IR and Raman. Sections of the BIRS course abstracted for Chem 524, Tim Keiderling

Transcription

1 IR and Raman Sections of the BIRS course abstracted for Chem 524, 2005 Tim Keiderling

2 Optical Spectroscopy - Processes Monitored UV/Fluorescence/IR/Raman/Circular Dichroism Excited State (distorted geometry) Absorption hν = E grd -E ex UV-vis absorp. & Fluorescence. move e - (change electronic state) high freq., intense Ground State (equil. geom.) ν 0 ν S Fluorescence hν = E ex -E grd Raman: E = hν 0 -hν s =hν vib CD circ. polarized absorption, UV or IR Raman nuclei, inelastic scatter very low intensity molec. coord. Infrared: E = hν vib Q IR move nuclei low freq. & inten.

3 Optical Spectroscopy Electronic, Example Absorption and Fluorescence Essentially a probe technique sensing changes in the local environment of fluorophores What do you see? Intrinsic fluorophores eg. Trp, Tyr Change with tertiary structure, compactness ε (M -1 cm -1 ) Amide absorption broad, Intense, featureless, far UV ~200 nm and below Fluorescence Intensity

4 UV absorption of peptides is featureless --except aromatics TrpZip peptide in water Rong Huang, unpublished

5 Optical Spectroscopy - IR Spectroscopy Protein and polypeptide secondary structural obtained from vibrational modes of amide (peptide bond) groups Aside: Raman is similar, but different amide I, little amide II, intense amide III What do you see? Model peptide IR Amide I ( cm -1 ) α Amide II ( cm -1 ) β rc Amide III ( cm -1 ) I II

6 Vibrational Spectroscopy Basic principles, introduction to theory for IR Raman Later with extend to advanced techniques VCD (Vibrational Circular Dichroism) ROA (Raman Optical Activity) Microscopy and Imaging Time dependence

7 Spectral Regions and Transitions Absorption of infrared (IR) radiation induces of bond stretching and bond angle deformation. For gas phase molecules each vibrational transition has many sharp rotational levels, in solution these are broadened out due to collisions and interactions. symmetrical vibration H-O-H asymmetrical vibration H-O-H

8 Vibrational States and Transitions For the simplest harmonic oscillator case (diatomic molecule), a classical Hook s law restoring force: F = k r yields a potential energy: V = ½ k( r) 2 Quantum mechanically this results in energy levels: E v = (v+1/2)hν, where ν is the vibration frequency ν = (1/2π)(k/µ) 1/2 k is the force constant (d 2 V/dq 2 ). In practice, the stronger the bond, the sharper the potential energy curve, resulting in a higher k, and higher frequency. µ is the reduced mass [m 1 m 2 / (m 1 +m 2 )]. In practice the heavier the atom that moves, the lower the frequency.

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

10 Harmonic Oscillator Model for vibrational spectroscopy E (virtual state) Raman r e v = hν r e r q v = 3 v = hν 5 2 hν E v = (v+½)hν v = ± 1 E = hν ν = (1/2π)(k/µ) ½ IR v = 1 v = 0 hν r e 3 2 hν 1 2 hν

11 Selection Rules For Spectral Transitions Harmonic oscillator: only one quantum can change on each excitation v i = ± 1, v j = 0; i j. These are fundamental vibrations Anharmonicity permits overtones and combinations Normally transitions will be seen from only v i = 0, since most excited states have little population. Population, n i, is determined by thermal equilibrium, from the Boltzmann relationship: n i = n 0 exp[-(e i -E 0 )/kt], where T is the temperature (ºK)

12 Vibrational Transition Selection Rules For IR transitions, the molecule must interact with the electric field of the light by changing its electric dipole moment, µ, in the transition dµ / dq i 0 (i.e. charge distribution must change) The normal modes leading to intense IR transitions can often be recognized by common sense. For example, in an amide bond, stretching the C=O or wagging the N-H should move partially charged atoms. Stretching the C-N bond is less likely to change the dipole moment very much. For Raman, the polarizability must change in the vibration, this is harder to picture, but modes affecting π-systems or large atoms with lone pairs (e.g. S, I) are intense dα / dq i 0

13 Symmetry Selection Rules (Dipole, etc.) Example: symmetric stretch O=C=O Infrared inactive δµ/δq = 0 Raman Intense δα/δq 0 asymmetric stretch O=C=O Infrared active δµ/δq 0 Raman inactive δα/δq = 0 bending O=C=O Infrared active δµ/δq 0 Raman inactive δα/δq = 0 Note: in biomolecules generally there is little symmetry, however, the residual, approximate symmetry has an impact. Raman and IR intensities often are complementary, for example amide II and III

14 Raman Vibrational Intensity Change in polarizability ellipsoids during vibrations of CO 2

15 Polyatomic Vibrational States For a molecule of N atoms, there are 3N degrees of freedom or (3N-6) vibrations. This system is very complex, but can be solved approximately by separating into (3N-6) equivalent equations by transforming internal coordinates (q i for each atom) to a new set of normal coordinates (combinations of these q i ) to simplify the potential energy V V = V 0 + Σ (dv/dq i ) 0 q i + (1/2) Σ (d 2 V/dq i q j ) 0 q i q j

16 Normal Mode Analysis In the Harmonic approximation it is possible to define: normal coordinates: Q j = Σ c ji q i simplify potential: V harm = (1/2) Σ (d 2 V/dQ j2 ) Q j 2 This summed potential is solved by a simple product wavefunction: Φ = Π φ i (Q i ) This results in an energy that is just the sum of the individual vibrational energies of each normal mode: E = Σ E i = Σ (v i + 1/2) hν i

17 Vibrational States and Transitions Each φ i (Q i ) gives a unique frequency, ν i. The general mass and bond strength characteristics still come through. As a result we have IR and Raman group frequencies, ν i, which are characteristic of bond types in the molecule. The frequency pattern forms a fingerprint for the molecule and its structure. The variations due to its conformational and environmental conditions can give added structural insight and are the prime tools of IR and Raman spectra of Proteins and Peptides.

18 Dipole Moment Interaction of light with matter can be described as the induction of dipoles, µ ind, by the light electric field, E: µ ind = α. E, where α is the polarizability IR absorption INTENSITY is proportional to <Ψ b µ Ψ a > 2, transition moment between Ψ a Ψ b To be observed in the IR, the molecule must change its electric dipole moment, µ, in the transition dµ / dq i 0 Raman intensity is related to the polarizability: <Ψ b α Ψ a > 2, similarly dα / dq i 0 for observation

19 Anharmonic Transitions Real molecules are anharmonic to some degree so other transitions do occur but are weak. These are termed overtones ( v i = ± 2,± 3,..) or combination bands ( v i = ± 1, v j = ± 1,..). [Diatomic model] E/D e D 0 dissociation energy E 02 = 2hν anhrm - overtone E 01 = hν anh --fundamental ( r - r e )/r e

20 Raman Selection Rules At its core, Raman also depends on dipolar interaction, but it is a two-photon process, so there are two µ s. <Ψ 0 µ Ψ i > <Ψ i µ Ψ n > α => need a change in POLARIZABILITY for Raman effect n 0 ν 0 ν s i n 0 ν vib = ν n - ν 0 ν vib = ν 0 - υ s

21 Raman Selection Rules Any mode which expands the molecule, particularly any delocalized bonds, will be intense. Aromatic groups and disulfides in protein side chains have strong Raman bands and the C=O and C-N stretches in a peptide are relatively strong, O-H and N-H and water are weak (strong IR). Bases indna are strong Raman scatterers

22 Complementarity: IR and Raman IR Raman If molecule is centrosymmetric, no overlap of IR and Raman

23 Raman and IR Spectra Raman and IR are Complementary - similar transitions, different sensitivities Raman and IR both provide chemical bond information. Raman uses visible or uv light, so optics and cells can be glass detectors can be high sensitivity Frequently, Strong IR Absorbers are Weak Raman Scatterers, and Vice Versa - Analysis of biological molecules in water becomes easier Variants of Raman, especially resonance and surface enhanced Raman are very sensitive Arbitrary Y IR transmission Silicone Vibrational Spectra Raman scattering Raman Shift (cm-1) File # 2 : SILICONE

24 Comparison of Raman and IR Intensities

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27 IR Spectra are Information Rich Peak Positions Depend Upon Molecular Structure. (This is why vibrational spectroscopy is useful) Peak Heights/Areas Depend Upon Molecular Concentrations. Abs. Conc. Peak Widths Depend Upon Chemical Environment 1.2 Absorbance Frequency (cm-1 )

28 Peak Positions ν (k/µ) ν = Wavenumber of Absorbance k = Force Constant (bond strength) µ = m 1 m 2 /(m 1 + m 2 ) Strong Bonds Absorb at High Wavenumber Weak Bonds Have Peaks at Low Wavenumber Light Atoms Give High cm -1 Peaks Heavy Atoms Have Low cm -1 Peaks Hydroxyapatite Spectrum

29 Peak Heights Beer-Lambert Law: A = εbc A = Absorbance ε = Absorptivity b = Pathlength c = Concentration An overlay of 5 spectra of Isopropanol (IPA) in water. IPA Conc. varies from 70% to 9%. Note how the absorbance changes with concentration. The Size of Absorbance Bands Depends Upon Molecular Concentration and Sample Thickness (pathlength) The Absorptivity (ε) is characteristic of a molecule in given solvent

30 Peak Widths Water Benzene Peak Width is Molecule Dependent Strong Molecular Interactions = Broad Bands Weak Molecular Interactions = Narrow Bands

31 IR Instrumentation

32 Techniques of Infrared Spectroscopy Infrared spectroscopy deals with absorption of radiation-- detect attenuation of beam by sample at detector radiation source Frequency selector Sample transmitted radiation detector Dispersive spectrometers measure transmission as a function of frequency (wavelength) - sequentially-- same as typical UV or CD These are rare now, except for special applications see later Interferometric ( FT ) spectrometers measure intensity as a function of mirror position, all frequencies simultaneously-- Multiplex advantage, plus other aspects FTIR dominate market

33 Principles of Infrared Spectroscopy When a frequency of light corresponds to a molecular vibration it is absorbed by the sample The fraction of light transmitted by the sample compared with the light incident as a function of frequency gives the infrared spectrum of the sample

34 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 dispersive spectrometer slit for same resolution, increases throughput. Jacquinot advantage Wavenumber Precision The wavenumber scale is locked to the frequency of an internal He-Ne reference laser, +/- 0.1 cm -1. Conne s advantage

35 Interference - Moving Mirror Encodes Wavenumber Single wavelength case: δ=2x=2vt, is called Optical Retardation or Path Difference Michelson Interferometer each arm sum

36 Fourier Transform Interferometry Broad band response Interferogram Data Points Fourier Transform δ=2x=2vt, is called Optical Retardation or Path Difference V, velocity x, Displacement Single Beam Intensity Michelson Interferometer Wavenumbers (cm -1 )

37 Acquisition of an Infrared Spectrum The light coming from the source goes through the interferometer; the movement of the mirrors causes an interference pattern which is called the Interferogram Next the light passes through the sample; the Interferogram of the source is modified as the light is absorbed by the sample The Interferogram is measured by the detector The Interferogram is transformed into a spectrum by a mathematical operation called the Fourier Transform algorithm

38 Interference - Moving Mirror Encodes Wavenumber Source Detector Paths equal all ν in phase Paths vary interfere vary for different ν

39 Typical Elements of FT-IR IR Source (with input collimator) Mid-IR: Silicon Carbide glowbar element, T c > 1000 o C; cm -1 Near IR: Tungsten Quartz Halogen lamp, T c > 2400 o C; cm -1 IR 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 N 2 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 accessories

40 Acquisition of an Infrared Spectrum Broad spectral region sharply peaked interferogram Narrow (single frequency) cosine wave interferogram Need to measure two spectra to interpret Measurements are Single Beam: To get useful spectrum must correct for the light throughput, divide Sample response by Background response (better by blank, cell plus solvent) Take log of transmission, A = - log 10 (I/I 0 )

41 Interferograms for different light sources

42 Acquisition of an Infrared Spectrum 3 Interferogram of the source 50 Spectrum of the source (blank) FT Interferogram of the source & sample 40 Spectrum of the source & sample 1 0 FT Fast Fourier transform done in computer Convert Interferogram to Spectrum (single beam)

43 Acquisition of an Infrared Spectrum Single beam sample Spectrum of source & sample 40 I Divide by: 30 I i.e. I/I 0 gives Single beam background Spectrum of source only (no sample present) Transmittance transmittance spectrum of the sample ( polystyrene film ) -log 10 T Absorbance Absorbance spectrum Transmittance (negative peak), Absorbance (positive) ~ concentration

44 FTIR optical layout look under the hood Source HeNe laser alignment Aperture Interferometer Voice coil Air Bearing Moving mirror Fixed mirror Detector Sample Polarizer (rotate, - typically wire grid)

45 Corner-Cube Michelson FT-IR Simpler design eliminates air-bearing, more compact, stable

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47 Useful Terminology and Relationships Resolution: Resolution is the measured full width at half the maximum (FWHM) absorbance intensity of a spectral line which is inherently very narrow Resolution in FT-IR depends on 3 factors: Distance traveled by the scanning mirror Size of image transmitted at the sample focus Apodization function applied

48 Useful Terminology and Relationships Apodization Function In FTIR spectrometry, the true spectrum is convolved with the sinc function f(v). This function has been called the instrument line shape (ILS) function, the instrument function or the apparatus function. The word apodization refers to the suppression of the side lobes(or feet) of ILS A technique of computationally modifying the interferogram before Fourier Transformation to insure that every sharp spectral feature will be represented by a smooth spectral curve. No apodization, which is often called boxcar apodization, results in an undesirable oscillatory line shape but provides the best resolution

49 Apodization Functions Boxcar Triangular Cosine

50 Elements of FT-IR IR Source (with input collimator) Silicon Carbide glowbar element at > 1000 o C; gives usable intensity from cm -1 (Mid-IR) Tungsten Quartz Halogen lamp at > 2400 o C gives usable intensity from cm -1 (Near-IR)

51 Synchrotron Light Sources the next big thing Brookhaven National Light Source Broad band, polarized well-collimated and very intense (and fixed in space!) Light beam output Where e-beam turns

52 Synchrotron advantage high brightness

53 Useful Terminology and Relationships Spectral Range: The usable spectral range is the range over which there is sufficient available intensity to make useful measurements. Restricted by density of data points measured, if once every HeNe λ, ~ cm -1. At low frequency, the beamsplitter and window optics of the FT-IR become opaque. Below this point there is no intensity available. The cutoff point depends on the optical material. At high frequency source intensity and beamsplitter efficiency diminish gradually so that a cutoff point is less obvious.

54 Useful Terminology and Relationships Phase Correction Removes the effects of phase lag in electronics and optics of the interferometer. It insures that the spectrum is not distorted by phase lag. Automatically corrected in most spectrometers using Mertz phase correction algorithm (problem for differential data)

55 Useful Terminology and Relationships Mirror scan pattern: In the wishbone interferometer, both cube corners are displaced; one towards the beamsplitter and the other away from the beamsplitter. Most commercial FT-IR s scan only one mirror. Double sided scan: the mirrors scan such that the interferogram has the same length on both sides of the center burst Permits more precise determination of the phase correction than when the sides are unequal. Double sided scanning can be useful for low resolution (e.g. biomolecule spectra), but research FT-IRs need single sided scanning for high resolution. Many commercial FT-IR s scan only in one direction and return the mirror quickly for the next scan. For kinetics, it is useful to collect data going both directions bidirectional scan.

56 Raman Instrumentation

57 Advantages of Raman Spectroscopy Generally non-destructive (but could burn proteins) Flexible sampling - any phase - any size - no preparation (generally needs higher concentration) 1 µm sample area spatial resolution possible with Raman microscopy Glass does not absorb so it is good material for cells Water - weak scatterer - excellent solvent

58 Disadvantages of Raman Spectroscopy Sensitivity to π-systems means side-chain contributions and DNA base contributions are large, water is small Fluorescence can be a problem, sometimes it can be burned out of the sample, but excitation with red laser can typically avoid it Particulate scatter (~ν 4 ) can be a significant problem, filtering (both chemical and optical) helps

59 Raman Shift IR absorption frequency = Raman shift from laser Reported as Raman shift, ν Raman with units of cm -1 ν Raman Shift = ν laser - ν scattered vis Stokes scatter transmission IR (laser) C=C C-H Raman transitions Wavenumber 3000 C-H 1500 C=C Infrared transitions 0 The absolute wavenumber of the Raman scattered light depends on the laser wavelength. Always measure a shift. It s the Raman Shift, the Change in Photon Energy, that is Sensitive to Molecular Structure Slide Courtesy Renishaw Inc.

60 Raman spectra are result of scattering Typically a laser (intense at ν 0 ) is focussed onto sample and scattered light is collected, often at 90 degree angle Like fluorescence, but no real state is excited by the laser virtual } states ν = ν 0 +/- ν scat = ν vib ν 0 ν 0 v=1 Stokes Anti-Stokes v=0 ν Anti-Stokes ν 0 Stokes Intensity less in anti-stokes due to less population in excited states Normally only collect Stokes scatter

61 Photon Molecule Interactions

62 Raman and IR Spectra Raman and IR are Complementary - similar transitions, different sensitivities Raman and IR both provide chemical bond information. Raman uses visible or uv light, so optics and cells can be glass, detectors can be high sensitivity Frequently, Strong IR Absorbers are Weak Raman Scatterers, and Vice Versa - Analysis of biological molecules in water becomes easier Variants of Raman, especially resonance and surface enhanced Raman are very sensitive Arbitrary Y IR transmission Silicone Vibrational Spectra Raman scattering Raman Shift (cm-1) File # 2 : SILICONE

63 Low Wavenumber Bands Low Wavenumber Region: IR vs. Raman IR Spectrum Raman provides : Low wavenumber vibrational bands Raman Spectrum Wavenumber (cm -1 ) or Raman Shift (cm -1 ) Slide Courtesy Renishaw Inc.

64 Conventional Raman Instrumentation Excitation source (generally a CW laser) Sample illumination and scattered light collection system Sample holder Monochromator or spectrograph Detection system (PMT, Photodiode array, CCD) Scattering efficiency for Raman can be 1 in photons! Need extremely high intensity source, spectrometer with high degree of discrimination against stray light, extremely sensitive detection system able to detect small numbers of photons over dark background

65 Dispersive Raman - Single or Multi-channel Eliminate the intense Rayleigh scattered & reflected light Sample -use filter or double monochromator Typically 10 8 stronger than the Raman light Polarizer Lens Filter Alternate geometry: Laser 180 o backscatter Laser 90 o excite Single, double or triple monochromator Disperse the light onto a detector to generate a spectrum Detector: PMT or CCD for multiplex

66 Typical Dispersive Raman System

67 Excitation Sources CW gas lasers (Ar, Kr, Ne, N 2 (337.1 nm UV, pulsed), CO 2 (9-11 µm IR, CS/pulsed), excimer XeCl, 308 nm UV, pulsed) Dye lasers Solid-state lasers Ruby (694.3 nm, pulsed), Nd:YAG (1064-nm near-ir, CW/pulsed), Diode ( cm -1 IR, CW/pulsed) Pulsed Nd:YAG laser harmonics (532 nm, 355 nm, 266 nm) for timeresolved and UV resonance Raman applications

68 CW Gas Lasers These lasers require high voltage input and water cooling. Wavelength is given in Ångstroms, output in watts.

69 Solid-State Lasers Millennia IIs (Spectra Physics): Nd:YVO 4 (neodymium yttrium vanadate) laser crystal, end pumped by single fiber-coupled diode bar module. Crystal absorbs diode pumped light, produces 1064 nm output, which is resonated in cavity and amplified through stimulated emission. A lithium triborate (LBO) doubling crystal converts 1064 nm light from laser crystal to green 532 nm output of the laser. Power requirement is 110 V, <10 A. Unit is air-cooled, weighs 22 lb.

70 Sample Illumination Proper focusing of laser and efficient collection of scattered radiation required to detect very weak Raman scattering 90 0 scattering back-scattering Laser focuses to ~ 1mm; F-number (F=f/D) of collection lens should be small for large light-gathering power and should be matched to F-number of monochromator (usually two lenses are used, a short focal length, low F-number to collect the largest solid angle, and the second to F-match the monochromator

71 Detection: CCD (now most typical) The CCD (charge-coupled device) is an optical array detector consisting of a two-dimensional array of silicon diodes. CCDs have low read-out noise and high quantum efficiency and sensitivity over wide wavelength range ( nm).

72 Frequency Calibration With CCD, monochromator (spectrograph) is in fixed position, but precise wavelength (scattered wavenumber) for each pixel is unknown and needs calibration. Dispersion is relatively linear. Internal standards to obtain ~1 cm -1 accuracy Laser plasma lines (detune laser and scatter from melting point tube) Neon emission lines from neon lamp Indene (vacuum distilled and sealed in capillary

73 Frequency Calibration

74 FT-Raman spectroscopy solves problems of fluorescence and sample degradation by excitation in the near-ir. Interferometric optics achieve multiplex measurement. FT-Raman Spectroscopy Quartz beam splitter, solid state laser, backscattering collection, Rayleigh line filter before interferometer (holographic notch filter, OD >6 at laser frequency), InGaAs or high purity Ge detector. Can measure down to 70 cm -1 Stokes shift.

75 Fourier Transform Raman (Schrader&Simon, 1987) Interferometer InGaAs Detector Aperture Nd-YAG Excitation Filtering of Excitation λ Optional FTIR optics and Sampling Raman Sample

76 FT Raman Detectors Near-IR detector with high sensitivity needed. InGaAs or Ge typical. Excite with Nd:YAG, these work for ~3000 cm -1