Raman Spectroscopy
and Chemical Imaging
2
Overview
• Fundamentals of Raman Spectroscopy
• Raman Microscopy
• Raman Imaging
• Raman-AFM
• Summary
Thermo ScientificTM
DXRTM SmartRaman
Thermo ScientificTM
DXRTM Raman Microscope
Thermo ScientificTM
DXRxiTM Raman
Imaging Microscope
3
Raman is a Complementary Technique to FT-IR
C=O C-O C=C
C=O
C=C C-O
C=C
FT
-IR
R
am
an
• Complementary information
• End functional groups dominant in IR spectrum
• Molecular backbone dominant in Raman spectrum
• Raman often useful for characterizing morphology
• Weak IR absorbers often strong Raman emitters and vice versa
• Aqueous solutions pose fewer challenges with Raman
trans-cinnamyl
acetate
4
Raman Spectroscopy – The Raman Effect
Rayleigh scattering (filtered out)
Raman scattering (Stokes shift)
LASER
200 400 600 800 1000 1200 1400 1600 1800 2000
Raman shift (cm-1)
Blo
ckin
g F
ilter
0 E
xc
itati
on
fre
qu
en
cy
V = 0
Rayle
igh s
ca
tte
rin
g
V = 1
Ram
an
sc
att
eri
ng
~~~~~~~~~~~~~~~ V = virtual state
5
Raman spectra Changes Information Examples
Characteristic
frequencies
Material
identification,
polymorphs
differentiation
rutile and anatase of
TiO2
PS, PET
Intensity
Quantity of material
analyte concentration
in aqueous solution;
thickness of
transparent coating
Change in
frequency
Stress/strain in
material
520 cm-1 peak shift in
Si upon strain
Change in width
Disorder or defects
crystalline 520 cm-1
and amorphous 480
cm-1 peak in Si;
D band in CM
Change in
frequencies,
intensities, widths
Thermal or
pressure
Impact
phase transformation,
melting, crystallization
Information from Raman Spectroscopy
6
Common Raman Microscopy Applications
Art Conservation and Archeology Identification and discrimination of paint pigments
using DXR microscopy and fiber optic analysis
Polymers and Packaging Subsurface analysis to identify inclusions and
verify layers without sample preparation
Geology and Gemstones Rapid non-destructive identification of fluid inclusions
in minerals using DXR confocal analysis
Photovoltaics Measurement of
silicon crystalline
fraction using
automated macros
for routine post
production analysis
7
Carbon Nanomaterials Determination of graphene layer thickness and
nanotube diameter populations
Common Raman Microscopy Applications
Life Science Applications Rapid high sensitivity detection of biomolecules
using the DXR SERS kit
Pharmaceuticals High speed polymorph screening and recrystallization studies Forensic Science
Identification of components in explosives residue
8
Dispersive Raman Instrument Fundamentals
• DXR System Basics:
• Laser
• 455 nm, 532 nm, 633 nm &
780 nm
• Rayleigh rejection filter
• 50 cm-1 cut-off
• Aperture
• Slit
• Pinhole (confocal mode)
• Grating
• Standard, 5 cm-1
• High Resolution, 2 cm-1
• Detector
• CCD, EMCCD
Sample
Grating
Aperture
Laser
Multichannel detector
Filter
50
2D
G
9
Sampling in Raman Spectroscopy
• Various Sample Types
• Solids, liquids, gases, powders, slurries, films,
etc.
• Aqueous samples
• Water has very weak Raman spectrum!
• Materials at high/low temperatures and
variable pressures
• Linkam and Harrick stages
• Bulk or microscopic samples even at
remote locations with little or no sample
preparation
• Analysis through many containers
• Glass bottles, Pyrex® reaction vessels, plastic
containers, blister packs, etc.
10
DXR Raman Microscope – High Spatial Resolution Capability
• Confocal Design
• High Spatial Resolution
• 0.5 µm (lateral), confocal depth
resolution – 2 µm (axial)
• Raman Mapping Capability
• Point, area (x,y) and depth (z)
• User Interchangeable
Components
• Laser, filter, gratings
• Proprietary Auto-Alignment
• Ocular or Video Image
Observation
• Class-I Laser Safety
Detector
Aperture
Objective
Focus
Plane
Sample
Lift sample
stage
11
NicoDerm CQ Transdermal Nicotine Patch
PET (~ 10 um)
Polyethylene (~ 25 um)
Ethylene/Vinyl acetate
copolymer (EVA) +
nicotine (~ 70 um)
Polyethylene (~ 20 um)
Polyisobutylene (PIB) (~ 20 um)
Poly(ethylene teraphthalate)
PET (~ 55 um)
Raman Z-map Video image of
the cross section
1
2
3
4
5
6
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Ease of Use with Built-In Raman Expertise
• Point and shoot – results with a single push of a button
Start Measurement
Go Button
Autofocus Autoexposure Optimal Spectral
Quality
Collected Raman Spectrum Library Search Results Final Report
Real-Time
Fluorescence
Correction
Title:
GloboChem Corp.
Laser: 532 nm Grating: 900 lines/mm Spectrograph aperture: 25 µm pinhole Laser power level: 10.0 mW
-0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
Ra
ma
n I
nte
nsity (
cp
s)
500 1000 1500 2000 2500 3000 3500
Raman shift (cm-1)
Tue May 27 13:58:26 2008 (GMT-05:00)
Sample #5-24-20081234A
*JC C07-3023 #1 RLL - run 1 - cocaine HCl
Matc h:95.98
Manufac tu re r: Sigma
Product #: C -5776
Lo t # : 83F-0562
Coc aine HC l
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50
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90
100
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180
Int
500 1000 1500 2000 2500 3000
Raman sh ift (c m-1 )
Load sample Move to Target
13
Raman Images of Multi-Layer Solid Bead Dosage Form
Raman images of the split bead constructed as correlations with spectra of reference materials.
1 mm diameter bead with 5 μm mapping steps: 2500 high quality spectra for 7 hours!
14
Introducing DXRxi Raman Imaging Microscope
• Ultra fast data collection
• Hundreds of spectra per second
(600 spectra/sec max)
• Image-centric instrument platform
• Real-time chemical image
generation
• Parameter optimization on the fly
• Laser power, exposure time,
number of scans, etc.
A completely new approach to Raman imaging!
15
DXR versus DXRxi Sensitivity -Test Conditions
• Sample – Polystyrene Puck
• 100X Objective
• 532 nm laser – 0.5 mW
• 25 micron pinhole
• Exposure Times 0.020 – 8 seconds (50 – 0.125 spectra / s)
• S/N Calculations:
• Peak Height 1001 cm-1
• Noise RMS 2400-2300 cm-1
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Collection Speed – 0.125 Spectra per Second
0.125 Spectra per Second - DXRxi
0.125 Spectra per Second - DXR
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Compare Fastest DXR Result to Fastest DXRxi Result
50 Spectra per Second – DXRxi
(DXRxi could still go faster)
10 Spectra per Second – DXR
(instrument limit)
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Video - Fast Imaging for a Full Tablet
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Fast Surface Imaging of an Entire Headache Tablet
Acetaminophen Caffeine Titanium Dioxide Aspirin
• 226,000 spectra, 25 mm pixel size
• Acquisition parameters: 550 Hz
(1.8 ms/spectrum)
• 8 minute collect time!!
Determine:
• Size of each domain
• Distribution of domains
• Overall composition of tablet
11 x 11 mm surface area
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Analysis of Pharmaceuticals
Acetaminophen Caffeine Titanium Dioxide Aspirin
11 x 11 mm surface area
532nm laser
5.4 million spectra!!! 0.5 mm pixel size
Acquisition parameters: 550 Hz (1.8
ms/spectrum)
~3 hour collect time!!
Component Calculated %
(Surface Area) Reported %
Aspirin 38.6 37
Acetaminophen 35.4 37
Caffeine 7.7 9.6
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MCR Analysis of Headache Tablet
Acetaminophen Caffeine Starch Aspirin
1.6 x 1.7 mm surface area
532nm laser, 50X objective
116,000 spectra, 5 mm pixel size
Acquisition parameters: 200 Hz (5
ms/spectrum)
55 minute collect time
22
High Resolution MCR Analysis of Pharmaceuticals
Sodium Lauryl Sulfate Microcrystalline Cellulose
Acetaminophen Caffeine Starch Aspirin
225 x 250 mm surface area
532nm laser, 100X objective
229,000 spectra, 0.5 mm pixel size
Acquisition parameters: 100 Hz (10
ms/spectrum)
3 hour collect time!!
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High Res Discrimination between Similar 1μm Particles
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Raman Imaging of Tissue Samples
•17,000 spectra
• Acquisition parameters: 40 Hz (25 ms/spectrum), 100 scans, 2.0 mm pixel size
Collagen Glass Slide Cell Nuclei
*Sample provided by Ihtesham ur Rehman, University of Sheffield*
532 nm laser, 5.4 mW, 50X objective
25
Video - Fast Multi-Region Imaging for CVD Graphene
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Characterizing Materials for Product Development
10 microns
Scale:
Single Layer Double Layer Triple Layer Multiple Layer
• Graphene Layer Thickness
• Graphene exhibits different properties
depending on how many layers are
present
• Simple application of a discriminant
analysis method quickly classifies the
number of layers.
27
MCR of Synthetic Volcanic Rock - Analysis of Volatiles
Chalcocite? Labradorite?
OH Glass CO2 Carbon
• The DXRxi maintains the same confocal
capability as the DXR
• Quantifying the volatiles in the sample
will provide clues about the nature of
volcanoes.
• 21,000 spectra
• Acquisition parameters: 40 Hz (25
ms/spectrum), 100 scans, 0.5 mm pixel
size
*Sample provided by Jenny Riker, University of Bristol*
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Identification of Minerals and Carbonaceous Species in Oil Shale
*Sample provided by Prof Asish Basu, University of Texas, Arlington*
Pyrite Marcasite
Calcite Anatase
D-b
an
d
G-b
an
d
Video image showing the features of the oil
shale sample. Shown in red square is the
area where Raman imaging was performed.
Superimposed view of video and Raman
images, showing the kerogen in red.
Raman images
showing the locations
for Pyrite, Marcasite,
Calcite and Anatase.
Representative
Raman spectra.
Collection
parameters: 532
nm laser, 30
ms/spectrum, 30
scans
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Imaging Thermal Maturity of Carbonaceous Species in Oil Shale
*Sample provided by Prof Asish Basu, University of Texas, Arlington*
Raman shift (cm-1)
Ra
ma
n in
ten
sit
y (
cp
s)
FWHM range
G-b
an
d
Representative spectra for the red,
orange and green regions, showing the
broadening the G-band from Green to
Red.
Raman image of CM maturity differences in G-
band FWHM values. Red is higher FWHM
value and lower maturity. Green is lower
FWHM and higher maturity. The orange
regions have intermediate value. The blue
regions mostly contain minerals and lack
kerogen.
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High-Resolution Imaging of Stress in Silicon
• 50,000 spectra
• Acquisition parameters: 100 Hz (10
ms/spectrum), 25 scans, 0.5 mm pixel size
• Sample is a Si substrate with a
layer (41 nm) of Si/Ge deposited
followed by an additional layer of
Silicon
• The presence of the Ge causes
stress in the second layer (23
nm) of Si (Red imaging)
455 nm laser, 1.0 mW, 100X objective
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Raman and AFM Integration
Co-Localized AFM Raman
• Simultaneous measurement two techniques
• Diffraction limited confocal Raman
• AFM topographical image
• Light confinement maintains focus on probe
tip
Tip Enhanced Raman Spectroscopy (TERS)
• Ag or Au-coated AFM tip acts as nano-
antenna
• Provide exceptional signal enhancement
• Spatial resolution beyond the diffraction limit • As high as 10 nm
X
Focused laser spot
AFM Probe
Focused laser spot
Enhanced
Raman signal
Nanoantenna
(Metal AFM probe)
~ λ / 2
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Topography Adhesion Stiffness
dC/dZ
(dielectric constant) dC/dV
(differential capacitance )
Raman
(peak position) Raman
(peak intensity)
White Light
Image
Co-Localized AFM-Raman for a Polymer Blend
33
Complementary AFM and
Raman data provides rich
picture of sample
150 x 150 point scan
0.3 sec exposure time
Co-Localized AFM-Raman for CVD Graphene
Raman map:
2D-band
Raman map:
G-band
AFM height image Characteristic single point spectrum
2D-band
G-band
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TERS maps of single layer CVD Graphene on
copper substrate • Green: pristine graphene (2D band intensity).
• Blue: CH-terminated graphene areas (CH-bands
intensity).
TERS map of mechanically
exfoliated single layer graphene
on Au substrate • Green: pristine graphene (2D band
intensity).
• Red: areas with strong defects (D-
band intensity)
Lateral resolution of Raman maps: <12 nm
TERS Mapping of Graphene
35
Summary
• Non-destructive
• Minimal sample preparation
• Sample through glass or plastic packaging
• Remote sampling capability
• Characterization of very small particles (sub-micron)
• Confocal sampling – looking below the surface of samples
• Fast and high-resolution imaging capability
• Integrated AFM – Raman and TERS solution – nano Raman imaging