Chlorophyll( - University of California,...
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ì Chlorophyll Analysis & Extrac/on
Week 1 Experiment Summary
ì Absorbance (A) Measurements
& Food Coloring
ì λmax Comparisons
ì Beer’s Law to find ε
ì Fluorescence (F) Measurements
ì Standards by Serial Dilu/on
ì Calibra/on Curve (similar to BL Plot)
Yellow & Blue
ì Sketch blue food coloring’s absorp/on spectrum:
ì Sketch yellow food coloring’s absorp/on spectrum:
Why are plants green?
ì Plants contain chlorophyll, which is green.
ì Sketch chlorophyll’s absorp/on spectrum:
Wavelength (nm) 400 750
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?
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
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?
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
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.
ì Dipole-‐Dipole Forces ì electrosta/c ahrac/on between the par/ally posi/ve & nega/ve ends of
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
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.
What chemicals are in ?
ì Chlorophyll a & b
ì Pheophy/n a & b
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?
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?
3 Classes of “Chemicals” Silica SiO2
ì Phase? Sta1onary
POLAR Si-‐O-‐H (silanol) groups on outside of silica par/cles
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
ì Defini/on? Mixture of 2 or more chemicals to be separated
NONPOLAR to intermediate polar
How does TLC separate these chemicals?
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?
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
at 2 λs… low & high