Chlorophyll( - University of California,...

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Chlorophyll Analysis & Extrac/on Chlorophyll a

Transcript of Chlorophyll( - University of California,...

Page 1: Chlorophyll( - University of California, Irvinefaculty.sites.uci.edu/.../04/A07DISCChlorophyllwk1and2.pdf · 2015-11-09 · Yellow(&(Blue(! Sketch!blue!food!coloring’s! absorp/on!spectrum:!!

ì  Chlorophyll  Analysis    &  Extrac/on  

Chlorophyll  a  

Page 2: Chlorophyll( - University of California, Irvinefaculty.sites.uci.edu/.../04/A07DISCChlorophyllwk1and2.pdf · 2015-11-09 · Yellow(&(Blue(! Sketch!blue!food!coloring’s! absorp/on!spectrum:!!

Week  1  Experiment  Summary  

ì  Absorbance  (A)  Measurements  

 &   Food  Coloring  

 Standard  

ì  λmax  Comparisons  

ì  Beer’s  Law  to  find  ε

ì  Fluorescence  (F)  Measurements  

 Standard  

ì  Standards  by  Serial  Dilu/on    

ì  Calibra/on  Curve  (similar  to  BL  Plot)  

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Yellow  &  Blue  

ì  Sketch  blue  food  coloring’s  absorp/on  spectrum:  

Absorbance  

Wavelength  (nm)  

400   750  

400  nm  

430  nm  

480  nm  

560  nm  

590  nm  

630  nm  

750  nm  

ì  Sketch  yellow  food  coloring’s  absorp/on  spectrum:  

Absorbance  

Wavelength  (nm)  

400   750  

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Why  are  plants  green?  

ì  Plants  contain  chlorophyll,  which  is  green.      

ì  Sketch  chlorophyll’s  absorp/on  spectrum:  

Absorbance  

Wavelength  (nm)  400   750  

400  nm  

430  nm  

480  nm  

560  nm  

590  nm  

630  nm  

750  nm  

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   Stoke’s  Shift  

ì  What  is  the  rela/onship  between  the  fluorescence  and  absorp/on  spectra  of  the  same  chemical?  

ì  The  spectrometer  used  in  lab  has  2  excita<on  wavelengths:  405  nm  &  500  nm.    Which  should  be  used?  

HOMO

LUMO

Energy

hνa hνe

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Concentration  Units  (ppm)  

ì  If  an  aqueous  solu/on  is  6.0%  (pph)  by  mass  NaCl,  what  is  the  concentra/on  of  the  solu/on  in  Molarity?    

ì  6.0  g  NaCl  x  1  mol  NaCl  x  1.0  g  H2O  x    103  mL  =  1.0  M              100  g  H2O          58.44  g                          1  mL                          1L  

ì  If  an  aqueous  solu/on  is  6.0  ppm  by  mass  NaCl,  what  is  the  concentra/on  of  the  solu/on  in  Molarity?    

ì  6.0  g  NaCl  x  1  mol  NaCl  x  1.0  g  H2O  x    103  mL  =  1.0  x  10-­‐4  M              106  g  H2O                58.44  g                  1  mL                              1L  

ì  What  assump/on  was  made  in  this  calcula/on?  Vwater  =  Vsoln  

Page 7: Chlorophyll( - University of California, Irvinefaculty.sites.uci.edu/.../04/A07DISCChlorophyllwk1and2.pdf · 2015-11-09 · Yellow(&(Blue(! Sketch!blue!food!coloring’s! absorp/on!spectrum:!!

Fluorescence  Calibration  Curve  

ì  Chlorophyll  stock  solu/on:  6.0  ppm.    Create  5  standard  solu/ons,  each  ½  the  concentra/on  of  the  solu/on  before  it.    Direc<ons:    Add  2.0  mL  the  chlorophyll  stock/standard  solu/on  to  a  10  mL  graduated  cylinder,  then  add  2  mL  of  heptane.    

ì  What  does  a  fluorescence  calibra/on  curve  will  look  like?  

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Week  2  Experiment  Summary  

ì  Spinach  Extrac/on  ì  Grind  w/  acetone,  add  heptane,  then  water.    Separate  layers  &  

add  MgSO4  to  heptane  layer  

ì  TLC  of  Extract  ì  Developing  Chamber  ì  Rf  Calcula/on  

ì  Absorbance  &  Fluorescence  Measurement  of  Extract  ì  Determina/on  of  [Chlorophyll]  from  A  &  F  data  

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Which  of  the  following  is  false?  

A.  Electronega/vity  is  an  atom’s  ability  to  ahract  shared  electrons  to  itself.  

B.  A  bond  dipole  arises  when  a  electronega/vity  difference  exists  between  two  atoms.  

C.  A  significant  bond  dipole  exists  between  C-­‐H  in  heptane.  

D.  CCl4  has  a  molecular  dipole.  

Page 10: Chlorophyll( - University of California, Irvinefaculty.sites.uci.edu/.../04/A07DISCChlorophyllwk1and2.pdf · 2015-11-09 · Yellow(&(Blue(! Sketch!blue!food!coloring’s! absorp/on!spectrum:!!

Intermolecular  Forces  

ì  Dipole-­‐Dipole  Forces  ì  electrosta/c  ahrac/on  between  the  par/ally  posi/ve  &  nega/ve  ends  of  

polar  molecules  

ì  Hydrogen  Bonding  ì  dipole-­‐dipole  forces  for  polar  molecules  with  hydrogen  atoms  bound  to  

oxygen,  nitrogen,  or  fluorine  atoms.  

ì  Dispersion  Forces  ì  Transitory  dipole  forma/on:  random  e-­‐  shij  results  in  δ+  &  δ-­‐  

ends  of  a  molecule/atom    ì  Disturbing  other  molecules/  atoms  to  form  more  transitory  

dipoles  &  weak  electrosta/c  ahrac/on  

Page 11: Chlorophyll( - University of California, Irvinefaculty.sites.uci.edu/.../04/A07DISCChlorophyllwk1and2.pdf · 2015-11-09 · Yellow(&(Blue(! Sketch!blue!food!coloring’s! absorp/on!spectrum:!!

Which  of  the  following  is  false?  

A.  Hydrogen  bonding  occurs  between  molecules  containing  C-­‐H  bonds.  

B.  Dipole  Dipole  forces  exist  between  water  molecules.  

C.  Dispersion  Forces  exist  between  acetone  molecules.  

D.  Intermolecular  forces  between  nonpolar  molecules  decrease  with  molecular  weight.  

Page 12: Chlorophyll( - University of California, Irvinefaculty.sites.uci.edu/.../04/A07DISCChlorophyllwk1and2.pdf · 2015-11-09 · Yellow(&(Blue(! Sketch!blue!food!coloring’s! absorp/on!spectrum:!!

What  chemicals  are  in   ?  

ì  Chlorophyll  a  &  b  

ì  Pheophy/n  a  &  b  

ì  Xanthophylls  

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Extraction  /  Separatory  Funnels  

ì  How  do  you  use  a  separatory  (sep)  funnel?  

ì  What  is  the  iden/ty  of  the  layers?  

ì  Which  layer  contains  “  ”?  

ì  What  is  MgSO4  used  for?  

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Thin  Layer  Chromatography  (TLC)  

ì  What  is  the  TLC  process?  

ì  Developing  Chamber  

ì  Prep  &  Spot  Plate  

ì  Develop  Plate  

ì  Measure  Rf  =  xorigintocpd                                                      xorigintofront  

What  are  the  Rfs  for  A,  B,  &  C?      Does  the  size  of  plate  maHer?  

Page 15: Chlorophyll( - University of California, Irvinefaculty.sites.uci.edu/.../04/A07DISCChlorophyllwk1and2.pdf · 2015-11-09 · Yellow(&(Blue(! Sketch!blue!food!coloring’s! absorp/on!spectrum:!!

3  Classes  of  “Chemicals”  Silica  SiO2  

ì  Phase?  Sta1onary  

ì  Polarity?  

POLAR  Si-­‐O-­‐H  (silanol)  groups    on  outside  of  silica  par/cles      

Eluent  

ì  Phase?    Mobile  

ì  Polarity?  NONPOLAR  to  intermed.  polar    Eluent  use  wrt  polarity?  in  order  of  ñpolarity  

Use  order  of  these  eluents?acetone,  CH3(CH2)4CH3,  CH2Cl2,  CH3CH2OCH2CH3  

Analyte  

ì  Defini/on?  Mixture  of  2  or  more  chemicals  to  be  separated  

ì  Polarity?  

NONPOLAR  to        intermediate  polar  

 

Page 16: Chlorophyll( - University of California, Irvinefaculty.sites.uci.edu/.../04/A07DISCChlorophyllwk1and2.pdf · 2015-11-09 · Yellow(&(Blue(! Sketch!blue!food!coloring’s! absorp/on!spectrum:!!

How  does  TLC  separate  these  chemicals?  

By  Polarity…  

ì  Is  A  polar,  intermediate  polar,  or  nonpolar?  

ì  Which  chemical  is  the  most  strongly  adsorbed  to  the  silica  plate?    What  can  be  said  about  its  polarity?  

ì  Which  eluent  is  most  likely  responsible  for  the  separa/on  seen  to  the  right:  pure  acetone,  1:1  mix  of  acetone  &  heptane,  pure  heptane?      

A  B  

C  

D  

Page 17: Chlorophyll( - University of California, Irvinefaculty.sites.uci.edu/.../04/A07DISCChlorophyllwk1and2.pdf · 2015-11-09 · Yellow(&(Blue(! Sketch!blue!food!coloring’s! absorp/on!spectrum:!!

What  is  [Chlorophyll  a]  in   ?  

ì  Remember:  The  extract  is  a  mix  of  compounds…  the  standard  only  contains  chlorophyll!  

Use  the  data  from  week  1  to  find  [chlorophyll  a]:  

ì  from  Absorbance…    

ì  from  Fluorescence…  

ì  Which  method  is  beHer  to  calculate  concentra<on?  

Beer’s  Law  Eqn:  A  =  εCl  

Calibra1on  Curve  

at  2  λs…    low  &  high