Mid-IR Sampling Techniques for Biological Molecules

Transcription

1 Mid-IR Sampling Techniques for Biological Molecules

2 Mid-IR Sampling Techniques LIQUIDS Transmission ATR (Attenuated Total Reflectance) Solids Transmission (KBr pellets, Mulls) ATR Diffuse Reflectance

3 Sampling Techniques in Transmission: Liquids Liquids are analyzed as a thin film sandwiched between two windows in a liquid cell. The type of cell, choice of window material, and pathlength is determined by the sample, experiment and spectral regions--significant variable in IR spectra. Samples can be analyzed pure or diluted with an appropriate solvent (typical for biopolymers). In order to perform quantitative analyses, the sample must be analyzed in a cell with a known pathlength often not trivial, especially short path.

4 Sampling Techniques in Transmission: solids Disposable IR cards: Thin polymer films can be analyzed by using a film holder. (or solution can be evaporated on surface) MULL Make a mull by grinding a powdered sample with liquid paraffin, such as Nujol, and placing it between two infrared transparent windows. KBr pellet: The sample and an infrared transparent matrix, such as KBr, are ground together and the resulting powder is then pressed into a disc. Diamond compression cell: To study single fibers and other micro samples.

5 Sampling Techniques: Diffuse Reflectance: Solids IR energy penetrates into the powdered sample and then emerges in all directions. The optics collect this scattered radiation and direct it to the infrared detector. (This is the trick, need mirrors that surround the surface.) The diffusely scattered light can be collected from a sample directly or by using an abrasive sampling pad for intractable samples. Sometimes called DRIFTS

6 Internal Reflectance Light is Focused Upon Crystal of High Refractive Index Material Crystals typically ZnSe, Diamond, Silicon, or Germanium Light Refracts Towards Upper Surface What Happens When Light Encounters an Internal Surface? Depends upon the crystal s critical angle, θ c If θ < θ c light refracts and exits the crystal (dashed line) If θ > θ c light reflects off of internal surface, Internal Reflection (solid line) Single bounce concept, if external reflection, then IRRAS (later) Internally Reflected Beam θ > θ c Refracted Beam θ < θ c IR Beam Angle of Incidence, θ

7 The ATR Experiment ATR (Attenuated Total Reflectance) Spectra are obtained with dedicated accessories containing focusing mirrors and a crystal holder that mount in the sample compartment of a spectrometer Could be arbitrary number of bounces, depends on design

8 Conventional multi-reflection ATR IR beam totally reflects inside crystal. Multiple bounces (~10-20) inside until emerge. Sample surface (~1µ) provides absorption For liquids and solids: Use with materials which are either too thick or too strongly absorbing to be analyzed by transmission spectroscopy or when only the surface of the material is of interest. ATR is a technique for obtaining infrared spectra of samples that are difficult to deal with, such as solids limited solubility, films, threads, pastes, adhesives, and powders. out in

9 Attenuated Total Reflectance (ATR) At point of internal reflectance, incident and reflected beams constructively interfere Electric field of IR wave decays above crystal surface, can penetrate sample in close contact (attenuate) About 1-10 microns above surface IR radiation hanging out in space, but not transmitted This place on crystal surface called a Hot Spot Bring Sample into Contact with Hot Spot to Get Spectrum Not a point, more an area Hot Spot due to IR beam IR Beam

10 ATR SPECTROSCOPY FACTORS AFFECTING ATR ANALYSIS Wavelength of IR radiation Refractive Index of the IRE and sample Depth of Penetration Effective Pathlength Angle of Incidence Efficiency of Sample contact ATR Crystal Material

11 ATR SPECTROSCOPY Depth of Penetration, d p distance from the crystal-sample interface where the intensity of the evanescent wave decays to 1/e (approximately 37%) of its original value. (In other words a measure of how far IR beam penetrates into a sample). It is calculated by: d p =1 / [2πνn c (sin 2 θ -n sc2 ) 1/2 ] Where, ν = Wavenumber n c = Refractive index of ATR crystal θ = Angle of incidence n sc = n sample /n crystal

12 Depth of Penetration: Implications Variables in the d p equation impact how ATR works And are all in the denominator! As ν d p Low wavenumber (long λ) light penetrates further than high wavenumber (short λ) light In other sampling methods, all wavenumbers see same pathlength The relative intensities in an ATR spectrum are different than those obtained with other sampling methods Samples closes to the surface contribute most to the spectrum (absorbance), so surface binding can be an important issue in interpretation Difficult to compare ATR spectra to transmission spectra

13 Depth of Penetration: More Implications As n c d p Crystals with different refractive indices give different pathlengths Can take spectra at different depths non-destructively = Depth Profiling Properties of Common ATR Crystal Materials Material n c Range (cm -1 ) Color ph Range KRS , Red 5-8 ZnSe , Yellow 5-9 Si Grey 1-12 Ge Gray 1-14 Diamond , Clear

14 Depth of Penetration: Yet More As θ d p Adjusting mirror position on ATR accessory changes θ Can dial-in θ and hence d p d p is Essentially Sample Independent Only sample dependent parameter effecting d p is n sample To a first approximation, most organic (and hence biological ) materials have a constant refractive index (~1.4 to 1.5). Not true for inorganics. So, to a first approximation, d p is sample independent Great for quantitative work Don t Forget Pressure as a Variable

15 ATR SPECTROSCOPY EFFECTIVE PATHLENGTH = (Effective Penetration) x Number of reflections (N), Where, N = l/t cotθ; l=length of crystal; t= thickness of crystal; θ=angle of incidence

16 Relative Intensities are Affected by ATR Top: ATR Spectrum of Sucrose Bottom: DRIFTS Spectrum of Sucrose High cm -1 peaks have less relative intensity in ATR spectrum than DRIFTS spectrum

17 Pathlengths Vary with ATR Crystal ATR Spectra of Sucrose Obtained with Diamond and Ge Crystals Diamond n c = 2.4, Germanium n c = 4.0 Beam penetrates further into diamond, hence more intense absorbance bands

18 ATR Applications Semi-Solids Semi-Solids Thick, viscous fluids such as olive oil or maple syrup Things that are part liquid/part solid. Emulsions, colloids, mixtures. Things like butter, soap, grease, and peanut butter

19 ATR Applications- Solids and Powders In theory, any solid brought into contact with the ATR crystal can have its spectrum measured Traditionally, powders have been difficult because of scratching and crystal contact Diamond ATR: Diamond won t scratch, and can press hard enough to flatten anything Biological materials are normally soft anyway

20 ATR - Advantages Practically no Sample Prep. Its Fast and Easy Put in on, wipe it off (small few bounce setup) Non-Destructive Useful if have little sample Good for Quantitation Wide Variety of Applications Depth profiling Polymers Solids, Powders Liquids, Semi-solids

21 ATR Disadvantages Crystal Care Crystal must be kept clean and scratch free to insure good contact with IR beam If you drop it on the floor, it will break. Replacement crystals >$600 Limited Wavenumber Range Some crystals don t go below 700 cm -1 Sensitivity Shallow d p gives small absorbances Typically, analyte conc. must be > 0.1% Not good for trace analysis Despite these problems, a VERY Useful IR Sampling Technique!

22 Few -bounce diamond ATR Liquids: 9-bounce ATR No Pressure applied Cover to avoid evaporation Solids: 1 or 3-bounce ATR In many cases need to grind to minimize large particles to avoid scattering and achieve better reproducibility

23 A Few -bounces ATR - Types available Often diamond over ZnSe Provides very hard surface Can apply pressure with rod above 1-3 bounces, signal can be small solution difficult Good for membranes, solids Easy to clean 9-bounce dip to hold liquid Design to fit your FTIR Several manufacturers

24 Aqueous sample spectra collection Biological samples are often in water Peptides are often prepared by solid state synthesis on a resin, TFA impurity Solvent corrections normally done by subtraction these pose special problems for peptides/proteins in particular, somewhat less for nucleic acids

25 Bio-IR: Experimental - D 2 O vs. H 2 O H-O-H bending mode at ~ 1645 cm -1 overlaps the amide I band of peptides/proteins D 2 O % Transmittance Wavenumbers /cm -1 H 2 O

26 Protein Studies: Experimental D 2 O vs. H 2 O early studies done in D 2 O problem : need to deuterate protein can cause different frequency shifts possible conformational changes

27 Protein and H 2 O.6 protein water (buffer) Frequency (cm-1)

28 Protein Studies: Experimental If experiment is done in water, then solvent water spectrum must be carefully subtracted from the spectrum of protein. Environmental water vapor adds sharp peaks to the spectra in the amide I region. These peaks can severely reduce spectral quality and must be subtracted Side-chain absorbance spectrum might have to be subtracted, to keep focus on amide secondary structure

29 Protein Studies: Experimental LIQUID SAMPLES: Protein Concentration: 0.5 mg/ml - saturated Pathlength: 3-7 µ µ for H 2 O studies for D 2 O studies Amount of sample required: 5-30 µl Resolution: 4cm -1

30 Protein Studies: Experimental SOLID STATE IR spectroscopy is the only method that is capable of studying proteins in the solid, liqiud (solution) and vapor (use??) states Films of particular interest along with membranes containing proteins/peptides Also cells and tissue are being studied Solid state spectra can be collected using several techniques: KBr pellets ATR Diffuse reflectance Microscopy

31 Steps Involved in a Measurement Measure cell + buffer/solvent empty cell or cell holder cell + buffer/solvent + protein Data processing Buffer/solvent subtraction, Vapor Subtraction, Sidechain subtraction Spectral enhancement (later): FSD (Fourier Self-Deconvolution), Derivative Band fitting Secondary structure analysis Factor or Principle component analyses of variance (perturbation - temperatue, ph, ligand, denaturant, etc.)

32 Buffer / Solvent Subtraction.6 Buffer I Protein.4 II.2 Protein-Buffer Absorbance / Wav enumber (cm-1) Y-Zoom SCROLL File # 2 : BV1227A 12/ 27/ 96 2:23 PM Res=4 cm-1 buf f er f or 2cab,carbonic anhy drase,2 time,12/27/96

33 Water vapor subtraction Protein Water vapor Absorbance / Wav enumber (cm-1) Y-Zoom SCROLL Must eliminate the sharp vapor transitions by subtraction, direct overlap suggests study of wings (high wavenumber)

34 Water vapor subtraction -80E Protein Water vapor Absorbance / Wav enumber (cm-1) Y-Zoom CURSOR Subtract vapor spectrum until protein spectrum is flat (featureless)

35 Perfect FT-IR spectrum of protein: H 2 O solution c I d,e a I II a II b III Protein Spectrum Must Have a. Amide I/II ratio: b. Presence of Amide III bands c. Presence of C-H stretching modes d. Flat baseline between cm -1 e. Gradual baseline rise below 1800 cm -1 f. No vapor bands.4.2 Empirical formula for success

36 Transmission vs. ATR: Danger of measurements with ATR II I Published spectrum of aqueous solution measured using ATR (dashed line): notice incorrect ratio of Amide I/II intensities => mistake due to protein adsorption to the surface of ATR crystal (solid line is transmission spectrum)

37 Comparison of solution vs. solid can we do this? bounce diamond ATR - protein powder Note: Transmission - protein in solution bounce diamond ATR - protein powder 1. Change in FWHH (bandwidth) from 35 cm -1 for solution to 58 cm -1 for solid 2. Frequency shift for both Amide I and II Transmission - protein in solution Ratio difference for Amide I/II Key compare transmission with trans. and ATR with ATR