Introduction to Raman Spectroscopy

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What is Raman Spectroscopy?

A Versatile Vibrational Spectroscopy Technique
Applicable to
Organics and inorganics Solids, liquids, and occasionally gases Micro and macro sampling Qualitative and quantitative analysis

R H

H R

Raman Effect

Virtual Energy States

Rayleigh Scattering Stokes Raman Scattering Excitation Energy Anti-Stokes Raman Scattering

4 3 2

IR Absorbance

1 0

Vibrational Energy States

Raman Spectra
Sample excitation yields an entire Raman vibrational spectrum
Excitation Frequency/ Rayleigh Scattering

Stokes Raman Scattering

Anti-Stokes Raman Scattering

cm-1

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0 Frequency of Vibrational Transitions

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Measurement Wavelengths
Excitation Frequency/ Rayleigh Scattering

Stokes Raman Scattering

Anti-Stokes Raman Scattering

cm-1

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0 Frequency of Vibrational Transitions

3500

nm 400 Visible

800 Near Infrared

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1400

Shifted Raman Spectra

Stokes Raman Spectrum

Anti-Stokes Raman Spectrum

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3500

nm 400 Visible

800 Near Infrared

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1400

Conceptual Raman Spectrometer

Excitation Laser

Mechanism for Frequency Differentiation Detector

Line Filter

Presentation/ Collection Optics

Rayleigh Rejection Filter

Rayleigh and Raman Scattered light

Sample

How Raman Spectroscopy is Practiced

Reflection Technique
Sample geometry not important Sample surface exposed to sample Size of sample can be as small as 1 m

Applicable Samples
Solids Liquids Occasionally gases
usually need to be pressurized

Advantages Offered by Raman

Complementary to IR
Both techniques provide information-rich spectra Weak IR absorbers often strong Raman emitters

Raman provides easy access to FIR vibrations

Typical spectrum extends to 100 cm-1 Great for analysis of organics and inorganics

Raman bands typically sharper than IR

Often more suited to interpretation

Useful for conformational analyses

Drug polymorphs Material stress

Advantages Offered by Raman

Sample preparation usually trivial
Samples analyzed neat Analysis possible through glass and plastic packaging

Useful for analysis of aqueous solutions

Water bands weaker

Remote sampling with fiber-optics

Common quartz fibers can be used Fibers can be stretched 100s of meters

Useful for microscopy

High spatial resolution possible
less than 1 m with visible excitation

Depth profiling possible with confocal optics

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Vibrational Techniques of Choice

Why doesnt everyone use Raman all the time?
At one point they did

Historical Raman use

1928 Raman discovered by C.V. Raman
Raman developed considerable popularity during the 1930s

By 1939 Raman had become a principle analysis technique 1945 more sensitive IR detectors had been developed
IR becomes relatively inexpensive and uncomplicated IR gradually eclipses Raman as the vibrational technique of choice

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Current State of Raman Spectroscopy

Significant developments made a dramatic impact on Raman
1965 Laser recognized as ideal light source 1986 CCD arrays available for Raman use

Recent developments
Rapid development in solid state and diode lasers Rapid development in optical filters

Raman currently reemerging as the technique of choice in some areas

New generation of highly automated Raman instruments appearing today
Serving as very productive investigative tools

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Drawbacks of Raman
Raman spectroscopy still has some drawbacks More expensive than IR
IR most cost effective for routine sampling

Fluorescence
Serious obstacle to collecting Raman with some samples

Potential of laser damage

Some samples very sensitive to laser energy

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Fluorescence
Fluorescence
Broad band emissions Stokes emissions (lower energy than excitation energy) Results from excitation to higher electronic states followed very quickly by decay to lower electronic states producing emission Considerably more intense than Raman emissions
Can completely obscure Raman emissions in some cases

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Fluorescence Avoidance
Spectral correction after collection
Several specialized baseline correction algorithms exist Not viable in cases where fluorescence saturates detector

Confocal optics
Can work well when the source of fluorescence is the substrate rather than the sample

Excitation laser change

Most reliable means of avoiding fluorescence Switch to an excitation frequency that does not stimulate fluorescence in the sample
Typically this means switching to longer (NIR) wavelengths FT-Raman operating with 1064 excitation rarely exhibits fluorescence

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Fluorescence Avoidance
Example of fluorescing pharmaceutical

Dispersive Raman, 532 nm laser

Dispersive Raman, 785 nm laser

FT-Raman, 1064 nm laser

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Accuracy of Raman Intensity

Raman is an emission technique
Emission measurements are absolute measurements

IR is an absorbance technique
Absorbance is relative measurement insensitive to instrument Always ratioed to background reference
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Emissivity

35 30 25 20 15 101700 5 4000

Transmittance

90

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3000

Wavenumbers

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2000

1500

80 500 1000 70 60 50 40 30 20 4000 3500 3000

Ratio
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Emissivity

35 30 25 20 15 10 5 4000 3500 3000

Wavenumbers

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Wavenumbers

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Accuracy of Raman Intensity

Emission measurements start with raw emissions from sample
Intensity is further attenuated by wavelength dependence of
Detector response Optical throughput

Intensity differs between excitation lasers and optical designs

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Accuracy of Raman Intensity

Raman spectra of the same sample collected under different conditions can be quite different in appearance
785 nm laser

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Raman shift (cm-1) 633 nm laser

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Raman shift (cm-1) 19

White Light Correction

Intensity differences can be corrected for
White light correction is an intensity normalization procedure
Utilize a white light black body standard to develop a wavelength dependent scaling factor that can be applied to correct intensity NIST also has several standards available with emission characterized at specific excitation frequencies.

Important when comparing to common reference spectra

785 nm laser

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2000 1500 1000 Raman shift (cm-1)

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785 nm laser white light corrected

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1000 2000 1500 Raman shift (cm-1)

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White Light Correction

Comparison of white light corrected spectra
785 nm laser white light corrected

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Raman shift (cm-1)

633 nm laser white light corrected

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Raman shift (cm-1) 21

Selecting Between IR and Raman

Factors to consider
Sensitivity differences with some compounds Differences in sample preparation Interferences from water Are low frequency vibrations significant? Is microscopy important?

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Sensitivity Differences
Compounds for which Raman offers increased sensitivity
Weak IR Absorbers often strong Raman emitters Symmetric bonds represented more (S-S, C-C, etc.) Molecular backbone emphasized more End groups de-emphasized Spectral range offers more information on inorganics

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Lack of Sample Preparation

Sample prep advantages obtained because
Raman emission is weak
Samples can be analyzed neat

Measurement typically performed with visible or NIR wavelengths

Sample through glass and plastic packaging Utilize remote fiber-optic sampling easily

Saves time and can be important to the analysis

Example: pharmaceutical polymorph analysis

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Interference from Water

Less interference from liquid water
More applicable to solution studies Water still emits and can still be a significant interference

Water vapor usually insignificant

Allows humidity cells for environmental studies

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Access to Low Frequency Vibrations

Access to low frequencies is routine with Raman
Generally limited only by Rayleigh rejection mechanism Filters allow access to 100 cm-1 or lower
Often can be tuned to 50 cm-1

Atmospheric water vapor not a concern

217.78 150.67

81.56

48.75

300

250

244.61

Sulfur spectrum collected on Almega XR with 532 nm laser

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Raman shift (cm-1) 26

Raman Microscopy
Typically performed with visible wavelengths
Diffraction limit is much smaller than IR
Typical limit for IR instrumentation is 10 m Raman can typically get to 1 m or smaller

Provides higher spatial resolution Depth profiling possible with confocal optics
Usually little absorbance of Raman frequencies to within 2 m
Detector

Objective

Adjust z-axis Sample with scope focus knob

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Raman Microscopy - Mapping

Effervescent cold medicine tablet
Large area captured with video mosaic - spliced video images Collected on Almega XR with 785 nm Laser

Investigate the distribution of active and inactive ingredients

Pain reliever Antacid Dye

350 Microns

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350 Microns

Raman Microscopy - Mapping

Multiple compounds identified by library search and mapped using spectral correlation
Point 1, 4-Acetamidophenol

Point 42, Sodium Bicarbonate

Point 171, FD&C Yellow #6 Powder

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Raman shift (cm-1) 29

Emission Technique
Emission technique means no reference necessary for Raman
IR requires a reference No reference needed for Raman
Good Raman spectra can be collected on nearly any substrate metal glass paper polymers silicon

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Reflectance Technique
Reflectance requires minimal sample preparation IR reflectance capabilities
Direct Reflectance limited to thin samples on metal substrates ATR more versatile but still limited
Can not access very small samples in crevices Requires good sample contact Requires physical contact with the sample

Raman collected as direct reflectance

No sample contact Can sample deep in crevices Microwell plate sampling is routine

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Performance of Raman Systems - SNR

Highly dependent on how you are sampling Signal-to-Noise Ratios (SNR) a function of
Energy on sample
Laser power density at sample depends on laser power how tightly focused it is

Detector sensitivity range (CCD, Ge, InGaAs) Raman emission strength Confocal apertures
Trade off between SNR and resolution Larger apertures let in more energy but also more stray light

Throughput of optics

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Raman Detectors
Detector Sensitivity Range
Si CCD most sensitive
Only sensitive in visible range Best response typically with 532 nm and 633 nm lasers

Ge and InGaAs sensitive in NIR

Ge more sensitive but Liquid N2 cooled

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Intensity of Raman Emissions

Raman emission is excitation wavelength dependent
Stronger emissions with shorter excitation wavelengths Raman emission proportional to (1/)4
Intensity of Raman Emissions relative to 1064 nm excitation at 780 nm - 3.4X stronger at 633 nm - 8.0X stronger at 532 nm - 16.0X stronger at 473 nm - 25.6X stronger

Actual measurements further attenuated by wavelength dependent collection efficiency

Theoretical emission gains often not fully realized

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Intensity of Raman Emissions

Relative Emissions strength and Stokes spectral range with some common Raman Lasers
244 266 325 458 473 488 514 532 633 780/785 830 1064 200 400 600 800 1000 1200 Wavelength (nm) 1400 1600 1800 0 to +3300 cm-1 ranges for selected Raman lasers

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Selection of Excitation Lasers

Factors to consider in excitation laser selection
Fluorescence
Organic fluorescence commonly between 400 and 800 nm Glass fluorescence can be significant between 850 and 1000 nm

Visible CCD performance falls off below 500 nm Glass optics transmission falls off below 350 nm Higher spatial resolution obtainable with shorter wavelengths Detector technology creates a division at border of visible and NIR
Si CCDs and dispersive technology used in visible Offer higher sensitivity but more susceptible to fluorescence Ge and InGaAs detectors and FT technology used in NIR Sensitivity less, but nearly always fluorescence free

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Selection of Excitation Lasers

Special Regions of Interest Resonance Raman Fluorescent Organics

Glass Fluorescence Water Absorbance

Arbitrary Scale

Glass Optics

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800 1000 1200 Wavelength (nm)

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Dispersive vs. FT Technology

Selection dictated by detector behavior
InGaAs and Ge detectors limited by 1/f noise
Benefit from having more frequencies/energy on the detector FT measures all frequencies simultaneously

Si CCD detectors limited by combination of dark current and quantum efficiency

No benefit provided by measuring frequencies simultaneously Dispersive technology discriminates frequencies before detection

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Dispersive Technology
Dispersive systems utilize a grating to break light into component frequencies before it arrives at the detector
Torroidal Mirrors

Aperture

Shutter Grating

Detector

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FT Technology
Fourier Transform (FT) technology obtains frequency discrimination by introducing an interference pattern with an interferometer which allows individual frequencies to be differentiated after detection with the Fourier Transform. All frequencies are measured by the detector simultaneously. Fixed mirror Laser

Beamsplitter

Notch Filter Sample

x 0 -x Moving mirror

He-Ne laser Laser diode

Detector
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Dispersive vs. FT Technology

FT-Raman
Interferometer Near Infrared laser Detector is InGaAs (room temperature) or Germanium (LN2 Cooled)

Dispersive Raman
Grating spectrograph Visible lasers Silicon CCD array detector

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Spectral Range
High cm-1 spectral range typically limited by detector cutoff
Si CCDs limited to 1050 nm
At 830 nm excitation this is 2500 cm-1 Stokes At 780 nm excitation this is 3300 cm-1 Stokes At 633 nm excitation this is 6275 cm-1 Stokes

Bands above 4000 cm-1 are almost exclusively overtone and combination modes with very weak Raman emissions AntiStokes high end limited by distribution of excited state molecules
Generally this only goes a few hundred cm-1

Low cm-1 spectral range limited by Rayleigh blocking mechanism

Usually between 100 cm-1 and 50 cm-1 for filters Premonochrometers can extend this limit
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Resolution
Spectral resolution limited by
Wavelength discrimination mechanism
Dispersive Grating resolution CCD pixel spacing Focal length of spectrograph FT Optical retardation introduced by interferometer

Excitation laser line width

Most Raman lasers have line width approaching 1 cm-1 Defines the effective limit for Raman spectroscopy at 1 cm-1 for all but the most specialized applications

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Quantitative Raman
Raman emission proportional to concentration Requires a calibration set specific to the analysis and experimental conditions Applicable for use with a number of quantitative algorithms
Beers Law CLS MLR PLS

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Example of Quantitative Raman

FT-Raman spectra collected directly through gelcap walls Calibration using known concentrations of active ingredient

Additional known samples collected for method validation

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Gel-Caps with Ketoprofen active ingredient

Pure Drug spectrum

Raman intensity

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6 4 2 3500 3000 2500 2000 1500 1000 500

Raman shift (cm-1)

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Example of Quantitative Raman

Predicted % Ketoprofen

8 6 4 2 0 0 2 4 6 8 10
R = 0.9993 RMSEC = 0.0310 SMLR at 999 cm-1

Calibration Prediction

Actual % Ketoprofen

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Summary
Raman spectroscopy is a powerful tool for a wide range of chemical analyses.

Raman offers a number of benefits over other vibrational techniques with specific samples
Most labs with an investigative focus can benefit from Raman technology

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