BioMolecular Vibrational Spectroscopy:

Part 1: Principles of Infrared, Raman

Spectra and Techniques
Lectures for Warwick CD Workshop, Dec. 2011

Tim Keiderling
University of Illinois
at Chicago
tak@uic.edu
T

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 methodssolution, solid
Protein Sampling Techniques (aqueous), ATR

Part II:
Application Examples
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Structural Biology
Optical Spectroscopy is limited for determining
structure lacks site specificity
but often fits important QUESTIONS

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

Electro-Magnetic Spectrum
Electron
Excitation
Wavenumber (cm-1)

107

Spectral
Regions

X-ray

14,285

Near-IR

Electron
Transition
106

105

Molecular
Vibration
104

103

Ultraviolet

Infrared

4,000

400

Mid-IR

Molecular
Rotation
102

10

Microwave
100

Far-IR

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

Optical Spectroscopy - Processes Monitored

UV/ Fluorescence/ IR/ Raman/ Circular Dichroism
Excited
State
(distorted
geometry)

Ground
State (equil.
geom.)

Diatomic Model

Analytical Methods
Absorption UV-vis absorp.
hn = E - E & Fluorescence.
grd

n0 nS

ex

Fluorescence
hn = Eex - Egrd
Raman: DE = hn0-hns
= hnvib

Infrared: DE = hnvib
Q
molec. coord.

move e- (change
electronic state)
high freq., intense

CD circ. polarized
absorption, UV or IR

Raman nuclei,
inelastic scatter
very low intensity

IR move nuclei
low freq. & inten.
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Optical Spectroscopy Electronic,

Example Absorption and Fluorescence
Essentially a probe technique sensing changes in the local environment of fluorophores

What do you see?

(typical protein)

eg. Trp, Tyr

Change with tertiary

structure, compactness

(M-1 cm-1)

Intrinsic fluorophores

Amide absorption broad,

Intense, featureless, far UV
~200 nm and below
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ROA

1340
1300

935

I -I

Optical Spectroscopy - 0IR Spectroscopy

1665

1640

4.3 x 10

I + II + I

Protein and polypeptide secondary structural obtained from

b) jack bean concanavalin A
vibrational modes of amide (peptide bond) groups
LR

a) human serum albumin

Aside: Raman is similar, but different What do you see? LOTS!
amide I, little amide II, intense amide III 2.5 x 109
4

1340

-4

935
0.6

4.7 x 10

Absorbance

0.5

1220
1241

1658
1345

1640

II
III

LR

0.1

6.3 x 10

0.0
2000

1800

1600

1400

1200

1000

1299 Wavenumbers
1342
(cm-11665
)
ROA
9 serum albumin
1554
a)
human
1683
2.5 x 10
1462
1677
1295 1316
ROA
5
8

1426

2.6 x 10
1240
9.0 x 10
800
1200
1400
1340
5 1000 1220
ROA
4.7 x 10935
1300
1241
-1
1345
wavenumber / cm

Goaltry to give this meaning

L

1665

1300

0.2
b) jack bean concanavalin
A

LL

L RR

I - I II -+I I

Amide III
(1300-1230 cm-1)

1677

1295 1316

0.4
c) hen lysozyme
5
4.3 x 10
0.3

I + II + I

Amide II
(1580-1480 cm-1)

0 ROA

-2

L
R

L R

I -I I -I

Amide I
(1700-1600 cm-1)

ROA
8
9.0 x 10

D x 105

1641

1658
1600
1665

Spectroscopic Process (covered)

Molecules contain distribution of charges (electrons and
nuclei, charges from protons) which is dynamically
changed 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 that
emerges from the sample (absorption, scattering, fluorescence,
photochemistry, etc.) so we can detect it

Spectroscopic Process (covered)

Molecules contain distribution of charges (electrons and
nuclei, charges from protons) which is dynamically
changed 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 that
emerges from the sample (absorption, scattering, fluorescence,
photochemistry, etc.) so we can detect it

Quantum mechanical picture

Full Hamiltonian describes electron and nuclear motion
H = - Sab [2/2Maa2 - 2/2mei2 - Zae2/ria + e2/rij + 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) = cu (R) fel (r,R) -- product fct. solves sum H
Electronic Schrdinger Equation issue for CD (done prev.)
Hel fel (r,R) = Uel (R) fe (r,R) electron soln nucl. pot.
Vn(R) = Sab [Uel(R) + ZaZbe2/Rab] nuclear potential energy

Nuclear Schrdinger Equation

Hn cu(R) = -[Sa (2/2Ma) a2 + 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 series expansion:

V(R) = V(Re) + Sab V/RaRe(Ra-Re) +
Sab 2V/RaRbRe(Ra Re)(Rb Re) +

3rd term non-zero / non-const. - harmonic kx2

Ra, Rb mixed Solution Normal coordinates
Qi = Sjcij qj

H = -Si [2/2 2/Qi2+ kQiQi2] = Si hi (Qi)

hi ci(Qi) = Ei ci(Qi) Ej = (uj + ) hnj solve as if independent

Diatomic: n = (1/2p) k/m k force const. m = MAMB/(MA + MB)

Harmonic Oscillator
Model for vibrational spectroscopy
E
re

r
e

v=3

5 hn
2
3 hn
2
1hn
2

v=2

v=1

Ev = (v+)hn
IR
v=0
Dv = 1
DE = hn
n = (1/2p)(k/m)

Raman
9hn
2
7
hn
2

v=4
r

(virtual
state)

hn

re
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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

symmetrical
deformation
(H-O-H bend)

asymmetrical
stretch
H-O-H
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Characteristic vibrations and structure

heavier molecules bigger m - lower frequency
H2 ~4000 cm-1 CH ~2900 cm-1 CD ~2100 cm-1
HF ~4141 cm-1 HCl ~2988 cm-1
F2

892 cm-1 Cl2

564 cm-1 II ~214 cm-1

stronger bonds higher k - higher frequency

CC ~2200 cm-1 C=C ~1600 cm-1 CC ~1000 cm-1
O=O 1555 cm-1 N O 1876 cm-1 NN 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

a

Amide I
(1700-1600 cm-1)

Amide II
(1580-1480 cm-1)

rc
Amide III
(1300-1230 cm-1)

II

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 vj = 0; i j .
These are fundamental 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 (K) (note: kT at room temp ~200 cm-1)
T

Anharmonic Transitions
Real molecules are anharmonic to some degree so other transitions do
occur but are weak. These are termed overtones (D vi = 2, 3, . .) or
combination bands (D vi = 1, D vj = 1, . .). [Diatomic model]
E/De

D0dissociation energy
DE02 = 2hnanhrm - overtone

DE01 = hnanh--fundamental
( r - re )/re

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

~ |<Yf |m| Yi>|2, transition moment between Yi

Yf

To be observed in the IR, the molecule must change its electric

dipole moment, , in the transitionleads to selection rules

d / dQi 0 relatively easy, ex. C=O str. intense

Raman intensity is related to the polarizability,
I ~ <Yb |a| Ya>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

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 molecules absorbance at a given

wavenumber normalized to correct for concentration and pathlength but as
shown can be concentration dependent if molecules interact
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Peak Widths
Water
Water

Benzene

Peak Width is Molecule Dependent

Strong Molecular Interactions = Broad Bands

Weak Molecular Interactions = Narrow Bands
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Atomic resolution

Ca chain

Structural
Biology
Level of structure
determination needed
depends on the
problem

Secondary structure

Segment 23
fold (tertiary)

Chain conformation depends on f, y angles

If (f,y) repeat, they determine secondary structure

Polymer analysis
Study the repeat units

Detection requires method sensitive to amide coupling

Far UV absorbance broad, little fluorescencecoupling impact small

Physical method of detection must sense

secondary structure e.g. couple amides
IR/Raman coupling comparable to band width, intensity
maximum is characteristic of structure frequency basis

Circular dichroism --dipole and through-bond chiral coupling of

local modes (excitations) circularly polarized transitions,
DA = AL-AR - Develops characteristic band shapes (intensity)

Theoretically try to understand spectra/structure relation

IR ~ D=m.m~|dm/dQ|2 (Raman ~ |da/dQ|2) Major activity,
ECD, VCD ~ R = Im(m.m)
for analysis!

Computable with ab initio QM techniques, ECD needs excited states

IR & VCD relatively easy, Raman more basis set sensitive

Characteristic Amide Vibrations

~3300 cm-1

A often obscured
by solvent
I - Most useful;

~1650 cm-1 IR intense, less interference

(by solvent, other modes,etc)
Less mix (with other modes)

1500-50 cm-1 II - IR intense

mix
1300-1250 cm-1

700 cm-1

III - Raman Intense

IV VII difficult
to detect, discriminate
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absorbance spectra of selected model peptides

Model polypeptide IR spectra -- Amide I and II

Absorbance

Helixsmall frequency
dispersion, central ones
most intense, amide I,
higher ones for amide II

helix

II

b-structure

Sheetlarge frequency
dispersion, characteristic
split amide I, broad amide II

1
random
coil

Coilless well-defined
broad amide I and II

0
1750 1700 1650 1600 1550 1500 1450
-1

Frequency based

Wavenumbers (cm )

Differentiation of conformations mostly due to coupling of amides

not to H-bonds or other factors, although they contribute

Monitoring structural change - temperature

Temperature dependent IR
spectra of the helical peptide

Temperature dependence of
amide I frequency
unfolded

folded

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

Tyr92
Tyr115

Tyr97

Tyr73
H1
H2

Ribonuclease A
combined
uv-CD and
FTIR study

H3
Tyr25

Tyr76

Simona Stelea,
Prot Sci 2001

124 amino acid residues, 1 domain, MW= 13.7 KDa

3 a-helices

6 b-strands
in6an

b AP b-sheet b sheet
6 Tyr residues (no Trp), 4 Pro residues, (2
2 cis,) 2 trans)
29
Optical spectra senses dynamic equilibrium - unstructured
systems

0.06

FTIR

Absorbance

0.05

Ribonuclease A

0.04
0.03
0.02

FTIRamide I

0.01

Loss of b-sheet

0.00

1720

1700

1680

1660

1640

1620

1600

Wavenumber (cm-1)
0

Ellipticity (mdeg)

-2

Near uv CD

-4
-6
-8

Loss of tertiary struct.

-10
-12
-14

Near-UV CD

-16
260

280

300

320

Wavelength (nm)

Far-uv CD

Ellipticity (mdeg)

Loss of a-helix

-5

-10

Far-UV CD
-15
190

200

210

220

Wavelength (nm)

230

240

250

Spectral Change
Temperature
30
10-70oC

Ribonuclease A

-6.4

1.0

Ci1 (x102)

0.5

-7.2

0.0

-7.6

-0.5

-8.0

-1.0

-5

PC/FA loadings
Temp. variation
Ci2 (x10)

FTIR

-6.8

FTIR (a,b)

10

-7
5

-5

-13
-15

-10

-17

-15

-10

5
0

-11

-5

Far-UV CD

Ci1

Near-uv CD
(tertiary)

Ci2

Near-UV CD

-11

-10

-12

-15

Far-uv CD
(a-helix)

Ci2

Ci1

-9

-20
-13

-25

Temp.
Pre-transition evident in far-uv CD and FTIR, 31
not near-uv CD
0

20

40

60

80

-30
100

Temperature (oC)

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

Other biopolymers
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

Combining Techniques: Vibrational CD

CD in the infrared region
Vibrational chirality Many transitions / Spectrally resolved / Local
Technology in place DA ~10-5 - limits S/N / Difficult < 700 cm-1
Same transitions as IR
same frequencies, same resolution
Band 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,
Recentpeptide VCD can be simulated theoretically

Poly Lysine in D2O Amide ISecondary structure

VCD
2

10

(b)

(a)

0
VCD

0
-5

VCD

-4
-15

1.0

1.0

Absorbance

IR
Absorbance

(c)

-10

-10

0.5

0.0
1750

VCD

1.0

IR

IR
Absorbance

-5

-2

DA x 105

DA x 105

DA x 105

0.5

0.0
1700

1650

1600

Wavenumber (cm-1)

High pH helix

1750

1700

1650

1600

0.5

0.0
1750

1700

1650

1600

Wavenubmer (cm-1)

Wavenumber (cm-1)

High pH, heating sheet

Neutral pH - coil

VCD of DNA, vary A-T to G-C ratio

base deformations

sym PO2- stretches

-1

big variation

little effect

All B-DNA forms

DNA VCD of PO2- modes in B- to Z-form transition

B, A

Z
A

Experimental

Theoretical
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Protein RAMAN & ROA spectra

III

hSA

I +I

a) human serum albumin

9.0 x 10

1340
935

1665

1300

I -I

ROA
0

1640

4.3 x 10

Con A

I +I

b) jack bean concanavalin A

2.5 x 10

1295

1677

1316

I -I

ROA
0

1220
1241

4.7 x 10

1658
1345

c) hen lysozyme

I +I

HEWL

6.3 x 10

1299

ROA

1462

1665
1683

I -I

1342
1554

ROA sign
patterns
stable but
frequencies
shift.
Chirality
selects out
amide modes
but Raman
spectra
dominated by
aromatics
Barron data

1426

2.6 x 10
800

1641

1240

1000

1200

1400

1600

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
(Notemore on sampling variations later)

Techniques of Infrared Spectroscopy

Infrared spectroscopy deals with absorption of radiation-detect attenuation of beam by sample at detector
radiation
source

Frequency
selector

transmitted
radiation

detector

Sample

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
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Comparison of IR Methods
Dispersive & Fourier Transform

But add to this now many laser-based technologies!

Nicolet/Thermo drawings

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

Major Fourier Transform Advantages

Multiplex Advantage
All spectral elements are measured at the same time,
simultaneous data aquisition.
Felgetts 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. Connes advantage

Typical Elements of FT-IR

IR Source (with input collimator)
Mid-IR: Silicon Carbide glowbar element, Tc > 1000oC; 200 - 5000 cm-1
Near IR: Tungsten Quartz Halogen lamp, Tc > 2400oC; 2500 - 12000 cm-1

IR Detectors:
DTGS: deuterated triglycine sulfate - pyroelectric bolometer (thermal)
Slow response, broad wavenumber detection
MCT: mercury cadmium telluride - photo conducting diode (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
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Interference - Moving Mirror Encodes Wavenumber

Source

Detector

Paths equal all

n in phase
Paths vary
interfere vary for
different n
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Interferograms for different light sources

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

Polarizer
Sample

Lens

Filter

Scattered Raman - ns
Laser n0

Detector:
PMT or
CCD for
multiplex
Single, double or
triple monochromator
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Synchrotron Light Sources the next big thing

Brookhaven National
Light Source

Broad band, polarized

well-collimated and
very intense
(and fixed in space!)
Light beam output
Where e-beam turns