Vibrational Spectroscopy for Pharmaceutical Analysis

10/11/2005

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ENGINEERING RESEARCH CENTER FOR

STRUCTURED ORGANIC PARTICULATE SYSTEMS

RUTGERS UNIVERSITYPURDUE UNIVERSITYNEW JERSEY INSTITUTE OF TECHNOLOGYUNIVERSITY OF PUERTO RICO AT MAYAGÜEZ

Vibrational Spectroscopy for Vibrational Spectroscopy for Pharmaceutical Analysis Pharmaceutical Analysis

Part VII. Introduction to Raman Spectroscopy

Rodolfo J. Romañach, Ph.D.

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ScatteringScattering

• Mid-IR and NIR require absorption of radiation from a ground level to an excited state, requires matching of radiation from source with difference in energy states.

• Raman spectroscopy involves scattering of radiation (matching of radiation is not required).

E. Smith and G. Dent, “Modern Raman Spectroscopy. A Practical Approach.”, Wiley 2005, pages 3 – 5.

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Raman SpectroscopyRaman Spectroscopy

• A single frequency of radiation irradiates the molecule and the radiation distorts (polarizes) the cloud of electrons surrounding the nuclei to form a short-lived state called a “virtual state”. This state is not stable and the photon is quickly re-radiated.

E. Smith and G. Dent, “Modern Raman Spectroscopy. A Practical Approach.”, Wiley 2005, pages 3 – 5.

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What is Raman Spectroscopy?What is Raman Spectroscopy?

Rayleigh scattering:Elastic scatter

200

400

600

800

1000

1200

1400

Ram

an In

tens

ity-400 -200 0 200 400

Raman Shift (cm-1)

Raman : Stokes Anti-StokesInelastic scatter

LASERLASER

Raman is a scattering technique

Slide courtesy Kaiser Optical Systems.

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Raman ScatteringRaman Scattering from Molecular Vibrations from Molecular Vibrations

2c1

= (k)

½ = Vibrational frequencyk = Spring force constant = Reduced mass of atoms, m1m2/(m1+m2)

Higher vibrational frequency with stronger chemical bond and lighter atoms.

Rayleigh – Elastic

Strongest Component

Anti-Stokes –

Photon Gains Energy

Stokes – Photon has less energy

Adapted from Kaiser Optical Systems slide

Only one in 106 or 108 photons is Raman scattered.

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Quantum Mechanical Modelof Raman Scattering

Stokes Rayleigh Anti-Stokes

E0

v=3v=2v=1v=0

Virtual state

hvex hvexhvex hvex

h(vex-vv) h(vex+vv)

E1

Courtesy Kaiser Optical Systems.

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Raman ScatteringRaman Scattering

• The difference in wavelength between the incident and scattered visible radiation corresponds to wavelengths in the mid-infrared region.

• An Indian physicist C.V. Raman discovered this effect in 1928.

• This has been considered an experimentally difficult technique for many years; but in recent years a number of advances in instrumentation has made it more available to non-specialized labs.

Courtesy Kaiser Optical Systems.

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• Sample is irradiated with intense monochromatic radiation usually in the visible or NIR region of the spectrum.

• The wavelength is well away from any absorption peaks of the analyte.

• The abscissa in the spectra are in terms of wavenumber shift Δυ between the observe radiation and that of the source, and we speak of Raman shift instead of frequency of absorption.

Skoog Holler Niemann, p. 429-433, 435 – 441.

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Raman Scattering and PolarizabilityRaman Scattering and Polarizability

Electric field of radiation: E = E0cos (2πυext) When it interacts with an electron cloud of an analyte bond, it induces a dipole moment m in the bond that is given by: m = αE = αE0cos (2πυext) Where α is a proportionality constant called the polarizability of the bond.

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Stokes ScatteringStokes Scattering

• Stokes scattering is, by convention, positive-shifted Raman scatter. Most Analytical work is done in this region.

• Represents inelastic scattering to a region of lower energy. This means that the energy of the detected radiation is higher in wavelength relative to the laser.

• The scattered spectrum appears similar to an IR spectrum and is interpreted similar IR spectrum.


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• C=C, and C≡C, C≡N bonds are strong scatterers, bonds undergo polarization.

• Symmetric stretches undergo greater changes in polarization, and are stronger in Raman than asymmetric stretches.

E. Smith and G. Dent, Wiley 2005, page 6.

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Advantages of Raman Spectroscopy – Advantages of Raman Spectroscopy – Chemical InformationChemical Information

• Raman bands can provide structural information (presence of functional groups).

• Raman spectroscopy can be used to measure bands of symmetric linkages which are weak in an infrared spectrum (e.g. -S-S-, -C-S-, -C=C-).

• The standard spectral range reaches well below 400 cm-

1, making the technique ideal for both organic and inorganic species.

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Advantages of Raman Spectroscopy – Ease of Use for Advantages of Raman Spectroscopy – Ease of Use for

Process MeasurementsProcess Measurements

• Fiber optics (up to 100's of meters in length) can be used for remote analyses.

• Purging of sample chamber is unnecessary since Water and CO2 vapors

are very weak scatterers. • Little or no sample preparation is required • Water is a weak scatterer - no special accessories are needed for

measuring aqueous solutions • Inexpensive glass sample holders, non-invasive probes and immersion

probes are ideal in most cases

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Disadvantages of Raman SpectroscopyDisadvantages of Raman Spectroscopy

• Inherently not sensitive (need ~ 1 million incident

photons to generate 1 Raman scattered photon)• Fluorescence is a common background issue• Typical detection limits in the parts per thousand

range• Fluorescence Probability versus Probability of

Raman Scatter ( 1 in 103-105 vs 1 in 107-1010)• Requires expensive lasers, detectors and filters.• Small sample volume can make it difficult to obtain

a representative sample.

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Complementary Nature of IR and Raman SpectroscopyComplementary Nature of IR and Raman Spectroscopy

• IR absorption intensities are proportional to the change in dipole moment as the molecule vibrates.

• Raman scattering intensities are proportional to the change in molecular polarizabilities upon vibrational excitation.

• For molecules with a center of inversion IR and Raman and mutually exclusive.

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• Need to emphasize complementarity with more specific examples.

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Placzek's EquationPlaczek's Equationfor Raman Scattering Intensityfor Raman Scattering Intensity

• IR proportional to IL• IR proportional to N• IR stronger at shorter

wavelength• Statistical factor:

(1-e-h/kT)

(45)(32)c4

243

(1-e-h/kT)hILN(0-)4

[45(a')2+7(a')

2]=IR

Where

c = speed of lighth = Planck's constantIL = laser intensityN = number of scattering molecules = molecular vibrational frequency in HzL = laser excitation frequency, in Hz = reduced mass of the vibrating atomsk = Boltzmann's constantT = absolute temperaturea' = mean value invariant of the polarizability tensora' = anisotropy invariant of the polarizability tensor

courtesy Kaiser Optical Systems

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ILCI

IRaman intensity

= Raman cross section

L = Pathlength

C = Concentration

I = Instrument parameters

Analytical Raman Spectroscopy

Sample

courtesy Kaiser Optical Systems

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Raman Scattering is Stronger from Some Vibrations than from Raman Scattering is Stronger from Some Vibrations than from OthersOthers

• 3N-6 vibrations possible, many have no Raman bands• Change in polarizability during a molecular vibration leads

to Raman scattering.– Covalent bonds more polarizable than ionic bonds– Intensity from stretching vibration increases with bond

order– Intensity tends to increase with increasing atomic

number– Symmetry-forbidden vibrations


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Raman Scattering is Stronger from Some Vibrations than from Raman Scattering is Stronger from Some Vibrations than from OthersOthers

• Stretching bands often stronger than bending ones

• Symmetric bands often stronger than anti-symmetric ones

• Crystalline materials often have stronger Raman bands than non-crystalline materials


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-0.25

0.75

1.75

2.75

3.75

300500700900110013001500Wavenumber (cm-1)

animal source Aanimal source B Vegetable source

C-H Rocking

-0.5

1.5

3.5

5.5

28002850290029503000

SN

V R

am

an

In

ten

sity

animal source Aanimal source BVegetable source

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CaHPO4

0.5

0.9

28002850290029503000Wavenumber(cm-1)

Vec

tor

Nor

mal

izat

ion 0.13% MgSt

0.25% MgSt0% MgSt0.5% MgSt

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Kaiser Optical Systems Rxn-1-785 nm Raman Spectrometer.

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FluorescenceFluorescence

• Properties– Very efficient conversion of laser photons into unwanted light– Emission spectrum usually changes little, if at all, with changing

laser wavelength– Fluorescence lifetime typically 1 to 10 nanoseconds

• Sources– impurities– additives

• Elimination– Near-infrared wavelength excitation– Far-UV wavelength excitation– Photobleaching– Spectral subtraction methods– Time-resolved detection

En

ergy

Fluorescence EnergyLevel Diagram

Stokes Anti-Stokes

Twophoton

Slide Courtesy of Kaiser Optical Systems

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Example of a Raman SpectrumExample of a Raman Spectrum

0

2

4

6

400 600 800 1000 1200 1400 1600 1800

4 component mixture: omp-xylene and ethylbenzene

Raman shift in wavenumbers from the laser line

Inte

nsit

y in

dete

cte

d p

hoto

ns x

10

-5

pm

o

e p

o

m

ep

p

m, e

m, e

op, e

om

omp ompe


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Glass/Amorphous MaterialsGlass/Amorphous Materials

• Stress on molecular groups from local environment changes vibrational energy.

• Discrete peaks become broad bands.


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Raman Scattering from CrystalsRaman Scattering from Crystals

• Periodicity of a crystalline lattice reduces the number of vibrations that Raman observes.

• Spectrum consists of narrow peaks.• Spectrum effected by orientation

X X XX X X X X X X

X X XX X X X X X X

Polarizabilitychanges

add together

Polarizabilitychanges

cancel out


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Theophylline Anhydrous vs. MonohydrateTheophylline Anhydrous vs. Monohydrate

1941.9 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 793.5

Raman Shift / cm-1

anhydrous

monohydrate

Slide Courtesy of Kaiser Optical Systems, work by Lynne Taylor’s group,.

Industrial & Physical Pharmacy, Purdue University

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Theophylline Phase Stability as a Theophylline Phase Stability as a Function of TemperatureFunction of Temperature

Literature Transition temperature for hydrate to anhydate is around 60°C

69°C

64°C

58°C

54°C

48°C

1816.1 1700 1600 1500 1400 1300 1200 1100 1000 877.4

Raman Shift / cm-1

Inte

nsit

y


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Components of a Raman Components of a Raman SpectrographSpectrograph

• Laser• Fiber optic sampling device• Notch filter• Grating• CCD Detector


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RamanRamanRxn1Rxn1 Schematic Schematic OverviewOverview

Imaging Spectrograph

HoloPlex

TE CooledCCD

Detector

Slit Notch

ControlElectronics

ProbeHead

Invictus NIR Laser

ProbeHead

Filtering Universal ProbeHead

Immersion and Non-Contact Sampling Optics

Axial Transmissive Spectrograph

HoloPlex Grating

TE Cooled CCD Detector

Invictus NIR Laser


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Innovative all refractive design!

HoloPlex Advantages: Quantitative Raman! Full Simultaneous Spectral Coverage High Throughput High Spectral Resolution No Moving Parts

Low 1.8 f/# means Higher Optical Throughput (~4X)

Improved Thermal Stability (5X)

Rugged Compact DesignHolographicTransmission

Grating

EntranceSlit

Multi-elementLenses

Output Plane

Axial Transmissive DesignAxial Transmissive Design


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Lasers Commonly used for RamanLasers Commonly used for Raman

• 1064 nm – Nd:YAG laser– FT Instrumentation– Out of range of CCD, must use InGaAs or Ge

• 830 nm– Not common but could help avoid fluorescence

• 785 nm– Diode laser– Most common laser used for Raman work– Good compromise between fluorescence and Raman

efficiency…makes it somewhat universal– Stable to environment– Electronically efficient


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Lasers Commonly used for RamanLasers Commonly used for Raman

• 633 nm – He-Ne laser– Longer lifetime

• 532 nm– Frequency doubled Nd:YAG laser– Good efficiency, low power– Watch out for fluorescence!– Sensitive to temperature

• 514 nm– Ar-ion laser

• 488 nm– Ar-ion laser

• UV lasers– Resonance Raman– EXPENSIVE


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Sampling OptionsSampling Options

Pilot Plant Trial Production Installation

Immersion Probe Non-ContactOptic

Stream Stream

532 nm excitationSlide Courtesy of Kaiser Optical Systems

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Purpose of Notch FiltersPurpose of Notch Filters

• Filter out non-informative radiation• In the case of Raman instrumentation,

this means filtering the Rayleigh scattered energy

• Holographic notch filters are the most common…in nearly every Raman instrument you will find a Kaiser notch filter

Slides by courtesy of: Mark Kemper, [email protected]

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Properties of Holographic Notch Properties of Holographic Notch FiltersFilters

• High attenuation• Narrow bandwidth• Sharp spectral edges• Good transmission• High damage

threshold• Environmentally

stable Center = 785 nmFWHM at 50%T = 12 nm0.3 to 4.0 OD edge = 7.1 nm

0

40

80

700 720 740 760 780 800 820 840

Wavelength (nm)

% T

ran

sm

issio

n


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CCD DetectorCCD Detector

• Multi element silicon detector (1024 x 128)• Maintained at low temperature (-40ºC)• Key reason for lack of moving parts• High sensitivity• Detection range 400 – 1050 nm

Slides by courtesy of: Mark Kemper, [email protected]

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To learn more about Raman To learn more about Raman Spectroscopy:Spectroscopy:• E. Smith and G. Dent, “Modern Raman

Spectroscopy A Practical Approach”, John Wiley & Sons Ltd; (Chichester, United Kingdom), 2005.

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Comparing FT-IR and RamanComparing FT-IR and RamanFT-IRFT-IR AbsorptionAbsorption Fundamental informationFundamental information Sample preparationSample preparation Process measurements Process measurements

difficultdifficult High spectral densityHigh spectral density OrganicsOrganics DipolesDipoles O-H, C=O, N-H Water a problem

RamanRaman EmissionEmission Fundamental informationFundamental information No sample preparationNo sample preparation Process measurementsProcess measurements High spectral densityHigh spectral density Sampling challengesSampling challenges Organics and inorganicsOrganics and inorganics PolarizabilityPolarizability Aromatics, C=CAromatics, C=C Water no problemWater no problem

Slide courtesy Kaiser Optical Systems.

Vibrational Spectroscopy for Pharmaceutical Analysis

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