Lasers, Spectrographs, and Detectors - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 4

Sample

Light
Source

Detection

Dispersion

Fig. 1.1. Simplified components of a Raman spectrometer

the phenyl ring would have a weak response, while the OH would be very
strong. However, in both infrared and Raman spectra, the exact frequency
and intensity of a given vibrational band will be effected by interactions with
the other vibrational modes present in the molecule. This means that the
spectrum for a given molecule is effectively a fingerprint of that molecule.
This is recognized in the US Pharmacopeia in that either the mid-IR or Ra-
man spectrum can be used as conclusive identification of a pure substance.
This also has the important implication in that Raman spectroscopy can be
used to determine specific molecules that may be markers for disease states,
biothreats, or environmental contaminants.
The Raman effect has also been broadly applied to online and bench-top
quantitative applications, such as determination of pharmaceutical materi-
als and process monitoring [4-6], in vivo clinical measurements [7], biological
materials [8, 9], to name only a few. Because the absolute Raman response is
dicult to measure accurately (sample presentation and delivered laser power
can vary), these measurements are almost always calculated as a percentage
with respect to the response from an internal standard. This standard is typ-
ically part of the sample matrix; in a drug product, the standard may be an
excipient; in a biological sample, it is commonly water.
This is represented graphically in Fig. 1.1. These spectra are from a cal-
ibration set consisting of differing levels of ethanol in decanol ranging from
0 to 10%. As described above, the spectra-to-spectra overall intensity varies
significantly. A wide variety of approaches can be taken to normalize the spec-
tra, suppress background interference, and enhance selectivity and accuracy.
Quantitative methods for Raman spectroscopy using univariate and multivari-
ate methods are treated extensively elsewhere [10]. In this example, a second
derivative was applied to the spectra, which removes baseline variation and
enhances band resolution, followed by a univariate intensity calibration. Al-
though limit of quantitation will vary widely depending on the analyte and
matrix, the results shown in Fig. 1.2 (quantitation in the ∼ 1% by weight

range) are typical.
Although both Raman and IR can be used to determine molecular identity
or quantify the presence of an analyte, there are critical differences in the way
that they can be applied. The extremely strong absorption of OH in the mid-
IR makes any measurement dicult in the presence of water - a ubiquitous
material in most biological samples. Subtle spectral differences that may be
apparent in the Raman spectrum tend to be obscured by the overwhelming
water absorption. This is shown in Fig. 1.3 for a solution of lactose in water;