides rapid and highly specific pharmaceutical ingredient identity testing. IR spectroscopy is used for quantitative and
qualitative analysis of solids and liquids, but the most common use for pharmaceuticals analysis is identity confirmation
of powdered ingredients. Three common sampling techniques are used: two involving transmission sampling and a third involving
attenuated total internal reflectance (ATR).
One approach in transmission sampling is the alkali halide disk method (often referred to as the KBr method), which involves
dispersing the sample in a nonabsorbing matrix (e.g., potassium bromide [KBr]) and pressing the mixture into a semitransparent
disk to be measured in transmission by passing the IR beam through the disc. Alternatively, in another transmission method,
the sample is mixed with a mineral oil and ground into a paste that is pressed into a thin layer between two IR-transparent
windows and measured. Both techniques require manual sample preparation, are time-consuming, and may be prone to error. Among
all the pharmacopeias, however, these two methods for IR solids sampling are most commonly used. Despite advances in FT–IR
technology to provide increased sensitivity, reproducibility, and reliability, developments in sampling apparatus for these
techniques have been unremarkable.
Advances in transmission sampling have been slow to develop in part because of the use of another approach, attenuated total
internal reflectance (ATR) techniques for IR measurement of solids. With ATR, the sample is pressed into contact with a high
refractive index crystal, such as germanium or diamond, and the sample is measured by reflection (see Figure 1). ATR systems
often are designed as a central component of the overall FT–IR system. Under ATR, users can more easily acquire good quality
IR data from a wide range of samples with minimal sample preparation. ATR technology has improved with respect to crystal
design and hardware coupling with FT–IR systems as well as with respect to the software performing automated system performance,
suitability, and contamination checks. ATR crystals also can be coupled with mid-IR fiber optics and light-guide systems,
where improved sample accessibility is required.
Figure 1 (FT–IR): Schematic for attenuated total internal reflectance for infrared (IR) measurement of solids. (FIGURES 1–3(FT–IR)
ARE COURTESY OF THE AUTHOR)
The level of acceptance of the ATR technique among the different pharmacopeias is somewhat mixed. For example, the US Pharmacopiea recognizes the technique as an acceptable alternative, but many other pharmacopeias do not mention ATR. The responsibility
is with the user to demonstrate equivalence when this method is chosen to replace existing transmission methods.
The acceptance of ATR in regulated environments requires a general understanding of ATR because new sources of variation not
encountered with conventional transmission measurements can affect the reliability of results. ATR is a reflection technique
and can show distinct surface and optical geometry effects that may need to be characterized (1). For example with ATR, IR
spectral-band intensities generally change with increasing force applied, and the effect is confounded by an increase in sample
penetration depth across the wavelength range measured. This variability can affect quantitative and qualitative IR measurements,
which rely on relative band intensities. An example of this can be seen in the ATR spectra of kaolin (see Figure 2) recorded
at two different contact pressures, where a silicon–oxygen band in the spectrum of the mineral has a significant shift attributed
to deformation of the crystal lattice. Assessing the effects of such variability is important for some materials, where altering
pressure can change the degree of sample crystallinity and polymorphic form. Consequently, some ATR devices are designed to
provide real-time display of force and spectral intensities before recording the IR spectra so that both can be checked before
measurement. Pharmaceutical packaging materials also are frequently analyzed using ATR.
Figure 2. (FT-IR): Attenuated total internal reflectance spectra of kaolin at different sample pressures. A is absorbance.
In cases where oriented samples are presented to the system, care should be taken to understand the effect or orientation
on the FT–IR spectra used in the analysis. This orientation effect is particularly true with molded or extruded polymers.
Figure 3 shows the spectra of the surface of a polylactic acid where the only difference between the two measurements is a
rotation of the sample on the ATR device. This difference is large enough to cause problems with routine measurements.
Figure 3: Attenuated total internal reflectance spectra of polylactic acid with rotation of sample. A is absorbance.
As IR use increases for routine measurements of pharmaceuticals, the acceptance of ATR among the pharmacopeias is likely to
increase. Using ATR as an alternative to existing transmission methods requires showing evidence of suitability. Controlling
and understanding the sources of measurement variation are important. IR instrument packages are being expanded with more
sophisticated software routines to improve confidence in ATR results and knowledge-based offerings to assist the user in method
development and validation.
1. R. Spragg, "Contact and Orientation Effects in FT–IR ATR Spectra, Spectrosc., http://spectroscopyonline.findanalytichem.com/spectroscopy/FT-IR+Spectroscopy/Contact-and-Orientation-Effects-in-FT-IR-ATR-Spect/ArticleStandard/Article/detail/735155 (Aug. 1, 2011).