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Absorption Spectroscopy

Topic 4 (abridged)
Biophysics
Energy and Absorption
 2 t 2 sin 2  E b  E a  w  t 
~
 b   a  E0     2
 
Pb  C b 
2
2
2  E b 
 w  t 
Ea
2 
    2

• Absorption is
proportional to
~ 
 a   b  E0

• Get resonance at hf
= ħw = Eb – Ea
www.andor.com/chemistry/?app=189
Absorption Broadening
•Every electronic state
(eg S0 and S1) has
several vibrational and
rotational substates.
•Each of the sharp peaks
is further broadened by
Doppler shifts and
solvent effects and
quantum probabilities.
pubs.rsc.org/ej/DT/2000/B002123G/
Extinction Coefficient
monochromator l I
I0
Sample with
Light Source pathlength l Detector
Or
l I1
I0
Beam Reference
monochromator Detector
Splitter

Light Source l I2
I0
Sample with
pathlength l Detector

A(l) = log (I0/I)


A(l) = Ce(l)l
Biopolymer absorption

Protein Secondary Structure Protein Aromatics

Blood
http://life.nthu.edu.tw/~lslpc/biophys/hyperchorism.gif
Chemical Kinetics
Zero Order
1.40E+00
1.20E+00
1.00E+00 Reaction of nitrite

Absorption
C 8.00E-01
6.00E-01
A
B
with
4.00E-01 deoxyhemoglobin
2.00E-01
0.00E+00
450 500 550 600 650 700
Wavelength (nm)

• Rate independent of concentrations


• -dC/dt = k
• C(t) = C0 – kt
Chemical Kinetics
First Order
ln(CA)

t
k
A  Z
• -dCA/dt = kCA , CA = CA0 e-kt
• t1/2 = ln(2)/k; t = 1/k = lifetime NO binding to Hb
Chemical Kinetics
Second Order
AB
 Pr oducts k

• -dCA/dt = -dCB/dt = kCACB


 CA 
ln  ( )  C0A
  CA  CB kt  ln 
0 0 
0 
 CB   C B

• Make one species in excess so get pseudofirst


order kinetics, kobs = kCB so
CA = CA0 exp(-kobst)
Hemoglobin

 Cooperative Binding of
Oxygen
Linked to quaternary
structure
Explained by MWC Model
On the Nature of Allosteric
Transitions:
A Plausible Model
Jacques Monod, Jeffries Wyman,
Jean-Pierre Changux
J. Mol. Biol. 1965
Time Resolved Optical Density
TROD
Stopped-flow Pump-probe

http://fig.cox.miami.edu/~cmallery/255/255enz/ecb3x28.jpg

Perturb System, measure change


in absorption over time.
SVD and data analysis
(eg. for Hofrichter paper)
Basis Specta Time Course of Basis
Specta

www.math.tau.ac.il/~nin/Courses/BioInfo04/svd.ppt

• Truncated X (or A) data matrix is fit to exponentials or other model


• Get noise filtering
Circular Dichroism and
Optical Rotary Dispersion
CD ORD

http://upload.wikimedia.org/wikipedia/com
mons/thumb/d/d1/Circular_dichroism.png/6
22px-Circular_dichroism.png

• CD is the differential absorption of circularly


polarized light
• ORD rotates linearly polarized light (and other)
• Both result from chiral structures as can be used
as structural probes (eg Protein 2ndary Structure)
Polarized Absorption
 1  LD  LD' CD 
  LD 1 CB LB ' 
M  eA  
 LD'  CB 1  LB 
 
 CD  LB ' LB 1 
A  ln 10 ( Ah  Av ) / 2,
LB  2  ( nh  nv )l / l , LB'  2  ( n  n )l / l , CB  2  ( nl  nr )l / l ,
LD  ln 10 ( Ah  Av ) / 2, LD'  ln 10 ( A  A ) / 2, CD  ln 10 ( Al  Ar ) / 2,

Arrangements can be made to measure elements directly, using PEM or


using ellipsometric techniques.
Ellispometric Technique

• Key is crossed (or nearly crossed) polarizers as


in this schematic for TRORD.
• Used for protein folding (Goldbeck paper).