Feature Article
JY Division
Raman Instrumentation for the
Information
Raman
Pharmaceutical Industry
Gwenaelle Le Bourdon, Fran Adar
Abstract
Over the past 5 years our Raman business has seen continually accelerating growth. The biggest component to this
growth is in the pharmaceutical industry. Several factors contribute to this success. It is generally now known that
it is easy to record a Raman spectrum, and that the information derived complements that of XRD (X-ray diffraction)
and FTIR (Fourier Transform Infrared Spectroscopy). The spectral resolution at 1 µm is far greater than either of
these techniques. And spectra can be recorded inside of glass and plastic containers. Of greatest interest is the
differentiation between the various solid state forms, and distribution of the APIs (Active Pharmaceutical Ingredients)
in final products. All of these issues are discussed in this article, with illustrative examples.
Introduction
With the revolution and evolution of Raman
instrumentation over the past 10-12 years, the Raman
market has been growing at an accelerated pace.
Consequently we get more and more requests to provide
systems specialized for particular applications, and some
of our “special developments” evolve into becoming
standardized products.
The largest area of growth that we are seeing is in the
pharmaceutical industry. This is a result of several factors.
• It has become recognized that measuring a Raman
spectrum is an “easy” experiment.
• There is information in the Raman spectrum that
complements FTIR and XRD.
• There are several types of information of interest to the
pharmaceutical industry that can be derived from Raman:
Differentiating Excipient vs. Active Ingredient,
Differentiating polymorphic forms of the Active Ingredient,
Monitoring Chemical changes of intermediate forms, and
Measuring Composition of Solution.
Of particular interest to the pharmaceutical industry is
information on polymorphy *1 – for 2 reasons. Clearly the
polymorph of the active ingredient will effect things like
solubility and bioavailability. But, because of these
properties, the pharmaceutical companies have been able
to patent not only the chemical composition, but the
polymorph as well.
56
What is important to recognize is that the polymorphy
of small molecules is determined by chemical interactions.
That means that intra- and/or inter-molecular interactions
between specific molecular functional groups can determine
the conformation of the molecules as well as the intermolecular packing in crystals. While XRD can identify
different polymorphs, extraction of detailed information
requires a full fitting of the pattern. On the other hand,
many molecular functional groups exhibit identifiable
vibrational modes in the Raman and/or IR spectra, and these
modes will shift due to chemical associations.
This article will show examples of results of
measurements made on these types of materials that will
demonstrate the power of the Raman instrumentation.
*1 By polymorphy, we include related phenomena of
pseudomorphy in which waters of hydration are included in
the crystal, and salt forms in which positive or negative
functional groups are associated with anions or cations.
Excipients vs. Active Ingredients
– Identification and Mapping
Pharmaceuticals are rarely sold as pure substances.
APIs are mixed with excipients in order to provide bulk,
extend shelf life, control solid state chemical reactions,
and control the time of release (by wrapping APIs in
polymer), etc.
An important characteristic of solid mixtures is the
distribution of the species. In fact, the operation of mixing
English Edition No.7
Technical Reports
itself becomes critical. It has been shown that vibration of a
dry powder mixture of several species of particles of equal
size but different densities will result in completely separating
the species [1]! Thus, significant engineering and quality
control has to be exerted in guaranteeing tablet composition.
20
Length X (µm)
25
30
35
40
45
5µm
50
20
25
30
35
40
45
50
Length X (µm)
Intensity (a.u.)
(a)
1200
1000
1086.7
800
600
400
Raman
20
Length Y (µm)
(b)
Emission
10
(a)
Grating & OEM
Raman spectroscopy is an ideal tool for the
differentiation of API from excipient. In fact, one could
say it is custom tailored. Normally the API is a minor
ingredient. But because it almost always has at least one
aromatic group, its Raman signal is quite high compared
to the other components.
The first example that we will present represents a study
of a pharmaceutical tablet. Fig. 1 shows a microscopic
image of a tablet. While it is possible to “see” particles,
there is no chemical information in the picture.
(c)
15
200
30
400
600
800
1000
1200
Wavenumber (cm-1)
40
(b)
2000
10
20
30
40
50
60
70
Length X (µm)
Fig. 1 Optical Microscopic Image of a Tablet
1500
477.3
1000
500
400
600
800
1000
Wavenumber (cm-1)
1200
Thin Film
(c) 14000
12000
1003.0
10000
8000
Optical
Spectroscopy
6000
4000
2000
0
400
600
800
1000
Wavenumber (cm-1)
1200
Fig. 2 Raman Map of a Tablet
Forensic
Spectral examination indicated the presence of at least
three chemical species. It was decided to “map” the
region indicated by the blue outline. In this operation,
the sample is rastered in the X and Y directions. At each
position, a full spectrum is acquired. The collection of
spectra is used to map the position of each of the chemical
species; in the map each color represents material whose
spectrum is printed in the same color (Fig. 2). The two
excipients have been identified as lactose (Fig. 2(a))
and starch (Fig. 2(b)) following a search of the spectra
in a Raman spectral library. The third component,
component C, has not been identified, but the presence of
a strong line at ca. 1000 cm-1 indicates that it is an aromatic
(Fig. 2(c)). Note also the intensity axis of component C
is an order of magnitude higher than that of the other two
components which confirms the expectation that aromatic
API will produce more intense Raman spectra.
Intensity (a.u.)
0
Intensity (a.u.)
60
Fluorescence
50
(a) Raman Spectrum of Lactose
(b) Raman Spectrum of Wheat Starch + Component C
(c) Raman Spectrum of Component C
(assumed as aromatic series)
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Feature Article
Raman Instrumentation for the Pharmaceutical Industry
It was mentioned above that the identification of the
excipients was done using Spectral ID, library-searching
software. Fig. 3 shows the Raman spectra of lactose
and starch, the two excipients mentioned above.
Fig. 4 is a reproduction of the Spectral ID screen
showing the overlay of the top spectrum with that of a
library spectrum of starch.
Polymorphy
Control of polymorphy is a major issue in the
pharmaceutical industry, as mentioned above. To reiterate, solubility and bio-availability depend on polymorphy.
Consequently, pharmaceutical companies want to control
polymorphy, and patent-protect their products with
polymorph spefications. Classically, polymorphy, and its
variants as described above, were determined by XRD.
943.5
Intensity (a.u.)
478.3
2907.1
1086.2
3363.0
1462.0
Wheat Starch
hand, vibrational spectroscopy will indicate chemical
2887.4
359.1
interactions between identifiable functional groups. And
the spatial resolution of vibrational techniques is determined
2946.5
478.3
852.9
1086.7
1326.5
2977.7
3382.9
Lactose
500
1000
1500
2000
2500
However, XRD requires rather large samples, and does
not provide chemical information directly. On the other
3000
Wavenumber (cm-1)
Fig. 3 Raman Spectra of Lactose and Wheat Starch
3500
by the physical diffraction limit and is quite good compared
to XRD. For Raman, of course, the spatial resolution is
about 1 µm. For infrared spectroscopy (FTIR) the spatial
resolution is about 20 µm.
In the pharmaceutical industry, not only is the
identification of the polymorph important, but its control
during formulation of product is of great concern. Under
certain circumstances it can be quite easy to convert from
one polymorph to another. For instance, if relative humidity
is not controlled, the degree of hydration of a crystal form
can change, which changes its pseudopolymorphic phase.
A different set of conditions that could require control
might be the mixing of APIs with excipient and/or pressing
into tablets. Under such operations one could also imagine
the possibility of changing hydration or even salt forms.
The following spectra(Fig. 5) document the ability to
Fig. 4 Spectral ID Screen
identify polymorphy of Tylenol by Raman spectroscopy.
The spectra represent the A and B forms.
FTIR spectra of the same species have been recorded
on the LabRam IR and are shown in Fig. 6 – first the
fingerprint part, and then the CH/NH part.
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English Edition No.7
Technical Reports
(a)
(a)
3000
1731
0.3
Absorbance
Intensity (a.u.)
1729
0.4
2000
Form B
0.2
Form B
0.1
Form A
Form A
0
800
100
200
300
400
1000
500
(b)
1735
1650
Intensity (a.u.)
1500
1000
4000
1617
500
0
3000
1600
1650
1700
1750
1600
1800
3426
0.20
3460
0.15
3506
0.10
Emission
5000
1730
Absorbance
1724
2500
2000
1400
0.25
1668
1670
3000
1200
Wavenumber (cm-1)
Wavenumber (cm-1)
(b)
Grating & OEM
1000
Form B
0.05
Form A
2000
2600
Form B
2800
3000
3200
3400
3600
3800
Wavenumber (cm-1)
Raman
1000
Form A
0
600
800
1000
1200
Wavenumber
1400
Fig. 6 Infrared Absorption Spectrum of Tylenol Polymorphism
1600
(a) Fingerprint Region
(cm-1)
(b) CH/NH Region
(c)
7000
Intensity (a.u.)
how the interactions between functional groups can
determine the way the molecule crystallizes. Now we
5000
Form B
can see how vibrational spectra can provide information
4000
3000
2000
3434
3033
3460
on molecular associations. Table 1 tabulates the
frequencies of >NH/-OH (3300-3600 cm-1) and >C=O
3050
3502
(1710-1740 cm-1) bands.
1000
Form A
0
2800
3000
3200
Wavenumber
3400
(cm-1)
Table 1 Raman and FTIR Peak Wavenumber for Tylenol
Polymorphism
Raman
FTIR
Form A
3460/3502 cm-1
1730 (sh 1735) cm-1
3460/3506 cm-1
1731 cm-1
Form B
3434 cm-1
1724 cm-1
3426 cm-1
1729 cm-1
Optical
Spectroscopy
Fig. 5 Raman Spectrum of Tylenol Polymorphism
(a) Wavenumber Approx. 50 to 500 cm-1
(b) Wavenumber Approx. 500 to 1800 cm-1
(c) Wavenumber Approx. 2600 to 3700 cm-1
3600
Thin Film
3025
Fluorescence
Recall that in the introductory section, we described
6000
Forensic
59
Feature Article
Raman Instrumentation for the Pharmaceutical Industry
There is overall agreement between the FTIR and Raman
results. Form A has a doublet in the NH/OH region but a
singlet in Form B. The carbonyl band has a frequency
(a)
slightly higher in form A than form B. However, the
difference in frequency is more pronounced in the Raman
spectrum rather than the FTIR spectrum. This is
somewhat ironic because the IR is believed to be more
sensitive to carbonyl than Raman, which is, in fact,
supported by the relatively low Raman intensities of this
band. However, the insensitivity of the FTIR frequency
could possibly be a result of the larger width of IR band.
These differences in frequencies can be assigned to
phenomena such as hydrogen-bonding. A lower frequency
(b)
means more H-bonding, which is also often reflected in a
wider band. In this case form B is exhibiting stronger Hbonding.
High Throughput Screening
and the Multiwell LabRam
(c)
High Throughput Screening (HTS) is the latest approach
in analytical technology. Used particularly in the
pharmaceutical industry and in life science laboratories,
this method enables systematic screening of drug design
and high throughput analysis of batch production.
The LabRam software has been adapted for automated
study of Multiwell Plates. All of the phenomena discussed
above are amenable to automated analysis for High
Throughput Screening. Using a user-defined preprogrammed protocol for analysis, the software will output
information in the form of an Excel spreadsheet. For
example, the spreadsheet can report concentrations of API
vs. excipient or different polymorphic forms as a function
of well position. Fig. 7 shows the Raman Multiwell
Fig. 7 Raman Multiwell Analyzer
(a) Multiwell LabRam
(b) Multiwell Plate
(c) Spectrum Screen
Analyzer.
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English Edition No.7
Technical Reports
Conclusion
Developments of the last 75 years have paved the way
for the implementation of sophisticated, automated Raman
instruments in the analytical laboratory. These
developments are in fact, two-pronged. On the one hand,
Grating & OEM
the interpretation of spectra has to enable use of the
phenomenon to extract useful information. On the other
hand, the equipment has to produce the information with
a minimum of effort. As this has converged, the
pharmaceutical industry is now poised to take advantage
of these developments to aid in the development of their
Emission
products at all stages.
Reference
Raman
[1] Tom Mullin, “Mixing and De-mixing,” Science 295,
p.1851 (8 March, 2002)
Fluorescence
Thin Film
Optical
Spectroscopy
Gwenaelle Le Bourdon, PhD
Jobin Yvon S.A.S
Raman Division
Application Scientist
Jobin Yvon Inc.
Raman Worldwide Senior Application Scientist
61
Forensic
Fran Adar, PhD