Tak Warrick.12 2011 Vib Princ Final

8/18/2019 Tak Warrick.12 2011 Vib Princ Final

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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

[email protected]


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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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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!)

Tak Warrick.12 2011 Vib Princ Final

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