Discovery and Formulation - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 224

order of magnitude greater than anticipated. Complementary studies [55] have
shown that roughening sample surfaces reduces the laser penetration into the
sample, but that the Raman collection volume is still larger than expected.
Coupled with the fact that both the refractive index and the 3D shape of each
material in a sample can affect the response in mapping experiments, there
will always be a degree of uncertainty in the domain sizes indicated by Raman
chemical images.
A further limitation of Raman mapping experiments is that the technique
is inherently slow. Experiments can take many hours or even days to produce
enough data to allow meaningful conclusions to be made. Several manufactur-
ers now offer systems that make measurements at significantly faster sampling
rates, but as yet there is no body of literature related to pharmaceutical sam-
ples to allow their effectiveness to be evaluated. Speed can also be obtained
through Raman imaging, but as the laser is defocused the resultant chemical
image is generally of poorer quality than that obtained via Raman mapping.
Raman imaging generally works best with spatially resolved samples.
Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerg-
ing technology. By tuning a pump laser and a Stokes laser to a Raman-active
molecular vibration, molecular selectivity and faster measurement speed can
be obtained. This approach has been used to track the phase segregation,
crystallisation and dissolution of paclitaxel from biocompatible excipients
and films providing kinetic data not achievable through standard Raman mi-
croscopy methods [56].
A complementary approach is that of FT-NIR mapping. With more rel-
ative energy being available from NIR sources and having a larger spot size,
these instruments map considerably faster over a larger sample area than
Raman systems though significantly sacrifice spectral and spatial resolution.
This will be important when particle domains of < 10 m are being investi-

gated. Similarly, NIR microscopy methods may forfeit sensitivity particularly
for compounds present at low concentrations. It is clear that when a chemical
image of a sample is required careful consideration must be given to what
information is required and then in matching the appropriate technology to
deliver the required data.
Irrespective of the method chosen, meaningful data can only be obtained
if the appropriate level of signal to noise (S/N) is reached in the spectrum of
each analyte. This has been achieved for Raman measurements through short
data acquisition times ( < 1 s) and application of mathematical approaches such
as K -harmonic means clustering (KHMC), factor analysis [57] and principal
component analysis (PCA) [58] to the data set. Ultimately the sample re-
sponse to the excitation energy determines the speed that a measurement can
be made.
Despite these limitations the ability to obtain information from tablets
(and other solid dosage forms) about the location and form of excipients and
API non-destructively using Raman mapping approaches is second to none.

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