BioMolecular Optical Spectroscopy: Part 1: Infrared And Raman .

BioMolecular Optical Spectroscopy:Part 1: Infrared and RamanVibrational Spectra BackgroundSpecial Lectures for Chem 344Fall, 2007Tim KeiderlingUniversity of Illinois at Chicagotak@uic.eduT

Vibrational Spectroscopy - Biological ApplicationsThere are many purposes for adapting IR or Ramanvibrational spectroscopies to the biochemical,biophysical and bioanalytical laboratory Prime role has been for determination of structure. We willfocus early on secondary structure of peptides and proteins,but there are more – especially DNA and lipids Also used for following processes, such as enzyme-substrateinteractions, protein folding, DNA unwinding More recently for quality control, in pharma and biotech New applications in imaging now developing, here sensitivityand discrimination among all tissue/cell components are vitalT

Spectral Regions and Transitions Infrared radiation excites molecular vibrations, i.e.stretching of bonds and deformation of bond angles.Molecule has 3N-6 internal degrees of freedom, N atoms.States characterize the bound ground state. Radiation in the visible (Vis) and ultraviolet (UV) regions ,will excite electrons from the bound (ground) state tomore weakly bound and dissociative (excited) states,involving valence electrons.– Changes in both the vibrational and rotational states of themolecule can be associated with this, causing the spectra tobecome broadened or have fine structure. Radiation in vacuum UV and x-ray correspond tochanges in electronic structure to either very highexcited states or from core electrons (ionization)T

Optical Spectroscopy - Processes MonitoredUV/ Fluorescence/ IR/ Raman/ Circular e (equil.geom.)Diatomic ModelAnalytical MethodsAbsorption UV-vis absorp.hν E - E & Fluorescence.grdν0νSexFluorescencehν Eex - EgrdRaman: E hν0-hνs hνvibInfrared: E hνvibQ0 molec. coord.move e- (changeelectronic state)high freq., intenseCD – circ. polarizedabsorption, UV or IRRaman –nuclei,inelastic scattervery low intensityIR – move nucleilow freq. & inten.T

Spectroscopy Study of the consequences of the interaction of electromagneticradiation (light) with molecules. Light beam characteristics - wavelength (frequency), intensity,polarization - determine types of transitions and informationaccessed.MyAbsorbance:E-M wave maintain phase,attenuate intensity in samplehνT

Optical Spectroscopy – Electronic,Example Absorption and FluorescenceEssentially a probe technique sensing changes in the local environment of fluorophoreseg. Trp, TyrChange with tertiarystructure, compactnessε (M-1 cm-1)Intrinsic fluorophoresAmide absorption broad,Intense, featureless, far UV 200 nm and belowFluorescence IntensityWhat do you see?(typical protein)T

UV absorption of peptides is featureless --except aromaticsAmideπ π* and n-π*TrpZip peptide in waterRong Huang, unpublishedTrp – aromatic bandsT

Time out—states and transitionsReview of Vibrational Spectra Theory and CharacteristicsSpectroscopy—transitions between energy states of amolecule excited by absorption or emission of a photonhν E Ei - EfEnergy levels due to interactions between parts ofmolecule (atoms, electrons and nucleii) as described byquantum mechanics, and arecharacteristic of components involved, i.e. electrondistributions (orbitals), bond strengths and types plusmolecular geometries and atomic masses involvedT

Spectroscopy Study of the consequences of the interaction of electromagneticradiation (light) with molecules. Light beam characteristics - wavelength (frequency), intensity,polarization - determine types of transitions and informationaccessed.IntensityI E 2zB EE zB x Polarization}yk yxλWavelengthν c/λFrequencyT

Properties of light – probes of structure Frequency matches change in energy, type of motionÆE hν, where ν c/λ(in sec-1) Intensity increases the transition probability—ÆI ε2 –where ε is the radiation Electric Field strengthLinear Polarization (absorption) aligns with direction of dipole change—(scattering to the polarizability)ÆI [δµ/δQ]2 where Q is the coordinate of the motionCircular Polarization results from an interference:ÆIm(µ m) µ and m are electric and magnetic dipoleIntensity(Absorbance)Absorbance1.2νIR ofvegetableoil.8.4040003000Frequency (cm )2000-11000λT

Optical Spectroscopy - IR SpectroscopyProtein and polypeptide secondary structural obtained fromvibrational modes of amide (peptide bond) groupsAside: Raman is similar, but differentamide I, little amide II, intense amide IIIWhat do you see?Model peptide IRAmide I(1700-1600 cm-1)αβAmide II(1580-1480 cm-1)rcAmide III(1300-1230 cm-1)IIIT

Vibrational States and Transitions The simplest case is a diatomic molecule. Rotations (2) andtranslations (3) leave only one vibrational degree of freedom, thebond length change, r, a one dimensional harmonic oscillator One can solve this problem exactly as a classical Hook’s lawproblem with–a restoring force: F –k r– potential energy: V 1/2 Quantum mechanically:andk( r)2Ev (v 1/2)hν ,ν is the vibration frequencyT

Harmonic OscillatorModel for vibrational spectroscopy(virtualstate)ERamanrerreqv 49hν2v 37hν2v 25 hν2v 13 hν21hν2Ev (v ½)hνIRv 0 v 1 E hνν (1/2π)(k/µ)½hνreT

Vibrational States and Transitions for the simplest harmonic oscillator case (diatomic):ν (1/2π)(k/µ)1/2where k is the force constant (d2V/dq2).– In practice, stronger bonds have sharper (more curvature)potential energy curves, result: higher k, and higher frequency.and µ is the reduced mass [m1m2 / (m1 m2)].– In practice, heavier atom moving, have lower frequency.Thus vibrational frequencies reflect structure, bonds and atomsT

Vibrational States and Transitions Summary: high mass low frequencystrong bond high frequency Some simple examples (stretches in polyatomics):-C-C- 1000 cm-1C-H 2800 cm-1-C C- 1600 cm-1C-D 2200 cm-1-C C- 2200 cm-1C---N 1300 cm-1T

Polyatomic Vibrational States For a molecule of N atoms, there are (3N-6) vibrational degrees offreedom. This complex problem can be solved the harmonicapproximation by transforming to a new set of normal coordinates(combinations of the internal coordinates, qi) to simplify the potentialenergy V—method unimportant for this course– V V0 Σ (dV/dqi)0qi (½) Σ (d2V/dqiqj)0qiqj . . . . . This results in a molecular energy that is just the sum of theindividual vibrational energies of each normal mode:– E Σ Ei Σ (vi ½) hνi As a result we have characteristic IR and Raman frequencies, νi,which are reflect bond types in the molecule. The frequency patternforms a “fingerprint” for the molecule and its structure. Variations due to conformation and environment give structuralinsight and are the prime tools for Protein - Peptide IR and Raman.T

Normal Mode Analysis In the Harmonic approximation it is possible to define:– normal coordinates: Qj Σ cji qi– simplify potential: Vharm (1/2) Σ (d2V/dQj2) Qj2 This summed potential is solved by a simple product wavefunction:Φ Π φi(Qi) This results in an energy that is just the sum of the individualvibrational energies of each normal mode:E Σ Ei Σ (vi 1/2) hνiT

Vibrational States and Transitions Each φi(Qi) gives a unique frequency, νi. The general massand bond strength characteristics still come through. As a result we have IR and Raman group frequencies, νi,which are characteristic of bond types in the molecule. Thefrequency pattern forms a “fingerprint” for the molecule andits structure. The variations due to its conformational and environmentalconditions can give added structural insight and are theprime tools of IR and Raman spectra of Proteins andPeptides.T

Vibrational Transition Selection RulesHarmonic oscillator: only one quantum can change on each excitation vi 1, vj 0; i j .These are fundamental vibrationsAnharmonicity permits overtones and combinationsNormally transitions will be seen from only vi 0, since most excitedstates have little population.Population, ni, is determined by thermal equilibrium, from the Boltzmanrelationship:ni n0 exp[-(Ei-E0)/kT],where T is the temperature (ºK) – (note: kT at room temp 200 cm-1)T

Dipole Moment Interaction of light with matter can be described as the induction ofdipoles, µind , by the light electric field, E:¾µind α . Ewhere α is the polarizability IR absorption strength is proportional to¾Α Ψf µ Ψi 2,transition moment between Ψi Ψf To be observed in the IR, the molecule must change its electricdipole moment, µ , in the transition—leads to selection rulesdµ / dQi 0 Raman intensity is related to the polarizability,¾Ι Ψb α Ψa 2, similarly dα / dQi 0 for Raman observationT

Raman Selection RulesAny mode which expands the molecule ,particularly any delocalized bonds, will be intense.Aromatic groups and disulfides in protein side chains have strongRaman bands and the C O and C-N stretches in a peptide arerelatively strong, O-H and N-H and water are weak (strong IR).Bases in DNA are strong Raman scatterersT

IR vs. Raman Selection Rules At its core, Raman also depends on dipolar interaction,but it is a two-photon process, excite with ν0 and detectνs, where νvib ν0 - νs, so there are two µ’s. Ψ0 µ Ψi Ψi µ Ψn α need a change in POLARIZABILITY for Raman effectn0νvib νn- ν0 E / hν0νsin0νvib ν0- νsT

Symmetry Selection Rules (Dipole, etc.)Example:symmetric stretchO C OInfrared inactiveδµ/δQ 0Raman Intenseasymmetric stretchO C OInfrared activeRaman inactivebendingO C OInfrared activeRaman inactiveδα/δQ 0δµ/δQ 0δα/δQ 0δµ/δQ 0δα/δQ 0Note: in biomolecules generally there is little symmetry, however,the residual, approximate symmetry has an impact. Raman and IRintensities often are complementary, for example amide II and IIIT

Anharmonic TransitionsReal molecules are anharmonic to some degree so other transitions dooccur but are weak. These are termed overtones ( vi 2, 3, . .) orcombination bands ( vi 1, vj 1, . .). [Diatomic model]E/DeD0—dissociation energy E02 2hνanhrm - overtone E01 hνanh--fundamental( r - re )/reT

Complementarity: IR and RamanIRRamanIf molecule is centrosymmetric, no overlap of IR and RamanT

Comparison of Raman and IR IntensitiesT

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IR & Raman Instrumentation - Outline Principles of infrared spectroscopy FT advantages Elements of FTIR spectrometer Acquisition of a spectrum Useful Terminology Mid-IR sampling techniques– Transmission– Solids Raman instrumentation comparison (Note—more on sampling variations later)T

Techniques of Infrared SpectroscopyInfrared spectroscopy deals with absorption of radiation-detect attenuation of beam by sample at dradiationdetectorSampleDispersive spectrometers (old) measure transmission as a functionof frequency (wavelength) - sequentially--same as typical UV-visInterferometric spectrometers measure intensity as a function ofmirror position, all frequencies simultaneously--Multiplex advantageT

Major Fourier Transform Advantages Multiplex Advantage– All spectral elements are measured at the same time,simultaneous data aquisition.Felgett’s advantage. Throughput Advantage– Circular aperture typically large area compared to dispersivespectrometer slit for same resolution, increases throughput.Jacquinot advantage Wavenumber Precision– The wavenumber scale is locked to the frequency of an internalHe-Ne reference laser, /- 0.1 cm-1. Conne’s advantageT

Typical Elements of FT-IRIR Source (with input collimator)– Mid-IR: Silicon Carbide glowbar element, Tc 1000oC; 200 - 5000 cm-1– Near IR: Tungsten Quartz Halogen lamp, Tc 2400oC; 2500 - 12000 cm-1IR Detectors:– DTGS: deuterated triglycine sulfate - pyroelectric bolometer (thermal) Slow response, broad wavenumber detection– MCT: mercury cadmium telluride - photo conducting diode (quantum) must be cooled to liquid N2 temperatures (77 K) mirror velocity (scan speed) should be high (20Khz)Sample Compartment– IR beam focused ( 6 mm), permits measurement of small samples.– Enclosed with space in compartment for sampling accessoriesT