Vibrational Spectroscopy for
Pharmaceutical Analysis
Part II. Introduction to Applications and Theory.
Rodolfo J. Romañach, Ph.D.
ENGINEERING RESEARCH CENTER FOR
STRUCTURED ORGANIC PARTICULATE SYSTEMS
RUTGERS UNIVERSITY
PURDUE UNIVERSITY
NEW JERSEY INSTITUTE OF TECHNOLOGY
UNIVERSITY OF PUERTO RICO AT MAYAGÜEZ
10/11/2005
1
Spectroscopy
• Spectroscopy – is the study of the interaction
between radiation and matter.
• In spectroscopy the absorption or reflection of
radiation by matter (solids, liquids, gases) is
studied.
• Spectroscopists study the interaction of radiation
with practically the entire electromagnetic
spectrum.
2
Electromagnetic Spectrum
12,500 cm-1 (800 nm)
108
107
106
4,000 cm -1 (2500 nm)
105
104
103
102
101
1
10-1
10-2
10-3
Interaction
Region
Frequency
(cm-1)
-Ray
Nuclear
Transitions
X – Ray
v
i
s
i
b
l
e
Ultraviolet
NIR
Valance
Electron
Transitions
Inner
Shell
Electronic
Transitions
MIR
FIR
ESR
NMR
Infrared
Microwave
Radio, TV Waves
Molecular
Vibrations
Molecular
Rotations
Spin
Orientation in
Magnetic
Field
Wavelength
(m)
10-10
10-9
10-8
10-7
10-6
10-5
10-4
10-3
10-2
10-1
1
101
Slide Courtesy Bruker Optics
3
Spectroscopy Provides Structural
Information
The various spectroscopic techniques provide
information such as:
• Presence of functional groups.
• Differences in the crystal structure of compounds.
• Presence of elements (Fe, Ni, Pb) in a sample.
4
Modules Cover
• Mid-infrared spectroscopy.
• Near infrared spectroscopy.
• Raman spectroscopy
There are many other spectroscopic used in other in
analytical chemistry and materials science.
5
Mid-IR Spectroscopy Widely Used in:
• Identification of Pharmaceutical Raw Materials and Finished
Products. Principal method of material identification in a
pharmaceutical manufacturing company.
• Combination with MS and NMR to determine structure of process
impurities and degradation products.
• Characterization of natural products, use of GC/FT-IR.
• Forensic Analysis, IR-Microscopy.
• Environmental Analysis: GC/FT-IR.
• Surface Analysis, Diffuse Reflectance, Attenuated Total
Reflectance, Grazing Angle.
• Studies of Protein Structure and Dynamics.
6
Mid-infrared spectrum of acetone.
7
Units in IR Spectroscopy
• Mid-infrared range, wavelength is 2.5 – 25 μm.
Equivalent to 4000 – 400 cm-1.
• The cm-1 are termed wavenumbers or reciprocal
centimeters, not frequency (ν). Wavenumber is the
number of waves in a 1 cm wavetrain.
• Frequency is number of complete waves in each second.
• Wavenumber = ν/c = 1/λ
8
Units of spectra- nm, m, cm-1
Sometimes see cm-1 :
• 10,000 cm-1 = (1/10,000) cm or 0.0001 cm = 1
m = 1000 nm
• 6,000 cm-1 = (1/6000) cm or 0.000167cm = 1.67
m = 1670 nm
• 5,000 cm-1 = (1/5000) cm or 0.0002 cm = 2 m
= 2000 nm
• 4000 cm-1 = (1/4000) cm or 0.00025 cm = 2.5
m = 2500 nm.
9
Vibration Theory
The molecule can be thought of as mass m1 and m2 connected by a
spring. At equilibrium, the distance between the two masses is r0. If
the molecule is stretched by an amount r = x1 + x2, then a restoring
force, F, is produced. If the spring is released, the system will vibrate
around the equilibrium position. According to Hooke’s Law, for small
deflections the restoring force is proportional to the deflection:
F = -k . r
Since the force acts in a direction opposite to the deflection, the
proportionality constant, or force constant, k, is negative in sign. The
force constant is called the spring constant in the mechanical model,
whereas in a molecule the force constant is a measure of the bond
strength between the atoms.
Courtesy Bruker
Optics
10
Molecular Vibrations
M1
For a simple harmonic
oscillator it is possible to
calculate the vibrational
frequency, n, of a diatomic
molecule as follows:
M2
Simple harmonic oscillator:
n=
n - vibrational frequency in
wavenumber
k - vibrational force constant
c - speed of light
 - reduced mass = m1 m2 /(m1+m2)
1
2pc
k

1/2
( )
Slide Courtesy Bruker
Optics
11
Vibration Theory
On the basis of the equation above it is possible to state the following:
n~ the higher the vibrational
1) The higher the force constant k, i.e., the bond strength,
frequency (in wavenumbers).
3 absorption peaks for different force constants. Note that
by convention, in infrared spectroscopy wavenumbers are
plotted right-to-left; i.e., highest wavenumber to the left.
Slide Courtesy
Bruker Optics
12
Vibration Theory
2) The larger the vibrating atomic mass,
the lower the vibrational frequency in wavenumbers.
3 absorption peaks for different atomic masses. Note that by
convention, in infrared spectroscopy wavenumbers are
plotted right-to-left; i.e., highest wavenumber to the left.
Slide Courtesy
Bruker Optics
13
Vibration Theory
For the harmonic oscillator model, the
potential energy well is symmetric.
According
to
quantum-mechanical
principles molecular vibrations can only
occur at discrete, equally spaced,
vibrational levels, where the energy of the
vibration is given by:
Ev=(v + ½) h  n
v = 0, 1, 2, 3, ...
Where h is Planck’s constant and v is the
vibrational quantum number. Even in case
of v = 0, which is defined as the ground
vibrational level, a molecule does vibrate:
Ev= ½ h  n
Potential energy curve
for a harmonic oscillator
Based on Bruker Optics
Slide
14
Vibration Theory
• When absorption occurs,
the molecule acquires a
clearly defined amount
of energy, (E = h  n),
from the radiation and
moves up to the next
vibrational level (v =
+1).
• For a harmonic
oscillator, the only
transitions permitted by
quantum mechanics are
up or down to the next
vibrational level (v =
1).
Based on Bruker Optics Slide
If the molecule moves down
to the next vibrational level
(v = -1), a certain amount of
energy is emitted in the form
of radiation. This is called
emission.
15
A Molecule Absorbs Infrared Energy
when:
• A vibration occurs where the dipole moment of the molecule
changes, and the molecule is illuminated with radiation
equal to the frequency of vibration.
16
Change in Dipole Moment during Molecular Vibrations
• Must change for IR
absorption to occur.
• The dipole moment is
a measure of the
degree of polarity of
molecule (magnitude
of the separated
charges times the
distance between
them).
• A measurement of
degree of unequal
distribution of charges
in molecule.
-
+
+
-
-
+
H
Cl
17
Band Intensity in IR
• Band intensity depends on the rate of change of
dipole moment during absorption of IR light.
• Stronger bands occur when the change in dipole
moment is greatest.
18
A Mid-Infrared Spectrum
• A spectrum is a plot that shows the absorption or
reflection of radiation as wavelength or frequency
of the radiation is varied.
19
IR Spectrum of Grease used in
Pharmaceutical Industry
17
20
USP on Mid-IR Spectrosopy
The Unites States Pharmacopeia indicates:
“The IR spectrum is unique for any given
chemical compound with the exception of optical
isomers, which have identical spectra. However,
polymorphism may occasionally be responsible for
a difference in the IR spectrum of a given
compound in the solid state.”
USP section 851, “Spectrophotometry and Light Scattering”
21
211.84 Testing and approval or rejection of components, drug
product containers, and closures.
 (d) Samples shall be examined and tested as follows:
 (1) At least one test shall be conducted to verify the identity of each
component of a drug product. Specific identity tests, if they exist,
shall be used.
 (2) Each component shall be tested for conformity with all
appropriate written specifications for purity, strength, and quality.
In lieu of such testing by the manufacturer, a report of analysis may
be accepted from the supplier of a component, provided that at least
one specific identity test is conducted on such component by the
manufacturer, and provided that the manufacturer establishes the
reliability of the supplier's analyses through appropriate validation
of the supplier's test results at appropriate intervals.
22
Group Frequencies
 Characteristic of functional groups such as -O-H,
-CH3, -COCH3, and COOH.
 Determined empirically by studying the spectra of
many related molecules.
 Always found in the spectrum of a molecule
containing that group, and always occurs in the
same narrow frequency range.
 The form of the bands is nearly always the same
in every molecule containing that group.
23
Fingerprint Frequencies
 Highly Characteristic of the Specific Molecule.
 Due to Vibrations of the Molecule as a whole.
 The numerical values cannot be predicted in most
cases.
 Valuable in characterizing a molecule.
 Fingerprint region is useful for discriminating
between molecules that resemble each other.
24
Dividing Line
 1500 cm-1 dividing line: above 1500 cm-1 if a
band has a reasonable intensity, it is a group
frequency.
 Below 1500 cm-1 the band may be either a group
or fingerprint frequency.
 Below 1500 cm-1 called the fingerprint region.
25
IR Spectroscopy
 Excellent in identification.
 Every compound has a unique IR spectrum.
 Better than a fingerprint, provides structural
information.
 Helps in completing the “structure puzzle” when
coupled with NMR, and MS
(Detection of Compounds vs. Obtaining Structural
Information).
26
Benzoic Acid Spectrum
27
FINGERPRINT FREQUENCIES
HIGHLY USEFUL TO DISCRIMINATE BETWEEN
MOLECULES THAT RESEMBLE EACH OTHER.
CH3
CH3
28
Identity Test in Pharmaceutical
Manufacturing
• In an identity test at a pharmaceutical
manufacturing site, an analyst would compare the
spectrum of the material to be used in production
with that of a reference standard.
29
Typical Identification in Pharmaceutical
Industry with Mid-Infrared Spectroscopy
30
Checking Fingerprint Region.
31
32
Propylene Glycol – Ref. Std.
33
Mid-IR Spectrum Cellulose Acetate
34