ì Chlorophyll Analysis & Extrac/on Mg2+
Chlorophyll a Week 2 Experiment Summary ì  Spinach Extrac/on ì  Grind w/ acetone, add hexane, then water. Separate layers & add MgSO4 to hexane 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 aMract 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. Polarity Continuum δ+ δ-­‐ δ+ δ-­‐ δ+ δ-­‐ δ+ δ-­‐ + H-­‐H < H-­‐S < H-­‐Cl < H-­‐O < H-­‐F < Na -­‐ F ΔEN (2.1)(2.1) (2.1)(2.5) (2.1)(3.0) (2.1)(3.5) (2.1)(4.0) (0.9)(4.0) Dipole-­‐Dipole Forces ì  Defini/on: electrosta/c aMrac/on between the par/ally posi/ve & nega/ve ends of polar molecules ì  Energy compared to a covalent bond? Edip-­‐dip = 1 kJ/mol (~1% covalent bond energy) ì  Sketch the dipole-­‐dipole force that exists between two molecules of acetone. Hydrogen Bonding ì  Defini/on: dipole-­‐dipole forces for polar molecules with hydrogen atoms bound to oxygen, nitrogen, or fluorine atoms. ì  Energy compared to a covalent bond? EHbond = 10 kJ/mol (~5% covalent bond energy) ì  Sketch the hydrogen bonding force that exists between two molecules of water. London Dispersion Forces Also known as (aka): Induced Dipole Forces, van der Waals Forces ì  Defini/on (What happens?): ì  Transitory dipole forma/on: random e-­‐ shih results in δ+ & δ-­‐ ends of a molecule/atom ì  Disturbing other molecules/ atoms to form more transitory dipoles & weak electrosta/c aMrac/on ì  Sketch the Dispersion force that exists between four molecules of propane. (You’ll need 2 steps.) 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. Why is Chlorophyll’s structure special? Head ì  unsaturated & Tail conjugated ì  nonpolar ì  planar ì  saturated ì  intermediate polarity What chemicals are in ì  Chlorophyll a & b ì  Pheophy/n a & b ì  Xanthophylls ? 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 ma2er? 3 Classes of “Chemicals” Silica SiO2 ì  Phase? Sta0onary ì  Polarity? POLAR Si-­‐O-­‐H (silanol) groups on outside of silica par/cles Eluent Analyte ì  Phase? Mobile ì  Defini/on? Mixture of 2 or more chemicals to be separated ì  Polarity? NONPOLAR to intermed. polar Eluent use wrt polarity? in order of ñpolarity ì  Polarity? NONPOLAR to intermediate polar Use order of these eluents?
acetone, CH3(CH2)4CH3, CH2Cl2, CH3CH2OCH2CH3 Explain your answer. 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 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… at 2 λs… Beer’s Law Eqn: A = εCl low & high ì  from Fluorescence… Calibra0on Curve ì  Which method is be2er to calculate concentra=on?