Vibrational Spectroscopy 3

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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



Far-infrared spectroscopy

(Terahertz pulsed spectroscopy, TPS)



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



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



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



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.



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



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



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



Different solid-state forms

crystalline form A crystalline form B

solvate

amorphous phase



Applications of TPS


▫

Solid form control

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

Sulfathiazole

Lactose



Applications of TPS


▫

Solid form control, characterization and identification of drugs

Carbamazepine IndomethacinZeitler et al., Journal of Pharmacy and Pharmacology, 59, 2007.



Applications of TPS


▫

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



Applications of TPS


▫

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



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.



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




• 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

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

Light microscopy imagesTypical terahertz waveform from a single pixel of a tablet



Applications of terahertz pulsed imaging• Pharmaceutical applications


Ho et al., Journal of Controlled Release, 119, 2007.2D images µm




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

• Coating layer thickness

• Coating reproducibility

• Coating distribution and

uniformity

3D images



• Advantage:non-destructive



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



Raman spectroscopy



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



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



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



Instrumentation - dispersive Raman spectrometer

ControllerLaser785 nm

Quick

starttest kit

Detector

Probe

90 µm optical fibre


Sample10 mm

ControllerLaser785 nm

Quick

starttest kit

Detector

Probe



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)



Instrumentation - FT Raman spectrometer

Bruker MultiRAM

• Nd:YAG laser: 1064 nm

• InGaAs detector andnitrogen-cooled Ge detector



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



Band assignment

Raman shift / cm-1



Sampling techniques

Solids

Liquids

Gases

Gas cells

Sealed in a glasscapillary

Glass or metalsample holders

• Rotating sample set-upto minimize localsample heating



Raman spectrum of air

Raman shift / cm-1

I n t e n s i t y



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



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



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



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




Raman: 1335 cm–1

IR: 2349 cm–1

IR: 667 cm–1

CO2

Raman + IR: 3657 cm–1



H2O



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

Raman shift / cm-1 Raman shift / cm-1

Rare-earth elements, zircon





• Material science

▫ Corrosion and oxidation studies with copper

and steel


Raman shift / cm-1

I n t e n s i t y

• Smooth area: cuprous oxide (Cu2O)

• Rough area: cupric oxide (CuO)




• Art▫ Identify pigments

▫ Investigate authenticity

I n t e n s i t y





• 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



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



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



Pharmaceutical applications Manufacturing of solid-dosage forms

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



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



Recrystallization of amorphous fenofibrate



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



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



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

Vibrational Spectroscopy 3

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