Chem 257 Lecture for Exp. 8, 2004 1. Exp. 7 Report: To be handed in this week, along with: • Relevant notebook pages • Vial containing triphenyl carbinol labeled with your notebook code, i.e., JST-I-38a 2. Exp. 8 Quiz. Be prepared for Quiz. 3. Required Reading. Lab Manual 8.1-10. 4. Relevant Material in Wade (5th): Ultraviolet spectroscopy 15-13 (p 666-672), 16-15 p(711-713), 18-5E (p 785-6). Friedel Crafts Alkylation: 17-11 (p 746-748) & p 980. Exp. 8. Fluorescence and Chemiluminescence. Purpose of the experiment: 1. Synthetic Organic Chemistry • Acquaint you with Friedel-Crafts acylation chemistry 2. Physical Organic Chemistry • Introduce you to photophysics • Acquaint you with chemiluminescent reactions 1 Photochemical Terms and Principles. Useful to consider a ground and excited state energy diagram for an organic molecule. Energy Level Diagram ISC S = singlet S T = triplet 1 A = Absorption F = Fluorescence T IC 1 P = Phosphorescence A ISC = Intersystem crossing E F P IC = Internal Conversion ISC Absorption of a photon Emmission of a photon Vibrational energy loss So Singlet State (S) • Spin paired. • Ground state usually a singlet (So). • First excited singlet state = S1 So S1 • Nth excited singlet state = S n ground excited Triplet State (T) • Spin unpaired. • Lowest triplet state is usually an excited state (T1). • Nth excited triplet state = Tn T T Exceptions: 0 1 • Oxygen is a ground state Ground excited triplet! (O2) 2 Selection rules. • Spin allowed transitions do not involve a change in spin state (Sn → Sm, Tn → Tm). • Thus ground singlet states are most easily excited to their first excited singlet states (So → S1). • Changing between spin states is called intersystem crossing and is spin forbidden and inefficient (Sn→ Tm , Tn → Sm) A ISC P F, IC ISC S So S1 T1 o Excited State Lifetimes • The lifetime refers to the amount of time that a particular state exists. • Excited singlet states of organic molecules (spin allowed transition S1→So,) usually exist for on the order of nano (10-9 s) to microseconds (10-6 s) • Excited triplet states (spin forbidden transition T1 -3 →So) can last for milliseconds (10 s) or more at room temperature. A ISC P F, IC ISCIC S So S1 T1 o 3 Intersystem crossing (ISC). Crossing from one spin state to another, usually from the first ISC excited singlet state to the S1 lower energy first excited triplet state. Or T1 to So T1 Internal conversion (IC). E (Radiationless decay). A IC F Molecules in an excited ISC P state can go to a lower state of the same spin by loss of energy through S0 vibrational energy, and ultimately heat. Fluorescence (F). • Molecules in their 1st excited singlet state can loose their energy by loss of a photon. ISC • Because the excited S1 singlet state usually has time to relax to its T1 ground vibrational state, E fluorescence emission A IC F maximum is usually at a ISCIC P longer wavelength than its absorption maximum. S0 • Very short lifetime because spin allowed transition, singlet to singlet). 4 Frank Condon Principle • Excitation occurs faster S1 than a vibration (no change in geometry) S • Occurs from most E o hνA probable geometry of the ground state • Occurs from lowest vibrational state • Occurs from equilibrium bond geometry (center of potential curve) • Goes to vibrational level of the excited state with the highest probability of having Intensity the same geometry • Goes to a turning point Wavelength (edge of potential curve) Frank Condon Principle S1 • Emmission occurs faster than a vibration (no change in geometry) E So • Occurs between lowest hνE vibrational state of the excited electronic state • Occurs from equilibrium bond geometry (center of potential) • Goes to the vibrational state with the greatest probability of having same geometry (turning point) Intensity Wavelength 5 Frank Condon Principle S1 • Net result: Emmission occurs at a longer wavelength (lower E) than E So hνA absorption hνE • Emmission maximum is red-shifted compared to excitation • Thus lower wavelength light (UV) that is invisible to the naked eye can be converted to visible light by the process of fluorescence Intensity Wavelength Color of Emitted Light. (source Louden, pg 661) • Our eyes can only detect wavelenghts in the 400- 700 nm range (visible light) • Sunlight at sea level consists of wavelengths from 290 nm and up Range Type wavelength color <200 vacuum UV 400-420 violet 200-280 UVC (germicidal) 420-491 blue 280-320 UVB (tanning) 491-575 green 320-400 UVA (black light) 575-585 yellow 400-700 visible 585-647 orange 700- IR 647-700 red 6 Fluorescent Compounds for Home and Office Use • Optical brighteners (in clothes and computer paper) • Fluorescent compounds that absorb the <400 nm light in sunlight or fluorescent lights which are invisible to you • The absorbed light is re-emitted as visible light • Your clothes and term papers really are brighter! HO N H - O3S N N N H N N H N NSON - 3 Blankophor B H N optical brightner OH A Common Medicinal Fluorescent Compound • Quinine is an antimalarial alkaloid used to impart bitter taste to tonic water (gin and tonic a pleasing type of therapy) • Tonic water contains quinine which is fluorescent and will glow under black light (365 nm) • Black light derives its name because you cannot see it. • The blueish green glow from the excited quinine goes off instantly when the black light is turned off, as expected for fluorescence OCH3 N absorption maximum 335 nm emission maxium 370 nm with a H tail out to 460 nm and a OH lifetime 10 nsec H N 7 Fluorescent Labels and Indicators for Biology • Fluorescein is used to label DNA and proteins • Fluorescein dipivalate is used as a probe of living cells O esterases O lipases O Na + H2O O proteases O O CH3 CH OOO 3 Na OOO CH 3 CH3 CH3 CH3 fluorescein dipivalate basic form of fluorescein (not fluorescent) (fluorescent) Membrane permeable Membrane inpermeable hydrolyzed in cell trapped inside cell Green Fluorescent Protein (GFP) • Chemical reaction of a protein leading to a fluorescent protein which emits green light • Requires oxygen • Used to tag proteins in cells by making fusion proteins and observing by fluorescence microsopy • Unsaturation shifts emmission wavelength to the visible -H2O O -H2 -Phe-Ser-Tyr-Gly-Val- N Val69 N 64 69 O HO N H colorless Phe 64 GFP Chromophore 8 Phosphorescence (P). • Molecules in their 1st excited triplet state can loose their energy by loss of a photon. ISC • Because the excited S1 triplet state is usually lower than the singlet state, T1 phosphorescence E A maximum is usually much IC F ISCIC P longer than the fluorescence emission maximum. S0 • Very long lived because it is spin forbidden (triplet to singlet). Phosphorescent Paints and Objects • Contain a phophorescent compound • Typically ZnS doped with Cu • First you must expose object to light to get a lot of the phosphorescent compounds into the excited state • The phophorescence does not go off instantaneously when the light is turned off, but can take minutes 9 Chemical Reaction. Excited states can also lead to bond dissociation, bond rearrangements, and reactions with other molecules. Quantum Yield. • The quantum yield is a measure of efficiency of a particular event • Expressed as the fraction of the original photon absorbed that results in the particular event. • A quantum yield of 1 for a particular event, indicates that one of the particular event occurs for every photon absorbed. Fluorescence Resonance Energy Transfer (FRET). • Exchange of excited state energy between two molecules through dipolar mechanism at close distances • Allowed, as there is no change in spin state • Acceptor (A) must have a lower energy excited state than the donor (D) • Emitted light (FD) is of lower energy (lower frequency) than the light emitted from the donor (FA) • Used by biologists to detect interactions between biomolecules D D A D A A A D FRET FA FD So S So S1 So S1 o 10 Use of FRET to detect specific mRNA sequence In absence of complementary Donor Acceptor mRNA green emmission observed In presence of FRET complementary mRNA FRET Donor Acceptor occurs and yellow emmission observed mRNA Chemiluminescence. • Emission of a light as a result of a chemical reaction. • Occurs when the ground state of one molecule crosses with the excited state of a re-arranged or fragmented molecule. (Opposite of fluorescence where molecule goes from excited singlet state to ground state). S1 ∆G S0 hν Reaction Coordinate 11 Common Commercial Use of Chemiluminescence. • Light sticks. Last 12 h at RT, days in freezer (not as bright). Cyalume by American Cyanamide. Quantum yield of 25%. • Labeling biomolecules O O * Cl Cl O Cl O + H2O2 2 C O OO O O O Cl + Cl Cl * energy transfer Fluorescence light Bioluminescence • Emission of a photon as a result of a chemiluminescent reaction catalyzed by an enzyme in nature Glowing bacteria V. harveyi bacteria glowing in colonizing a squid response to signals from E. coli (top) or salmonela (bottom) 12 Luciferin - Luciferase System • Light emitting system of the firefly • Requires an organic substrate luciferin, oxygen and the enzyme luciferase • Used commercially in non-isotopic biomolecule detection systems O excite state Luciferase N N 2+ N N O* OH O2, Mg , ATP S S HO HO S S luciferin + CO2 O N N O O HO S S Exp. 8. Fluorescence and Chemiluminescence. Goals of Your Experiment: • To synthesize a widely used fluorescent molecule, fluorescein by a Friedel Crafts acylation reaction • To examine the pH dependence of fluorescein fluorescence and correlate with structure • To study the chemiluminescent