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B ioMolecu lar Vibrat ional Spectroscopy :
Part 1: Princ iples o f In frared , Raman
Spectra and Techn iques
Lectu res for Warwick CD Workshop, Dec. 2011
Tim Keider l ing
University of I l l inoisat Chicago
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Tentative Schedule — can vary with interests
Part I:
• Optical Spectroscopy (general)—low resolution, fast response• Vibrational Theory
– Biologically relevant Vibrational Modes
– IR and Raman spectra - structure (qualitative)
• IR Instrumentation; FTIR principles
• Raman Instumentation
• Practical Demonstrations (lab? Break? ) – background material
•
Peptide methods —solut ion, sol id
• Protein Sampling Techniqu es (aqueous), ATR
Part II:
• Application Examples
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Structural Biology
• often need to know just the conformation
• structural determination of fold family may suffice,generally not after atomic structure
• In BioTech processes one must monitor effect of
mutation and environmental changes
need to get this information rapidly and
in a cost effective manner
Measure all phases/types of samples
Look at fast time-scale events
Optical Spectroscopy is limited for determining
structure – lacks site specificitybut often fits important QUESTIONS
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Near-IR
Electro-Magnetic Spectrum
SpectralRegions
Wavenumber (cm-1)
ElectronExcitation
ElectronTransition
MolecularVibration
MolecularRotation
106 105 103 102104107 10 1
X-ray Ultraviolet Infrared Microwave
14,285 4,000 400 100
Mid-IR Far-IR
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Vibrational Spectroscopy - Biological Applications
There are many purposes for adapting IR or Raman
vibrational spectroscopies to the biochemical,biophysical and bioanalytical laboratory
• Prime role has been for determination of structure. We will
focus on secondary structure of peptides and proteins, but
there are more – especially DNA and lipids• Also used for following processes, such as enzyme-substrate
interactions, protein folding, DNA unwinding
• More recently for quality control, in pharma and biotech
• New applications in imaging now developing, here sensitivity
and discrimination among all tissue/cell components are vital
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Optical Spectroscopy - Processes Monitored
UV/ Fluorescence/ IR/ Raman/ Circular Dichroism
IR – move nuclei
low freq. & inten.
Raman – nuclei,inelastic scatter
very low intensity
CD – circ. polarizedabsorption, UV or IR
Raman: DE = hn0-hns
Infrared: DE = hnvib
= hnvib
Fluorescence
hn = Eex - Egrd
0
Abs
orption
hn = Egrd - Eex
ExcitedState
(distorted
geometry)
Ground
State (equil.
geom.)
Q
n0 nS
molec. coord.
UV-vis absorp.
& Fluorescence. move e- (change
electronic state)
high freq., intense
Analytical Methods
Diatomic Model
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Essentially a probe technique sensing changes in the local environment of fluorophores
Optical Spectroscopy Electronic
Example Absorption and Fluorescence
Intrinsic fluorophores
eg. Trp, Tyr
Change with tertiary
structure, compactness ( M - 1 c m - 1 )
What do you see?
(typical protein)
Amide absorption broad,
Intense, featureless, far UV
~200 nm and below
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Optical Spectroscopy - IR Spectroscopy
Protein and polypeptide secondary structural obtained from
vibrational modes of amide (peptide bond) groups
Amide I
(1700-1600 cm-1
)
Amide II
(1580-1480 cm-1)
Amide III
(1300-1230 cm-1)
Aside: Raman is similar, but different
amide I, little amide II, intense amide III
What do you see? – LOTS!
D x
1 0 5
-4
-2
0
2
2000 1800 1600 1400 1200 1000
Wavenumbers (cm-1)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
A b s o r b a n c e I
II
III
9.0 x 108
a) human serum albumin
I R +
I L
0
935
1640
16651300
1340
4.3 x 105
ROA
0
I R -
I L
800 1000 1200 1400 1600
wavenumber / cm-1
Goal — try to give this meaning
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Spectroscopic Process (covered)
• Molecules contain distribution of charges (electrons and
nuclei, charges from protons) which is dynamicallychanged when molecule is exposed to light
• In a spectroscopic experiment, light is used to probe a
sample. What we seek to understand is:
– the RATE at which the molecule responds to this perturbation(this is response or spectral intensity – probability of transition)
– why only certain wavelengths cause changes (this is spectrum,
the wavelength dependence of the response – energy levels)
–
the process by which the molecule alters the radiation thatemerges from the sample (absorption, scattering, fluorescence,
photochemistry, etc.) so we can detect it
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Spectroscopic Process (covered)
• Molecules contain distribution of charges (electrons and
nuclei, charges from protons) which is dynamicallychanged when molecule is exposed to light
• In a spectroscopic experiment, light is used to probe a
sample. What we seek to understand is:
– the RATE at which the molecule responds to this perturbation(this is response or spectral intensity – probability of transition)
– why only certain wavelengths cause changes (this is spectrum,
the wavelength dependence of the response – energy levels)
–
the process by which the molecule alters the radiation thatemerges from the sample (absorption, scattering, fluorescence,
photochemistry, etc.) so we can detect it
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Quantum mechanical picture
Full Hamiltonian describes electron and nuclear motion
H = -S
ab
[
2 /2Ma
a
2 - 2 /2me
i2 - Zae2 /r ia + e2 /r ij + ZaZbe2 /Rab ]
i.e. n-KE e-KE n-e attr. e-e repul. n-n repul
• Born-Oppenheimer approx. separate electron-nuclear w/f
y
(r,R) =c
u (R)f
el (r,R) -- product fct. solves sum H
• Electronic Schröding er Equation – issue for CD (do ne prev.)
H el fel (r,R) = Uel (R) fe (r,R) – electron sol’n – nucl. pot.
Vn(R) =S
ab [Uel(R) + ZaZbe2 /Rab] – nuclear potential energy
• Nuc lear Sch rödin ger Equation
H n cu(R) = -[Sa (ħ2 /2M
a
) a
2 + Vn (R)] cu(R) = Eu cu(R)
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Solving Vibrational QM
• Nuclear Hamiltonian is 3N dim. – N atom, move x,y,z
– Simplify Remove (a) Translation (b) Rotation
– Result: (3N – 6) internal coordinates vibration
• Harmonic Approximation – Taylor s eries expansio n:
V(R) = V(Re) +S
ab
V/
Ra
Re(Ra-Re) +
½ Sab 2V/Ra
Rb
Re(Ra – Re)(Rb – Re) + …
– 3rd term –non-zero / non-const. - harmonic – ½ kx2
– Ra, Rb mixed
Solution
“Normal coordinates”
Qi = S jcij q j H = -Si [ 2 /2 2 /Qi
2+½ kQiQi2] = Si h i (Qi)
hi ci(Qi) = Ei ci(Qi) E j = (u j + ½) hn j solve as if independent
Diatomic:n
= (1/2p
) √k/m
k – force const.m
= MAMB /(MA + MB)
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Harmonic Oscillator
Model for vibrational spectroscopy
r e
r
e
r q
v = 1
v = 2
v = 3
v = 4
v = 0hn 1
2hn
3
2
hn
5
2hn
7
2hn
9
2hn
E
r e
Ev = (v+½)hnDv = 1
DE = hn
n = (1/2p)(k /m)
½
(virtual
state)
Raman
IR
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Spectral Regions and Transitions
• Infrared radiation induces stretching of
bonds, and deformation of bond angles – • Couples like motions into molecular mode
• (ignore rotations for biomolecules in solution)
symmetrical
stretch
H-O-H
asymmetrical
stretch
H-O-H
symmetrical
deformation
(H-O-H bend)
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Characteristic vibrations and structure
• heavier molecules bigger m - lower frequency
• H2 ~4000 cm-1 C –H ~2900 cm-1 C –D ~2100 cm-1
• HF ~4141 cm-1 HCl ~2988 cm-1
• F2 892 cm-1 Cl2 564 cm
-1 I –I ~214 cm-1
• stronger bonds – higher k - higher frequency
• CC ~2200 cm-1 C=C ~1600 cm-1 C –C ~1000 cm-1
• O=O 1555 cm-1 N O 1876 cm-1 N
N 2358 cm-1
• frequency depends mass + bond strength
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Frequency structure, small and large molec.
Same for vibrational modes of amide (peptide bond) groups
Amide I
(1700-1600 cm-1)
Amide II(1580-1480 cm-1)
Amide III
(1300-1230 cm-1) I II
a
b
rc
For polymer -- repeated structural elements have overlap/coupled spectra
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Vibrational Transition Selection Rules
Harmonic oscillator : only one quantum can change
D
vi = ± 1, D v j = 0; i j .
These are fundamenta l vibrations
Anharmonicity permits overtones and combinations
Normally transitions will be seen from only vi = 0, since most excited
states have little population.
Population, ni
, is determined by thermal equilibrium, from the Boltzman
relationship:
ni = n0 exp[-(Ei-E0)/kT],
where T is the temperature (ºK) – (note: kT at room temp ~200 cm-1)
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T ( r - r e )/r e
E/De
DE01 = hnanh--fundamental
D0 — dissociation energy
Anharmonic Transitions
Real molecules are anharmonic to some degree so other transitions dooccur but are weak. These are termed overtones (D vi = ± 2,± 3, . .) or
combination bands (D vi = ± 1, D v j = ± 1, . .). [Diatomic model]
DE02 = 2hnanhrm - overtone
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Vibrational Selection Rules• Interaction of light with matter can be described as the
induction of dipoles , mind , by the light electric field, E:
mind = a . E where a is the polarizability
• IR absorption strength is proportional to
~ ||
2
,
transition moment betweenY
i Y
f
• To be observed in the IR, the molecule must change its electricdipole moment, µ , in the transition—leads to selection rules
dµ / dQi 0 relatively easy, ex. C=O str. intense
• Raman intensity is related to the polarizability,
I ~ 2
, where da / dQi 0 for Raman trans.
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Complementarity: IR and Raman
If molecule is centrosymmetric, no overlap of IR and Raman
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Peak Heights
• Beer-Lambert Law:
•A =
lc – A = Absorbance
– = Absorptivity
– l = Pathlength
– c = Concentration
An overlay of 5 spectra of Isopropanol (IPA) in water. IPA Conc.
varies from 70% to 9%. Note how the absorbance changes with
concentration.
• The size (intensity) of absorbance bands depend upon molecular
concentration and sample thickness (pathlength)
• The Absorptivity () is a measure of a molecule’s absorbance at a givenwavenumber normalized to correct for concentration and pathlength – but asshown can be concentration dependent if molecules interact
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Peak Widths
• Peak Width is Molecule Dependent
• Strong Molecular Interactions = Broad Bands
• Weak Molecular Interactions = Narrow Bands
WaterWater
Benzene
At i l ti
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Level of structure
determination neededdepends on the
problem
Atomic resolution Ca chain
Secondary structure Segment fold (tertiary)23
Structural
Biology
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Chain conformation depends on f, y angles
Far UV absorbance broad, l i ttle f luorescence —
coupling impact small
Detection requires method sensitive to amide coupling
If (f,y
repeat, they determine secondary structure
Polymer analysisStudy the repeat units
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Physical method of detection must sense
secondary structure — e.g. couple amides
IR/Raman — coupl ing comparable to band width , intensitymaximum is characteristic of structure – frequency basis
Circular dichroism --dipole and through-bond chiral coupling oflocal modes (excitations) circularly polarized transitions,
DA = AL-AR - Develops characteristic band shapes (intensity)
Theoretically try to understand spectra/structure relationIR ~ D=
m
.
m~|dm/d
Q|
2
(Raman ~ |da/dQ|2)
ECD, VCD ~ R = Im(m.m)
Computable with ab initio QM techniques, ECD needs excited states
IR & VCD relatively easy, Raman more basis set sensitive
Major activity,for analysis!}
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Characteristic Amide Vibrations
I - Most useful;
IR intense, less interference(by solvent, other modes,etc)
Less mix (with other modes)
II - IR intense
III - Raman Intense
A – often obscured
by solvent
IV – VII – difficult
to detect, discriminate
~3300 cm-1
~1650 cm-1
1500-50 cm-1
1300-1250 cm-1
700 cm-1
mix
M d l l tid IR t A id I d II
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Wavenumbers (cm-1
)
1450150015501600165017001750
A b s
o r
b a n c e
0
1
2
3 helix
-structure
randomcoil
Model polypeptide IR spectra -- Amide I and II
Differentiation of conformations mostly due to coupl ing of amides
not to H-bonds or other factors, although they contribute
Helix — small frequency
dispersion, central onesmost intense, amide I,
higher ones for amide II
Sheet — large frequency
dispersion, characteristic
split amide I, broad amide II
Coil — less well-defined
broad amide I and II
I II
Frequency based
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Temperature dependent IR
spectra of the helical peptide
Temperature dependence of
amide I’ frequency
IR frequency shift shows a sigmoidal curve and
spectra have an isobestic point for thermal unfolding
However, frequency shift is ~1635 ~1645 cm-1 – solvated helix
Monitoring structural change - temperature
folded
unfolded
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6 b b sheet
, 2 )
Tyr97
Tyr25
Tyr92
H1
H3H2
Tyr76
Tyr115
Tyr73
• 124 amino acid residues, 1 domain, MW= 13.7 KDa
• 3a
-helices
• 6b
-strands in an AP b
-sheet
• 6 Tyr residues (no Trp), 4 Pro residues (2 cis, 2 trans)
Ribonuclease A
combined
uv-CD and
FTIR study
Simona Stelea,Prot Sci 2001
Optical spectra senses dynamic equilibrium - unstructured systems29
0.06
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Wavelength (nm)
260 280 300 320
Ellipticity
(mdeg)
-16
-14
-12
-10
-8
-6
-4
-2
0
Near-UV CD
Wavenumber (cm-1)
1600162016401660168017001720
Absorbance
0.00
0.01
0.02
0.03
0.04
0.05FTIR
Wavelength (nm)190 200 210 220 230 240 250
Ellipticity(mdeg)
-15
-10
-5
0
5
Far-UV CD
Temperature 10-70oC
FTIR — amide I
Loss of b-sheet
Ribonuclease A
Far-uv CDLoss of a-helix
Near – uv CDLoss of tertiary struct.
Spectral Change
30
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C
i 1
(x10
2)
-8.0
-7.6
-7.2
-6.8
-6.4
-1.0
-0.5
0.0
0.5
1.0
FTIR
C i 1
-17
-15
-13
-11
-9
-7
-5
C i 2
-15
-10
-5
0
5
10
Near-UV CD
0 20 40 60 80 100
Ci1
-13
-12
-11
-10
Ci2
-30
-25
-20
-15
-10
-5
0
5
Far-UV CD
Ribonuclease A
PC/FA loadings
Temp. variation
FTIR (a,b)
Near-uv CD(tertiary)
Far-uv CD(a-helix)
Pre-transition evident in far-uv CD and FTIR, not near-uv CD
Temp.
31
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Nucleic acid IR
Nucleic Acids – less variation —helicity all about the same
a) – monitor ribose conformation
b) – single / duplex / triplex / quad – H-bond link bases
O h bi l
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Sugars – little done, spectra broad, some branch appl.
Lipids – monitor order – self assemble – polarization
Example is CH2 wag, but
also stretch and scissor
bend are characteristic
Self assemble to lipid
bilayer – membrane
Polarization can tell
orientation of lipid or
protein in membrane
Other biopolymers
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Combining Techniques: Vibrational CD “CD” in the infrared region
Vibrational chiralityMany transitions / Spectrally resolved / LocalTechnology in place DA ~10-5 - limits S/N / Difficult < 700 cm-1
Same transitions as IR
same frequencies, same resolutionBand Shape from spatial relationships
neighboring amides in peptides/proteins
Relatively short length dependence
AAn
oligomers VCD have DA/A ~ const with n
vibrational (Force Field) coupling plus dipole coupling
Development -- structure-spectra relationships
Small molecules – theory / Biomolecules -- empirical,
Recent — peptide VCD can be simulated theoretically
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Wavenumber (cm-1)
1600165017001750
Absorbance
0.0
0.5
1.0
DA
x105
-10
-5
0
5
10
VCD
IR
(a)
Wavenubmer (cm-1)
1600165017001750
Absorbance
0.0
0.5
1.0
DA
x105
-4
-2
0
2
IR
VCD
(b)
Poly Lysine in D2O – Amide I’– Secondary structure
VCD
High pH – helix High pH, heating – sheet Neutral pH - coil
Wavenumber (cm-1)
1600165017001750
Absorbance
0.0
0.5
1.0
DA
x105
-15
-10
-5
0
5
IR
VCD
(c)
VCD of DNA vary A T to G C ratio
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-1
VCD of DNA, vary A-T to G-C ratio
base deformations sym PO2- stretches
big variation little effect
All B-DNA forms
DNA VCD f PO d i B t Z f t iti
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A B
DNA VCD of PO2- modes in B- to Z-form transition
Experimental Theoretical
Z
B B, A
Z
Protein RAMAN & ROA spectra
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800 1000 1200 1400 1600
0
1683ROA
1240
1426
1462
15541299 1342
1641
1665
2.6 x 105
IR-IL
c) hen lysozyme
6.3 x 108
IR+IL
0
1220
13451241
1658
16771295 1316
4.7 x 105
ROA
IR-IL
2.5 x 109
b) jack bean concanavalin A
IR+
IL
0
935
1640
166513001340
4.3 x 105
ROA
0
9.0 x 108
a) human serum albumin
IR-IL
IR+IL
Protein RAMAN & ROA spectra
hSA
Con A
HEWL
I II
ROA sign patterns
stable but
frequencies
shift. Chirality
selects out
amide modes
but Raman
spectra
dominated by
aromatics
Barron data
IR & R I t t ti O tli
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IR & Raman Instrumentation - Outline
• Principles of infrared spectroscopy
• FT advantages
• Elements of FTIR spectrometer
• Acquisition of a spectrum
•
Useful Terminology
• Mid-IR sampling techniques
– Transmission
– Solids
• Raman instrumentation comparison
• (Note—more on sampling variations later)
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Dispersive spectrometers (old) measure transmission as a function
of frequency (wavelength) - sequentially--same as typical UV-vis
Interferometric spectrometers measure intensity as a function of
mirror position, all frequencies simultaneously--Multiplex advantage
Sample
radiation
sourcetransmitted
radiation
Techniques of Infrared Spectroscopy
Infrared spectroscopy deals with absorption of radiation--
detect attenuation of beam by sample at detector
Frequency
selector
detector
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T Nicolet/Thermo drawings
Comparison of IR Methods –
Dispersive & Fourier Transform
But add to this now many laser-based technologies!
N i li d i ill di i IR
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New specialized experiments still use dispersive IR
T/jump IR with
diode laser
Dispersive VCD for Bio Apps
2-D IR setup with 4-wave mixing
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Major Fourier Transform Advantages
• Multiplex Advantage
– All spectral elements are measured at the same time,
simultaneous data aquisition. Felgett’s advantage.
• Throughput Advantage
– Circular aperture typically large area compared to dispersive
spectrometer slit for same resolution, increases throughput.
Jacquinot advantage
• Wavenumber Precision
– The wavenumber scale is locked to the frequency of an internal
He-Ne reference laser, +/- 0.1 cm-1. Conne’s advantage
T i l El t f FT IR
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Typical Elements of FT-IR
IR Source (with input collimator)
–
Mid-IR: Silicon Carbide glowbar element, Tc > 1000
o
C; 200 - 5000 cm
-1
– Near IR: Tungsten Quartz Halogen lamp, Tc > 2400oC; 2500 - 12000 cm-1
IR Detectors:
– DTGS: deuterated triglycine sulfate - pyroelectr ic b olom eter (thermal)
• Slow response, broad wavenumber detection
– MCT: mercury cadmium telluride - photo conduct ing d iode (quantum)
• must be cooled to liquid N2 temperatures (77 K)
• mirror velocity (scan speed) should be high (20Khz)
Sample Compartment
– IR beam focused (< 6 mm), permits measurement of small samples.
– Enclosed with space in compartment for sampling accessories
Interference Moving Mirror Encodes Wavenumber
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Interference - Moving Mirror Encodes Wavenumber
Source
Detector
Paths equal all
n
in phase
Paths vary
interfere vary for
different n
Interferograms for different light sources
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Interferograms for different light sources
Dispersive Raman Single or Multi channel
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Single, double or
triple monochromator
Detector:
PMT or
CCD for
multiplexFilter
Polarizer
Lens
Sample
Laser – n0
Dispersive Raman - Single or Multi-channel
Eliminate the intense Rayleigh
scattered & reflected light
-use filter or double monochromator
–Typically 108 stronger than the
Raman light
•Disperse the light
onto a detector to
generate a
spectrum
Scattered Raman - ns
Synchrotron Light Sources – the next big thing
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Synchrotron Light Sources the next big thing
Broad band, polarized
well-collimated and
very intense
Light beam output
Where e-beam turns
Brookhaven National
Light Source
(and fixed in space!)