Overview

Vibrational spectroscopy
Principles of vibrational spectroscopy Mid-infrared spectroscopy (IR spectroscopy) Near-infrared spectroscopy (NIR spectroscopy) Far-infrared spectroscopy (Terahertz pulsed spectroscopy) Raman spectroscopy

Pharmaceutical applications Methods of data analysis

Far-infrared spectroscopy (Terahertz pulsed spectroscopy, TPS)

Terahertz pulsed spectroscopy

NIR

Molecular rotations (gas) Low frequency bond vibrations Crystalline phonon Vibrations (solid) Hydrogen-bonding stretches and torsions (liquids)

0.01

0.1

1 frequency / THz 10 100

visible

radio waves

microwaves

infrared

UV

X-rays

10

100

1000

Wavenumber / cm -1

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-energy transitions 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

Instrumentation

Using femtosecond lasers and specially designed photoconductive semiconductor antenna switches terahertz radiation can be generated and detected at room temperature

Bruker TPI spectra 1000

Zeitler et al., Journal of Pharmacy and Pharmacology, 59, 2007.

Sample preparation
As in IR spectroscopy, sample has to be mixed with a diluent and then compressed to a pellet Diluent materials: Polyethylene and poly(tetrafluorethylene) (PTFE) are transparent to terahertz radiation Compressed into a pellet with a thickness of approximately 0.5 mm to 3 mm and diameter between 5 mm and 30 mm Particle size of both sample and diluent is preferably below 100 m to minimize scattering Alternatively, if the sample material compacts well, sample pellets can be prepared by direct compression without any diluent
Kogermann et al., Applied Spectroscopy, 61(12), 2007.

Sampling techniques

Transmission Specular reflectance Attenuated total reflection

Sample powders or liquids in good optical contact with silicon crystal Spectrum can be acquired within seconds No sample preparation required

Band assignment
Terahertz spectra are still not understood very well and bands in most cases cannot be assigned to intermolecular vibrations Research groups working on terahertz spectroscopy have thus tried to model spectra for specific drug molecules using computational methods (quantum mechanical calculations) Based on an energy minimized rigid molecule crystal lattice Calculations allow predicting vibrational modes and describe molecular vibrations

Hydrogen-bonded carbamazepine dimer

Predicted terahertz spectrum for a carbamazepin monomer (top) and terahertz spectrum for carbamazepine form III (bottom)

Applications of TPS
Life sciences
Structure analysis: DNA, RNA, bovine serum albumine, oligopeptides, amino acids

Wavenumber / cm-1

Wavenumber / cm-1 Korter et al., Chemical Physical Letters, 418, 2006. Markelz et al., Chemical Physical Letters, 320, 2000.

Different solid-state forms

crystalline form A

crystalline form B

solvate

amorphous phase

Applications of TPS
Pharmaceutical applications
Solid form control

Sulfathiazole

Lactose
Zeitler et al., Journal of Pharmacy and Pharmacology, 59, 2007.

Applications of TPS
Pharmaceutical applications
Solid form control, characterization and identification of drugs

Carbamazepine

Indomethacin
Zeitler et al., Journal of Pharmacy and Pharmacology, 59, 2007.

Applications of TPS
Pharmaceutical applications
Analysis of solid-state transformations Quantification of solid-state forms
Physical mixtures of carbamazepine form III and form I

Isothermal conversion of carbamazepine form III to form I

Zeitler et al., Journal of Pharmacy and Pharmacology, 59, 2007.

Applications of TPS
Pharmaceutical applications
Analysis of solid-state transformations

Dehydration of theophylline monohydrate to theophylline anhydrate

Temperature-dependent conversion of carbamazepine form III to form I

Zeitler et al., Thermochimica acta, 436, 2005.

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 3 mm 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 the sample matrix enable the reconstruction of the internal sample structure The time delay of these reflections relative to the surface reflection is used to calculate 3D structural images of the sample
Zeitler et al., Journal of Pharmacy and Pharmacology, 59, 2007.

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

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

Applications of terahertz pulsed imaging

Pharmaceutical applications
Determining the coating thickness of sustained release tablets

Typical terahertz waveform from a single pixel of a tablet

Light microscopy images Ho et al., Journal of Controlled Release, 119, 2007.

Applications of terahertz pulsed imaging

Pharmaceutical applications
Determining the coating thickness of sustained release tablets

2D images

Ho et al., Journal of Controlled Release, 119, 2007.

Applications of terahertz pulsed imaging

Pharmaceutical applications
Determining the coating thickness of sustained release tablets
Coating layer thickness Coating reproducibility Coating distribution and uniformity Advantage: non-destructive

3D images Ho et al., Journal of Controlled Release, 119, 2007.

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 intramolecular vibrations 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 fully understood

Raman spectroscopy

Raman spectroscopy
Raman scattering discovered in 1928 by Indian physicist C. V. Raman

Jablonski energy level diagram

A: IR absorption B: NIR absorption C: NIR absorption D: elastic Rayleigh scattering ( = 0); over 99 % of the scattered radiation E: inelastic Stokes Raman scattering ( = 0-i) F: inelastic Anti-Stokes Raman scattering ( = 0+i)

=3 =2 =1 =0

S1

virtual state
=3 =2 =1 =0
A B C D E F

S0

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 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 and depending on its polarizability (deformability of the electron cloud) a dipole moment is induced The dipole undergoes oscillations of the frequency If the molecule shows some internal motion, such as vibration and rotation, polarizability changes periodically Incident radiation is most effectively scattered by molecules that contain largely delocalized electron systems, for instance non-polar and aromatic groups (in contrast to IR where mainly polar groups absorb) H2, O2, N2 in contrast to IR spectroscopy are Raman active

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 = 0 and = 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: Raman spectrum of
Polarizability change is zero or small for some vibrations Combination or overtone bands are present Some vibrations are highly degenerate
Intensity

cysteine

Bands overlap

Instrumentation - dispersive Raman spectrometer

Laser
785 nm

Detector

Controller

200 m optical fibre 90 m optical fibre Quick start test kit

Probe

Operated with silicon-based charged coupled device (CCD) multichannel detectors Laser sources in the ultraviolet, visible, or NIR range (Raman spectrometer on the photo: 785 nm)

10 mm Sample

Instrumentation - FT Raman spectrometer

Nd:YAG laser: 1064 nm InGaAs detector and nitrogen-cooled Ge detector Bruker MultiRAM

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 or photomultiplier 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, Thermo Association of lipid bodies in neutrophils

Band assignment

Raman shift / cm-1

Sampling techniques

Solids
Glass or metal sample holders

Liquids
Sealed in a glass capillary

Gases
Rotating sample set-up to minimize local sample heating Gas cells

Raman spectrum of air

Intensity

Raman shift / cm-1

Fluorescence
2nd Electronic excited state Impurity 25,000

Excitation Energy, / cm1

1st Electronic excited state

Stokes

Anti-Stokes Fluorescence Electronic ground state Fluorescence = Trouble

Fluorescence: Some atoms and molecules absorb light at a particular wavelength and subsequently emit light of longer wavelength after a brief interval, termed the fluorescence lifetime

emit Fluorescence

4,000 0 IR Raman = emit-

Raman spectra - fluorescence

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

Intensity

Raman shift / cm-1

Fluorescence photochemical bleaching

Poly (diallyl phthalate) lex = 514.5 nm
Raman intensity Without bleaching

After 2 hours bleaching

1000

2000

3000

Raman shift / cm-1

Raman spectroscopy vs. IR spectroscopy Infrared spectroscopy

Absorbance phenomenon Intensity related to dipole moment change during vibration Polar bonds are infrared active

Raman spectroscopy
Scattering phenomenon Intensity related to polarizability change during vibrations how easily electrons flow Non-polar bonds are Raman active

Techniques are complementary

Raman spectroscopy vs. IR spectroscopy

CO2
Raman: 1335 cm1 IR: 2349 cm1 IR: 667 cm1

H2O
Raman + IR: 3657 cm1 Raman + IR: 3756 cm1 Raman + IR: 1594 cm1

Raman spectroscopy vs. IR spectroscopy

Applications of Raman spectroscopy

Forensic science
Paint, drugs, fibers

Heroin

Morphine

Renishaw, Information note from the Spectroscopy Products Division

Applications of Raman spectroscopy

Geology and mineralogy
Investigation of heavy mineral sand

Rare-earth elements, zircon

Raman shift / cm-1

Raman shift / cm-1

Renishaw, Information note from the Spectroscopy Products Division

Applications of Raman spectroscopy

Material science
Corrosion and oxidation studies with copper and steel

Intensity

Raman shift / cm-1

Smooth area: cuprous oxide (Cu2O) Rough area: cupric oxide (CuO)
Renishaw, Information note from the Spectroscopy Products Division

Applications of Raman spectroscopy

Art
Identify pigments Investigate authenticity

Intensity

Renishaw, Information note from the Spectroscopy Products Division

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 and dehydration Quantification of solid forms in powder mixtures

Pharmaceutical applications

Carbamazepine
O

N NH2

Form III Intensity / a.u. Form I

500

1000

1500

2000
-1

2500

3000

Raman shift / cm

Pharmaceutical applications
Aromatic ring vibrations
1601
Form III

Carbamazepine
O

N NH2

C-H vibrations
3021 3044 3071

Form III

1025 1040

Intensity / a.u.

Intensity / a.u.

Form I 3025

3062

Form I

800

1000

1200

1400
-1

1600

2900

3000

3100
-1

Raman shift / cm

Raman shift / cm

Pharmaceutical applications
Manufacturing of solid-dosage forms

Rantanen et al., Journal of Pharmacy and Pharmacology, 59, 2007.

Data analysis

univariate analysis

multivariate 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

Recrystallization of amorphous fenofibrate

Recrystallization of amorphous fenofibrate

metastable crystalline form II 30 C to 55 C

amorphous -180 C to 25 C

melt > 80 C

stable crystalline form I 60 C to 75 C

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

IR, NIR, terahertz, and Raman spectroscopies

Mid-infrared
Fundamentals 4000 cm-1 to 400 cm-1 Overtones, combinations 12,500 cm-1 to 4000 cm-1

Near-infrared

Absorption technique Small sample thickness (m) Polar functionalities Sample preparation required (except ATR) High structural selectivity Fiber-optic probes unavailable Scattering technique Large sample thickness Homonuclear functionalities No sample preparation required

Absorption technique Large sample thickness (up to cm) CH/OH/NH functionalities No sample preparation required Low structural selectivity Fiber-optic probes available Absorption technique Large sample thickness (up to cm) Intermolecular vibrations Sample preparation required (except ATR) Low structural selectivity Fiber-optic probes unavailable

Raman
Fundamentals 4000 cm-1 to 400 cm-1

High structural selectivity Fiber-optic probes available

Terahertz
Intermolecular vibrations 130 cm-1 to 2 cm-1