vibrational spectroscopy 3
TRANSCRIPT
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Overview
• Vibrational spectroscopy
• Principles of vibrational spectroscopy• Mid-infrared spectroscopy (IR spectroscopy)
• Near-infrared spectroscopy (NIR spectroscopy)
• Far-infrared spectroscopy (Terahertz pulsedspectroscopy)
• Raman spectroscopy
• Pharmaceutical applications
• Methods of data analysis
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Far-infrared spectroscopy
(Terahertz pulsed spectroscopy, TPS)
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v i s i b
l eradio waves X-raysUVmicrowaves infrared
0.01 0.1 1 10 100
1 10 100 1000
frequency / THz
Wavenumber / cm -1
0.01 0.1 1 10 100
1 10 100 1000
frequency / THz
Wavenumber / cm -1
Low frequency
bond vibrations
Hydrogen-bonding stretchesand torsions (liquids)
Crystalline phonon
Vibrations (solid)
Molecular rotations (gas)
N
I R
Terahertz pulsed spectroscopy
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Principles of terahertz pulsed spectroscopy
• Based on the absorption of radiation in the far-infrared region of
the electromagnetic spectrum between 1.67 cm-1 to 133 cm-1 ( 0.05
THz to 4 THz)
• Absorption occurs when there is a change in the dipole moment
• In contrast to IR and NIR spectroscopy where intramolecular
vibrations are probed, terahertz radiation causes low-energytransitions of molecules such as hydrogen bonding stretches,
torsion vibrations, translations and liberations of molecules in the
crystal lattice (phonon vibrations)
Study of intermolecular vibrations and
hydrogen bonding networks
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Instrumentation
Bruker TPI spectra 1000Zeitler et al., Journal of Pharmacy and Pharmacology, 59, 2007.
• Using femtosecond lasers and specially designed photo-
conductive semiconductor antenna switches terahertz
radiation can be generated and detected at room temperature
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Sample preparation
• As in IR spectroscopy, sample has to be mixed with a diluent and thencompressed to a pellet
• Diluent materials: Polyethylene and poly(tetrafluorethylene) (PTFE) aretransparent to terahertz radiation
• Compressed into a pellet with a thickness of approximately 0.5 mm to 3mm and diameter between 5 mm and 30 mm
• Particle size of both sampleand diluent is preferably below100 µm to minimize scattering
•
Alternatively, if the samplematerial compacts well,sample pellets can be preparedby direct compression withoutany diluent
Kogermann et al., Applied Spectroscopy, 61(12), 2007.
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Sampling techniques
▫ Sample powders or liquids in good opticalcontact with silicon crystal
▫ Spectrum can be acquired within seconds
▫
No sample preparation required
Attenuated total
reflection
Transmission
Specular reflectance
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Band assignment
• Terahertz spectra are still not understood very well and bands in mostcases cannot be assigned to intermolecular vibrations
• Research groups working on terahertz spectroscopy have thus tried tomodel spectra for specific drug molecules using computational methods(quantum mechanical calculations)
• Based on an energy minimized
rigid molecule crystal lattice
• Calculations allow predictingvibrational modes and describemolecular vibrations
Predicted terahertz spectrum for a carbamazepin monomer (top)and terahertz spectrum for carbamazepine form III (bottom)Hydrogen-bonded carbamazepine dimer
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Applications of TPS
• Life sciences
▫ Structure analysis: DNA,
RNA, bovine serum albumine,oligopeptides, amino acids
Wavenumber / cm-1
Markelz et al., Chemical Physical Letters, 320, 2000.Korter et al., Chemical Physical Letters, 418, 2006.
Wavenumber / cm-1
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Different solid-state forms
crystalline form A crystalline form B
solvate
amorphous phase
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Applications of TPS
• Pharmaceutical applications
▫
Solid form control
Zeitler et al., Journal of Pharmacy and Pharmacology, 59, 2007.
Sulfathiazole
Lactose
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Applications of TPS
• Pharmaceutical applications
▫
Solid form control, characterization and identification of drugs
Carbamazepine IndomethacinZeitler et al., Journal of Pharmacy and Pharmacology, 59, 2007.
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Applications of TPS
• Pharmaceutical applications
▫
Analysis of solid-state transformations▫ Quantification of solid-state forms
Isothermal conversion of carbamazepine
form III to form IZeitler et al., Journal of Pharmacy and Pharmacology, 59, 2007.
Physical mixtures of carbamazepine form III and form I
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Applications of TPS
• Pharmaceutical applications
▫
Analysis of solid-statetransformations
Temperature-dependent conversion of
carbamazepine form III to form I
Zeitler et al., Thermochimica acta, 436, 2005.Dehydration of theophyllinemonohydrate to theophylline anhydrate
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Terahertz pulsed imaging• Reveals spatially resolved information from below the surface of the sample
• Excipients most commonly used for formulation of solid dosage forms are transparent
or semi-transparent to terahertz radiation; hence pulse of terahertz light can
penetrate into the sample matrix
• Penetration depths into typical pharmaceutical formulations are between 1 mm and 3mm depending on the material or the pulse of terahertz radiation
• All information obtained in single
scan of the sample surface• Reflections of the terahertz pulse
from interfaces due to changes
in refractive indices within thesample matrix enable the recon-
struction of the internal sample structure
• The time delay of these reflections
relative to the surface reflection isused to calculate 3D structural
images of the sample
Zeitler et al., Journal of Pharmacy and Pharmacology, 59, 2007.
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Applications of terahertz pulsed imaging
• Safety and security
Concealed weapon, 1.56 THz image
Detection of bombs, biological
weapons, chemical weapons.
knifes, razor blades through
envelopes, clothes, suitcases,
soil, briefcases
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Applications of terahertz pulsed imaging
• Medical applications
▫ Terahertz radiation is non-ionizing and non-destructive
▫ Biomedical cancer screening
▫ Screening of teeth to identify regions of decay
Basal cell cancer
Martin, M. C., Filling the terahertz gap, Lawrence Berkley National Laboratory, CA, USA
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Applications of terahertz pulsed imaging• Pharmaceutical applications
▫ Determining the coating thickness of sustained release tablets
Ho et al., Journal of Controlled Release, 119, 2007.
Light microscopy imagesTypical terahertz waveform from a single pixel of a tablet
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Applications of terahertz pulsed imaging• Pharmaceutical applications
▫ Determining the coating thickness of sustained release tablets
Ho et al., Journal of Controlled Release, 119, 2007.2D images µm
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Applications of terahertz pulsed imaging
Ho et al., Journal of Controlled Release, 119, 2007.
• Coating layer thickness
• Coating reproducibility
• Coating distribution and
uniformity
3D images
• Pharmaceutical applications
▫ Determining the coating thickness of sustained release tablets
• Advantage:non-destructive
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Advantages and disadvantages of TPS
Advantages:• Direct information about crystal structure since intermolecular vibrations
(lattice phonon modes) are probed▫ In contrast to IR and NIR where only indirect information about the
crystalline structure is obtained since spectra represent intramolecularvibrations and contain a lot of chemical information
• Rapid measurements• No sample preparation (in the case of ATR measurements)
Disadvantages:• Water affects terahertz spectra
• Diffuse reflectance set-up currently not available
• Spectra difficult to interpret because vibrational modes are not yet fullyunderstood
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Raman spectroscopy
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Raman spectroscopy
ν=3
ν=2
ν=1
Jablonski energy level diagram
ν=0
S1
ν=3
ν=2
ν=1
ν=0
S0
virtual state
A B C D E F
A: IR absorption
B: NIR absorption
C: NIR absorption
D: elastic Rayleigh scattering (ν = ν0);
over 99 % of the scattered radiation
E: inelastic Stokes Raman scattering (ν
= ν0-νi)
F: inelastic Anti-Stokes Raman
scattering (ν = ν0+νi)
Incident radiation of a specific frequency (monochromatic radiation)excites a transition of the molecule to virtual high energy states from
which it returns by either elastic Rayleigh scattering or inelastic Raman
scattering.
• Raman scattering discovered in 1928 by Indian physicist C. V. Raman
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Raman spectroscopy• Raman scattering requires a change in polarizability
in the structures under investigation
•
When a beam of radiation irradiated on a sample,the electric field experienced by each molecule changes
• Electron cloud of a diatomic molecule experiences distortion anddepending on its polarizability (deformability of the electron cloud) a
dipole moment is induced• The dipole undergoes oscillations of the frequency ν
• If the molecule shows some internal motion, such as vibration androtation, polarizability changes periodically
•
Incident radiation is most effectively scattered by molecules that containlargely delocalized electron systems, for instance non-polar andaromatic groups (in contrast to IR where mainly polar groups absorb)
• H2, O2, N2 in contrast to IR spectroscopy are Raman active
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The Raman spectrum• Both, Stokes scattering and anti-Stokes scattering result in lines in the Raman
spectrum of which the Stokes lines are stronger than the anti-Stokes lines since
typically more molecules exist in the ground state than in the excited state
• The position of the Stokes lines is known as Raman shift and corresponds to the
energy and wavenumber difference between ν = 0 and ν = 1
• Number of bands in a Raman spectrum: as for an IR spectrum, the number of
bands in the Raman spectrum for an N-atom non-linear molecule is seldom 3N-6,
because:
I n t e n s i t y
Raman spectrum of cysteine
• Polarizability change is zero
or small for some vibrations
• Bands overlap
• Combination or overtonebands are present
• Some vibrations are highly
degenerate
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Instrumentation - dispersive Raman spectrometer
ControllerLaser785 nm
Quick
starttest kit
Detector
Probe
90 µm optical fibre
200 µm optical fibre
Sample10 mm
ControllerLaser785 nm
Quick
starttest kit
Detector
Probe
90 µm optical fibre
200 µm optical fibre
Sample10 mm
• Operated with silicon-based charged
coupled device (CCD) multichanneldetectors
• Laser sources in the ultraviolet, visible, or
NIR range (Raman spectrometer on thephoto: 785 nm)
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Instrumentation - FT Raman spectrometer
Bruker MultiRAM
• Nd:YAG laser: 1064 nm
• InGaAs detector andnitrogen-cooled Ge detector
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Raman microscopy and imaging• Raman spectroscopy is a surface technique, however, can be adapted to record
spectra from beneath a surface
• Raman microscope consists of a standard optical microscope, an excitation laser, a
monochromator, a sensitive detector (such as a charge-coupled device orphotomultiplier tube)
• FT-Raman can also be used with microscopes
• Raman mapping (spectral data are collected from each point of the interrogated region sequentially) and Raman imaging
(spectral data are collected from each point simultaneously) is possible
Confocal Raman microscope, ThermoAssociation of lipid bodies in neutrophils
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Band assignment
Raman shift / cm-1
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Sampling techniques
Solids
Liquids
Gases
Gas cells
Sealed in a glasscapillary
Glass or metalsample holders
• Rotating sample set-upto minimize localsample heating
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Raman spectrum of air
Raman shift / cm-1
I n t e n s i t y
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Fluorescence
Electronicground state
1st Electronicexcited state
E x c i t a t i o n E n e r g y , σ / c m – 1
4,000
25,000
0
F l u o r e s c e n c e
IRσ
σ σemit
2nd Electronicexcited state
Raman
∆σ = σemit-σ
σ ∆σ
F l u o r e s c e n c e
I m p
u r i t y
Fluorescence= Trouble
Stokes Anti-Stokes
Fluorescence:Some atoms andmolecules absorblight at a particular
wavelength andsubsequentlyemit light of longerwavelengthafter a brief interval,termed
the fluorescencelifetime
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Raman spectra - fluorescence
Raman shift / cm-1
I n t e n
s i t y
• Occurs in particular when short wavelength
sources are used (785 nm laser and below)
• Can be overcome or at least be minimized
by photochemical bleaching (irradiating the
sample with laser for minutes or hours)
• However, local sample heating needs to be
taken into consideration
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Fluorescence – photochemical bleaching
1000 2000 3000
Raman shift / cm-1
R a m a
n i n t e n s i t y Without bleaching
After 2 hours bleaching
Poly (diallyl phthalate)
lex = 514.5 nm
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Raman spectroscopy vs. IR spectroscopy
• Raman spectroscopy
▫ Scattering phenomenon▫ Intensity related to
polarizability change
during vibrations ”howeasily electrons flow”
▫ Non-polar bonds are
Raman active
• Infrared spectroscopy
▫ Absorbancephenomenon
▫ Intensity related to
dipole moment changeduring vibration
▫ Polar bonds are infraredactive
Techniques are complementary
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Raman spectroscopy vs. IR spectroscopy
Raman: 1335 cm–1
IR: 2349 cm–1
IR: 667 cm–1
CO2
Raman + IR: 3657 cm–1
Raman + IR: 3756 cm–1
Raman + IR: 1594 cm–1
H2O
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Raman spectroscopy vs. IR spectroscopy
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Applications of Raman spectroscopy• Forensic science
▫ Paint, drugs, fibers
Heroin Morphine
Renishaw, Information note from the Spectroscopy Products Division
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Applications of Raman spectroscopy
• Geology and mineralogy▫ Investigation of heavy mineral
sand
Raman shift / cm-1 Raman shift / cm-1
Rare-earth elements, zircon
Renishaw, Information note from the Spectroscopy Products Division
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Applications of Raman spectroscopy
• Material science
▫ Corrosion and oxidation studies with copper
and steel
Renishaw, Information note from the Spectroscopy Products Division
Raman shift / cm-1
I n t e n s i t y
• Smooth area: cuprous oxide (Cu2O)
• Rough area: cupric oxide (CuO)
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Applications of Raman spectroscopy
• Art▫ Identify pigments
▫ Investigate authenticity
I n t e n s i t y
Renishaw, Information note from the Spectroscopy Products Division
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Applications of Raman spectroscopy
• Biomedical applications▫
Particularly in the diagnosis of arteriosclerosis and cancer▫ In the study of arteriosclerosis: quantification of cholesterol and
cholesteryl esters in the coronary artery
• Pharmaceutical applications▫ Identification and characterization of active pharmaceutical ingredients
▫ Monitor formulation of solid-dosage forms, emulsions, and gels
▫ Study interactions between excipients and drug
▫
Monitor solid-state transformations including hydrate formation anddehydration
▫ Quantification of solid forms in powder mixtures
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Pharmaceutical applications
500 1000 1500 2000 2500 3000
Raman shift / cm-1
Form I
I n t e n s i t y
/ a . u .
Form III
N
O NH2
Carbamazepine
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Pharmaceutical applications N
O NH2
Carbamazepine
800 1000 1200 1400 1600
1 6
0 1
1 0 2 5
Raman shift / cm-1
1 0 4 0
Form I
I n t e n s i t y /
a . u .
Form III
2900 3000 3100
3062
3025
Raman shift / cm-1
Form I
I n t e n s i t y /
a . u .
Form III3071
30213044
Aromatic ring vibrations C-H vibrations
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Pharmaceutical applications Manufacturing of solid-dosage forms
Rantanen et al., Journal of Pharmacy and Pharmacology, 59, 2007.
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Data analysis
multivariate analysisunivariate analysis
Identification of single
characteristic peaks
Quantification based on
peak area and height
Principal component analysis (PCA)
- Qualitative purposes
Partial least squares (PLS) regression
- Quantitative purposes
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Recrystallization of amorphous fenofibrate
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Recrystallization of amorphous fenofibrate
amorphous-180 °C to 25 °C
metastable crystallineform II
30 °C to 55 °C
stable crystalline form I60 °C to 75 °C
melt> 80 °C
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Advantages and disadvantages of Raman spectroscopy
• Advantages:
▫ Little or no sample preparation
▫ Rapid measurements▫ Non-destructive
▫ Use of fiber-optic probes
▫ Relatively insensitive to particle size
▫ Not affected by environmental water▫ Glass or quartz cells can be used (avoids the inconvenience of working
with NaCl or other atmospherically unstable materials)
• Disadvantages:▫ Sample fluorescence
▫ Local sample heating may damage or even destroy sample
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Mid-infrared Near-infrared
IR, NIR, terahertz, and Raman spectroscopiesFundamentals4000 cm-1 to 400 cm-1
Sample preparationrequired (except ATR)
Overtones, combinations12,500 cm-1 to 4000 cm-1
No sample preparationrequired
High structural selectivity
Low structural selectivityFiber-optic probes availableFiber-optic probes unavailable
Polar functionalities CH/OH/NH functionalities
Large sample thickness (up to cm)Small sample thickness (µm)
Sample preparationrequired (except ATR)
Low structural selectivity
Fiber-optic probes unavailable
Intermolecular vibrations
Large sample thickness (up to cm)
No sample preparationrequired
High structural selectivity
Fiber-optic probes available
Homonuclear functionalities
Large sample thickness
Raman Terahertz
Fundamentals
4000 cm-1 to 400 cm-1
Absorption technique
Scattering technique Absorption technique
Absorption technique
Intermolecular vibrations130 cm-1 to 2 cm-1