18/06/08

Applica'ons  of  
Spectrophotometry  

Video  –  Fluorescence  Microscopy

1
 18/06/08

Beer-­‐Lambert  Law  Revisited

𝐴=𝜀𝑙𝑐  

• The  molar  absorp'vity  (𝜀)  depends  not  only  

on  molecular  species,  but  also  on  wavelength.  

Image  credit:  William  Reusch,    

hJps://www2.chemistry.msu.edu/faculty/reusch/virJxtjml/Spectrpy/UV-­‐Vis/spectrum.htm#uv3  

Analysis  of  a  Mixture  

Amix(λ1)   Amix(λ1)  =  +    
 

Amix(λ2)  =  +    
Amix(λ2)    
AX(λ1)  
AY(λ2)    Obtained  by  measurement  
Two  equa'ons  to  solve  
two  unknowns,    and  !  
AY(λ1)   AX(λ2)  

• The  spectrum  must  not  change  when  the  2  

compounds  are  mixed.  
Image  credit:  David  Harvey,  Chemistry  LibreTexts,  UC  Davis  

2
 18/06/08

Isosbes'c  Point  
Lau  et  al.,  Sensors,  2006  

aA  +  bB  ⇌ cC  +  dD  +  eD  

𝑎𝜀A(λ1)+ 𝑏𝜀B(λ1)  =  
𝑐𝜀C(λ1)+𝑑𝜀D(λ1)+𝑒𝜀E(λ1)  
𝜀X(λ1)  =  𝜀Y(λ1)  

A  proof  that  only  two  major  species  are  present  

Isosbes'c  Point  
Lau  et  al.,  Sensors,  2006  

aA  +  bB  ⇌ cC  +  dD  +  eD  

𝑎𝜀A(λ1)+ 𝑏𝜀B(λ1)  =  
𝑐𝜀C(λ1)+𝑑𝜀D(λ1)+𝑒𝜀E(λ1)  
𝜀X(λ1)  =  𝜀Y(λ1)  

A  proof  that  the  stoichiometry  of  a  reac'on  is  

unchanged  throughout  the  measurement  

3
 18/06/08

The  Method  of  Con'nuous  

Varia'on  
• To  determine  the  stoichiometry  of  the  complex  
Absorbance  only  from  the  product,  PaXb!  
– P  +  X  ⇌ PX  
– P  +  2X  ⇌ PX2  
– P  +  3X  ⇌ PX3  
?
For  aP  +  bX  ⇌ PaXb  
 

Maximum  absorp'on  
occurs  when  the  mole  
frac'on  of  X  =  b  /  (a  +  b)  
D.  Harris,  Quan'ta've  Chemical  Analysis,  9t  ed.  

Immunoassay  
• Chemical  analysis  through  
an'gen-­‐an'body  reac'on  

Reporter  label  

Image  credit:  Nagoya  City  Science  Museum  

4
 18/06/08

Medical  Spectroscopy  
• Visualizing  autofluorescence  from  'ssues  

Hazewinkel  et  al.,  Nat.  Rev.  Gastroenterol.  Hepatol.,  2011  

Cho  et  al.,  Clin.  Endosc.,  2011  

Medical  Spectroscopy  
• Visualizing  autofluorescence  from  'ssues  

• Raman  spectroscopic  detec'on  of  malignant  

'ssues  
 

NOTE:  One  figure  here  is  deleted  

due  to  the  permission  restrict.    
Please  refer  to  the  lecture  video.  
Duraipandian  et  al.,  J.  Biomed.  Opt.,  2012  
hJp://www.bioeng.nus.edu.sg/people/PI/Huangzw/  

5
 18/06/08

Medical  Spectroscopy  
• Visualizing  autofluorescence  from  'ssues  

• Raman  spectroscopic  detec'on  of  malignant  

'ssues  

• Pulse  oximetry  

Nuclear  Magne'c  Resonance  

Spectroscopy
• The  principle:  
1. A  spinning  charge  generates  a  magne'c  moment.  

2. Magne'c  moment  realign  when  an  external  magne'c  

field  is  applied.  

3. Molecules  with  different  alignment  falls    

   at  different  energy  levels.  
ΔE  can  be  probed  by  radio    
frequency  radia'on!
Image  credit:  William  Reusch,    
hJps://www2.chemistry.msu.edu/faculty/reusch/virJxtjml/Spectrpy/nmr/nmr1.htm#nmr1  

6
 18/06/08

Nuclear  Magne'c  Resonance  

Spectroscopy

ΔE  can  be  probed  by  radio    

frequency  radia'on!

Magne'c  Resonance  Imaging  

Short T1

Robia  Pautler,  Physiology,  2004  

7
 18/06/08

Magne'c  Resonance  Imaging  

Short T1

Robia  Pautler,  Physiology,  2004  

Image  credit:  The  University  of  Tokyo  Hospital  

Take-­‐home  Message  

Spectrophotometry  probes  a  wide  

range  of  chemical  proper'es  and  is  
applied  to  various  research  fields!