Presentation on theme: "Obtaining an NMR Spectra"— Presentation transcript:
1 Obtaining an NMR Spectra Basic Requirements:NMR sample: compound of interest dissolved in ml of deuterated solvent.Higher the concentration higher the sensitivityMagnet: differentiate spin states (aligned/unaligned).Higher the field strength higher the sensitivity and resolutionRequires homogeneous field over the sampleRF electronics: generate RF pulse to perturb system equilibrium and observe NMR signal.Requires accurate control of pulse power and durationStability of pulseReceiver electronics: detection of induced current from nuclear precessonRequires high sensitivityConversion of analog signal to digital signal
3 Superconducting Magnet solenoid wound from superconducting niobium/tin or niobium/titanium wirekept at liquid helium temperature (4K), outer liquid N2 dewarnear zero resistance minimal current lose magnet stays at field for years without external power sourceCross-section of magnetmagnetspinnersample liftNMR TubeRF coilscryoshimsshimcoilsProbeSuperconducting solenoidUse up to 190 miles of wire!Liquid N2Liquid He
4 NMR Sample Factors to Consider: Maximize sample concentration Avoid precipitation or aggregationUse a single deuterated solventReference for lockAvoid heterogeneous samples distorts magnetic field homogeneityAvoid air bubbles, suspended particles, sample separationAvoid low quality NMR tubes distorts magnetic field homogeneityBreaks easily damage the NMR probeChose appropriate temperature for the sampleFreezing or boiling the sample may break the NMR tube and damage the NMR probe.Properly position NMR sample in the magnetPosition sample in homogeneous region of magnet and between detection and RF coilsAvoid positioning meniscus close to coil edge distorts magnetic field homogeneityFrequency of absorption: n = g Bo / 2p
6 Lock SystemNeed to constantly correct for the field drift during data collectionNMR probes contains an additional transmitter coil tuned to deuterium frequencychanges in the intensity of the reference absorption signal controls a feedback circuit; a frequency generator provides a fixed reference frequency for the lock signalif the observed lock signal differs from the reference frequency, a small current change occurs in a room-temperature shim coil (Z0) to create a small magnetic field to augment the main field to place the lock-signal back into resonanceLock Feedback CircuitLock Changes FromOff-resonance toOn-resonance
7 Lock SystemSimply, the lock system can be considered as a separate NMR spectrometer that is constantly collecting a deuterium spectrum and making sure the peak doesn’t move relative to a defined chemical shift
8 Lock System – things to consider Measures the resonance of the deuterated solventa number of common solvents (D2O, methanol, chloroform) have known deuterium resonanceCan only lock on one resonance, defined by user.Multiple deuterium resonances may confuse lock in automated acquisitionNMR sample needs to contain at least 5-10% volume of a deuterated solventConsequence of locking wrong solvent – wrong chemical shifts and missing peaks!
9 Lock System – things to consider Maximize lock signal indicates on-resonanceUse lock signal to shim sampleLoss of lock during experiment is problematic data not reliableNMR sample degradedInstrument problemStarted with weak lock signalIncrease lock signal by increasing lock gainAmplification of the detected lock signalIncreases both signal and noise, so higher lock gain noisier lock signalIncrease lock signal by increasing lock powerStrength of RF pulse to detect lock signalToo high and lock signal is saturated intensity of lock signal fluctuates up and downToo low and lock signal may not be observable
10 Superconducting Magnet Problems:Field is not constant over sample (spatial variation)Again:n = gBo/2p
11 Magnetic Field Homogeneity Frequency of absorption: n = g Bo / 2pPoor Homogeneity multiple peaks at different effective BoResonance depends on position in NMR sampleGood Homogeneity single peak with frequency dependent on Bo
12 Shim System Corrects for magnetic inhomogeneity Spatial arrangement of 20 or more coilschange current in each coil to “patch” differences in field and fix distortions in peak shapeactual shim coilsSketch of shim coils
13 Shim Coilselectric currents in the shim coils create small magnetic fields which compensate for inhomogenieties in the magnetshim coils vary in the geometric orientation and function (linear, parabolic, etc)Z0,Z1,Z2,Z3,Z4,Z5X, XZ,XZ2,X2Y2,XY,Y,YZ, YZ2, XZ3,X2Y2Z, YZ3,XYZ,X3,Y3
14 Shim CoilsOptimize shims by i) minimizing line-width, ii) maximizing lock signal or iii) maximizing FIDExamples of poor line-shapes due to shimming errors
15 Shim CoilsExamples of poor line-shapes due to shimming errors
16 Shim Coils Examples of poor FID shape due to shimming errors Perfectly Shimmed MagnetMis-shimmed Magnet
17 Spinning the SampleImproves effective magnetic field homogeneity by averaging inhomogeneities in the magnetZ – shims are also known as spinning shimsSpinning the sample causes symmetric side-bands at intervals related to spinning rateNon-spinning shims (X,Y) problemsSamples are never spun for multi-dimensional NMR experimentsCreates artifacts streaks or T1 ridges from spinning side-bands and spinning instabilitySpinning side-bands symmetric about peak
18 Gradient ShimmingUse pulse field gradients to automate the shimming (TopShim)Gradients - spatial changes to B0Gradients are used to probe (map) the Field (B0) profileA Shim Map is unique to each probeRequires a Strong Signal (Solvent)Requires H2O+D2O, CH3CN+D2O or CH3OH+D2O solventShim Map
19 Gradient Shimming Two General Approaches to Gradient Shimming 1D gradshim (Z-shims) seconds to minutes3D gradient shimming (all shims) 5 to 30 minutesShimming is accomplished by matching gradient shims for your sample to shim mapGradient shim (red) fit to shim map
20 Gradient Shimming Gradient Shimming Water resonance before and after Gradient ShimmingGradientShimming
21 Environment Stability Changes in the environment during data acquisition may have strong negative impacts on the quality of the NMR dataCommon causes of spectra artifacts are:Vibrations (building, HVAC, etc)Temperature changesThe longer the data acquisition, the more likely these issues will cause problemsThe lower the sample concentration (lower S/N) the more apparent these artifacts will beNoise peaks due to building vibrations
22 Peak Chemical Shift and Shape Change as Temperature Changes Environment StabilityPeak Chemical Shift and Shape Change as Temperature Changes
23 Sample ProbeHolds the sample in a fixed position in the magnetic fieldContains an air turbine to spin, insert and eject the sampleContains the coils for:transmitting the RF pulsedetecting the NMR signalobserving the lock signalcreating magnetic field gradientsThermocouples and heaters tomaintain a constant temperature
24 Sample ProbeImportant to note, because of the high magnetic field, the probe has to be built with non-magnetic material such as glass and plastics.Thus, probes tend to be fragile and easy to break
25 Tuning the ProbePlacing the sample into the probe affects the probe tuningSolvent, buffers, salt concentration, sample concentration and temperature all have significant impact on the probe tuningProbe is tuned by adjusting two capacitors: match and tuneGoal is to minimize the reflected power at the desired frequencyTuning capacitor changes resonance frequency of probeMatching capacitor matches the impedance to a 50 Ohm cablePower submitted to transmitter and receiver is maximized
26 Tune and Match SystemTune- corrects the differences between observed and desired frequencyMatch – correct impedance difference between resonant circuit and transmission line (should be 50W )Adjust two capacitors until the tuning and desired frequency match and you obtain a nullAffects:signal-to-noiseaccuracy of 90o pulsesample heatingchemical shift accuracy
27 Tune and Match capacitors for a Bruker Probe Tune and Match SystemTune and Match capacitors for a Bruker Probe
28 If turned too far will easily break!! Tune and Match SystemChanging the Distance Between the Plates or theAmount of Plate Surface Area which overlaps in a Variable CapacitorPhysical limits to how far the capacitor can be turned in either direction.If turned too far will easily break!!
29 Tuning the Probe Side Notes: Impedance Impedance – any electrical entity that impedes the flow of currenta resistance, reactance or bothResistance – material that resists the flow of electronsReactance – property of resisting or impeding the flow of ac current or ac voltage in inductors and capacitorsIllustration of matching impedanceConsider a 12V car battery attached to a car headlight12V car battery – low impedance high powerConsider 8 1.5V AA batteries (12 volt total) attached to a very low wattage light bulb8 1.5V AA batteries – high impedance low powerNow swap the arrangement What happens?Car battery can easily light the light bulb, but the headlight will quickly drain the AA batteries poor impedance match
30 Tuning the Probe Side Notes: Quality factor (Q) “Q” - dimensionless and important property of capacitors and inductorsQ - frequency of the resonant circuit divided by the half power bandwidthAll inductors exhibit some extra resistance to ac or rfQ is the reactance of the inductor divided by this ac or rf resistanceNMR probes Q > 300Higher the probe Q the greater the sensitivityHigh Q for an NMR probe is required for high Signal-to-NoiseSample can effect the Q of the probeThe sample increases losses in the resonant circuit by inducing eddy currents in the solventThe more conductive the sample the more the losses and the lower the probe Q.Water, high salt lower the Q of the probeLower Q longer pulse widthsX – reactance of circuit in OhmsRL – the series resistance of the circuit in Ohms
31 * = Pulse Generator & Receiver System tp Pulse length (time, tp) FT Radio-frequency generators and frequency synthesizers produce a signal at essentially a single frequency.RF pulses are typically short-duration (msecs)- produces bandwidth (1/4t) centered around single frequency- shorter pulse width broader frequency bandwidthHeisenberg Uncertainty Principal: Du.Dt ~ 1/2p- Shortest pulse length will depend on the probe Q and the sample property*=tpPulse length (time, tp)A radiofrequency pulse is a combination of a wave (cosine) of frequency wo and a step functionFTThe Fourier transform indicates the pulse covers a range of frequencies
32 Pulse Generator & Receiver System RF pulse width determines band-width of excitation- Not a flat profile- All nuclei within ±1/4PW Hz will be equally affected1H 6 ms 90o pulse ±41666 Hz ±69.4 ppm at 600 MHzMinimizes weaker perturbations of spins a edges of spectra- There are also null points at ±1/PW Hz where nuclei are unperturbed1H 6 ms 90o pulse first null at ±1.67e5 Hz ±277.8 ppm at 600 MHzMaximum affectNull, no affectInvert signal, 180o pulse
33 Pulse Generator & Receiver System RF pulse width determines band-width of excitation- These issues become a problem at high magnetic field strengths (800 & 900 MHz) for 13C spectra that that have a large chemical shift range (>200 ppm)13C 15 ms 90o pulse ±16666 Hz ±18.5 ppm at 900 MHzAlso, complex experiments (multiple pulses) depend on the accuracy and consistency of pulse widths- Selective pulse long pulse width (ms) narrow band-width.Maximum affectNull, no affectInvert signal, 180o pulse
34 Pulse Length Calibration Need to experimentally determine 90o pulse- Measure intenisty of major peak (solvent) in spectrum as the function of 90o pulse length (P1)Maximum at 900 and minimum at 360oUsually measure 90o pulse at 360o time point
35 Pulse Length Calibration 90o pulse (12 ms)180o pulse (24 ms)360o pulse (44 ms)The pulse width was arrayed from 2 ms to 60 ms in steps of 2 ms90o pulse is ~ 11 ms270o pulse (32 ms)
36 Pulse Generator & Receiver System A magnetic field perpendicular to a circular loop will induce a current in the loop.90o NMR pulses places the net magnetization perpendicular to the probe’s receiver coil resulting in an induced current in the nanovolt to microvolt rangepreamp mounted in probe amplifies the current to 0 to 10 Vno signal is observed if net magnetization is aligned along the Z or –Z axisRotates at the Larmor frequencyn = gBo/2p
37 Continuous Wave (CW) vs. Pulse/Fourier Transform Continuous Wave – sweep either magnetic field or frequency until resonance is observedabsorbance observed in frequency domainPulse/Fourier Transform – perturb and monitor all resonances at once– absorbance observed in the time domain
38 Continuous Wave (CW) vs. Pulse/Fourier Transform NMR Sensitivity IssueA frequency sweep (CW) to identify resonance is very slow (1-10 min.)Step through each individual frequency.Pulsed/FT collect all frequencies at once in time domain, fast (N x 1-10 sec)All modern spectrometers are FT-NMRs
39 Continuous Wave (CW) vs. Pulse/Fourier Transform Fourier Transform NMRObserve each individual resonance as it precesses at its Larmor frequency (wo) in the X,Y plane.Monitor changes in the induced current in the receiver coil as a function of time.FID – Free Induction Decay
40 Fourier Transform NMRSignal-to-noise increases as a function of the number of scans or transientsIncreases data collection timeThere are inherent limits:Gain in S/N will eventually plateauThe initial signal has to be strong enough to signal average.Increase signal-to-noise (S/N) by collecting multiple copies of FID and averaging signal.
41 Fourier Transform NMRIncrease signal-to-noise (S/N) by collecting multiple copies of FID and averaging signal.But, total experiment time is proportional to the number of scansexp. time ~ (number of scans) x (recycle delay; D1)
42 Relative S/N per unit time of data collection Fourier Transform NMRRecycle time (D1) – time increment between successive FID collectionMaximum signal requires waiting for the sample to fully relax to equilibrium (5 x T1)T1 – NMR relaxation parameter that will be discussed in detail later in the courseMost efficient recycle delay is 1.3 x T1Relative S/N per unit time of data collection1.3T1Repetition time (tT/T1)Optimize your repetition time …
43 Fourier Transform NMRRecycle time (D1) – time increment between successive FID collectionTypical T1’s for organic compounds range from 50 to 0.5 secondsT1 relaxation times also vary by nuclei, where 13C > 1HEither estimates from related compounds or experimental measurements of T1 is required to optimize data collection especially for long data acquisitions.
44 Continuous Wave (CW) vs. Pulse/Fourier Transform Fourier Transform NMRNMR signal is collected in Time Domain, but prefer Frequency DomainTransform from time domain to frequency domain using the Fourier functionFourier Transform is a mathematical procedure thattransforms time domain data into frequency domain
45 Sampling the NMR (Audio) Signal Collect Digital data by periodically sampling signal voltageADC – analog to digital converterContinuous FIDDigitized FID
46 Sampling the NMR (Audio) Signal Collect Digital data by periodically sampling signal voltageADC – analog to digital converterSample intensity of voltage induced in coil by y-vector of net magnetization precessing in x,y-plane
47 Sampling the NMR (Audio) Signal To correctly represent Cos/Sin wave, need to collect data at least twice as fast as the signal frequencyIf sampling is too slow, get folded or aliased peaksThe Nyquist Theorem says that we have to sample at least twice as fast as the fastest (higher frequency) signal.SR = 1 / (2 * SW)Sample Rate- Correct rate, correct frequency½ correct rate, ½ correct frequency Folded peaks!Wrong phase!SR – sampling rateSW – sweep width
48 Equal delay between points Digital Resolution – number of data pointsThe FID is digitizedEqual delay between points(dwell time)DT = 1 / (2 * SW)Want to maximize digital resolution,more data points increases acquisition time (AQ) and experimental time (ET):AQ = DT x NP ET = AQ x NSlarger spectral width (SW) requires more data points for the same resolution
49 Sampling the NMR (Audio) Signal Sweep width (Hz, ppm) needs to be set to cover the entire NMR spectraSweep Width(range of radio-frequencies monitored for nuclei absorptions)If SW is too small or sampling rate is too slow, than peaks are folded or aliased (note phase change)
50 Sampling the NMR (Audio) Signal SW is decreasedThe phase of folded peaks can vary: (a) negative phase, (b) dispersive or (c) positive phase.
51 Sampling the NMR (Audio) Signal Always set SW to be slightly larger than needed to cover the entire spectrum.Allow for blank space at both low and high chemical shifts.Correct SpectraSpectra with carrier offset resulting in peak folding or aliasing
52 Sampling the NMR (Audio) Signal NMR data sizeAnalog signal is digitized by periodically monitoring the induced current in the receiver coilHow many data points are collected?What is the time delay between data points?How long do you sample for?Sample too long collecting noise & wasting timeAll this noise added to spectraHigher Digital Resolution requires longer acquisition times
53 FID signal is truncated Sampling the NMR (Audio) SignalNMR data sizeHow long do you sample for?Sample too short don’t collect all the data, lose resolution & get artifactsFID signal is truncatedTruncated FID leadsto artifacts
54 Sampling the NMR (Audio) Signal NMR data sizeDigital Resolution (DR) – number of Hz per point in the FID for a given spectral width.DR = SW / TDwhere:SW – spectral width (Hz)TD – data size (points)Dwell time DWTD
55 Sampling the NMR (Audio) Signal NMR data sizeDwell Time (DW) – constant time interval between data points.SW = 1 / (2 * DW)From Nyquist Theorem, Sampling Rate (SR)SR = 1 / (2 * SW)DR, DW, SW, SR, TD are ALL Dependent ValuablesDwell time DWTD
56 Sampling the NMR (Audio) Signal NMR data sizeTwo Parameters that the spectroscopist needs to setSW – spectral sweep widthShould be just large enough to include the entire NMR spectraTD – total data pointsDetermines the digital resolutionContributes to the total experiment time (acquisition time)Should be large enough to collect entire FIDDwell time DWTDTotal Data Acquisition Time (AQ):Should be long enough toallow complete delay of FIDAQ = TD * DW= TD/2SWH
57 Sampling the NMR (Audio) Signal NMR data sizeIncrease in the number of data points increase in resolutionIncreases acquisition timeIncrease in data points, resolution and acquisition time
58 Sampling the NMR (Audio) Signal NMR data sizeUnder sampling the data truncated FIDBaseline distortions sinc wigglesFTSinc wiggles
59 Sampling the NMR (Audio) Signal NMR Data Processing SoftwareUniform Data SamplingTraditionally, NMR acquires EVERY data point with a uniform time-step (DW) between pointsavoids under-sampling frequenciesFT algorithms expect uniform spacing of digital dataReason why nD NMR experiments take so long to collectWhy FIDS are truncatedWhy spectra have low resolution and sensitivityNo reason why the all the points of the FID need to be collectedtimevoltage
60 Sampling the NMR (Audio) Signal NMR Data Processing SoftwareNon-uniform data samplingSignificant improvement in resolution and sensitivity for nD NMR dataDon’t need uniform sampling, just need alternative to FFT to process the data.The sampling non-uniform scheme is the primary decision and impact on the spectraexponential in t1 and linear in t2Exponential in both t1 and t2randomly sampled from an exponential distribution in t1 and t2Random in t1 and t2.Graham A. Webb (ed.), Modern Magnetic Resonance, 1305–1311.
61 Sampling the NMR (Audio) Signal NMR Data Processing SoftwareNon-uniform data samplingVERY IMPORTANT POINT, tn is no longer defined by DW and number of pointstn is now user defined since DW is no longer relevant.Avoid FID truncation, maximize resolutiontimevoltageTraditional NMRFID is truncated because number of points and DW determine how much of the FID can be collectedNUS NMRFID is under-sampled, but the entire FID is sampled
62 Sampling the NMR (Audio) Signal NMR Data Processing SoftwareNon-uniform data samplingBoth noise (N) and signal to noise (SNR) are proportional to the total evolution timeOptimal setting is 1.3T2 of the evolving coherenceMaximize sensitivityMagn. Reson. Chem. 2011, 49, 483–491
63 Sampling the NMR (Audio) Signal NMR Data Processing SoftwareNon-uniform data samplingWhat is the optimal sampling density?Increase enhancement by increase exponential bias, eventually regenerate truncated FIDHighly resolved spectra is pT2TSMP – time constant for the exponentialweighting of the sampling.– enhancementlw – line widthMagn. Reson. Chem. 2011, 49, 483–491
64 Sampling the NMR (Audio) Signal NMR Data Processing SoftwareNon-uniform data samplingA 1.5 to 2.0 bias to early data points and a 4x reduction yields a 2x enhancementOr a 3T2 with a 3x reduction yields a 1.7 enhancementSampling Density/LW = TSMP/T2Truncated FIDMagn. Reson. Chem. 2011, 49, 483–491
65 Sampling the NMR (Audio) Signal NMR Data Processing SoftwareNon-uniform data samplingDifferent sampling schemes have different performances at different sampling densitiesSinusoidal Poisson Gap is currently the best – random sampling, while minimizing gap size particularly at the beginning and end of the FIDSome drastic sampling densities at 1% or less.Top Curr Chem ; 316: 125–148
66 Sampling the NMR (Audio) Signal NMR Data Processing SoftwareNon-uniform data samplingDramatic gain in the quality of strychnine NMR spectrum with 25% sampling densityThe spectrum was collected 4x faster (10 min. vs. 40 min.)Uniform SamplingNon-Uniform SamplingNat. Prod. Rep :
67 Sampling the NMR (Audio) Signal NMR Data Processing SoftwareNon-uniform data samplingHow is the time-domain data processed?Use the partial data to reconstruct the full Nyquist grid then process as normalmaximum entropy reconstruction is a common approachforward maximum entropy (FM), fast maximum likelihood reconstruction (FMLR)multi-dimensional decomposition (MDD); and compressed sensing (CS)MddNMR:Newton:RNMRTK:mpiPipe: Available by contacting the Wagner Group
68 Sampling the NMR (Audio) Signal Adjusting the Receiver Gain (RG) – electronic amplification of the signalThere is an optimal setting guided by the limits of the ADC digitizerFID intensity changes as the number of transients increase during data acquisitionRG depends on NSDigitizer has a finite data rangeIncrease in FID Intensity with number of transients
69 Sampling the NMR (Audio) Signal Adjusting the Receiver Gain (RG) – electronic amplification of the signalIf RG set too high, the digitizer is full and the FID is clippedFourier transform of a clipped FID results in sinc wiggles in the spectrum baseline.
70 Sampling the NMR (Audio) Signal Adjusting the Receiver Gain (RG) – electronic amplification of the signalIf RG is set too low, the spectrum will be noisy.RG should be set as increments of 2, where there is a maximum limitRG may be set to higher values, but no effect on the spectra will be observedRG may be set to non-factors of two, but adjusted to nearest factor of 2.
71 Sampling the NMR (Audio) Signal Solvent suppressionsolvent concentration is significantly larger than the sample concentrationwater is 55M compared to typical mM – mM of compoundWith Solvent SuppressionWithout Solvent Suppression
72 Sampling the NMR (Audio) Signal Solvent suppressionstrong solvent signal can fill digitizer making it impossible to observe the sample signalDynamic range problem16K – 32K range of intensitiesNeed to suppress intense solvent signals with selective saturation pulsewill discuss different NMR pulses in detail latterThe most intense peak is set to the largest value in the digitizer and every other peak is scaled accordingly
73 Peak intensity has to fit between range of 1:215 Sampling the NMR (Audio) SignalDynamic rangedefines the range of signal amplitudes (peak intensities) observed in the spectrumTypically 16 bit or 18 bit digitizers16 bit digitizer – FID amplitudes range from -215 to 215peak smaller than 1/32768 (16 bit) or 1/ (18 bit) of most intense peak is lost!!32768Want to “see” weak peaks in the presence of intense peaksPeak intensity has to fit between range of 1:2151
74 Quadrature detection carrier Frequency of B1 (carrier) is set to the center of the spectrum.Small pulse length to excite the entire spectrumMinimizes folded noisecarriersame frequency relative to the carrier, but opposite sign.PW excites a corresponding bandwidth of frequencies
75 Quadrature detection carrier Frequency of B1 (carrier) is set to the center of the spectra.Rate of precession in X,Y plane is related to carrier frequencyPrecession is difference from carrier frequencyPossible to have resonances with same frequency but opposite directionClockwise – magnetization traveling faster than rotating frameCounter clockwise – magnetization traveling slower than rotating framecarriersame frequency relative to the carrier, but opposite sign.
76 Quadrature detection carrier How to differentiate between peaks upfield and downfield from carrier?observed peak frequencies are all relative to the carrier frequencySame Frequency!Opposite signcarrierHow to differentiate between magnetization that precesses clockwise and counter clockwise?
77 Quadrature detection carrier If carrier at edge of spectrum, peaks are all positive or negative relative to carrierExcite twice as much noise, decrease S/NHalf of the digital resolutionHalf of the spectrum is irrelevant noisePW excites a corresponding bandwidth of frequencies centered on carriercarrierAll this noise added to spectrum
78 Quadrature detection PH = 0 B F B Use two detectors 90o out of phase. w (B1)PH = 90FPH = 0FBPhase of Peaksare different.PH = 90FB
79 (imaginary component of FT) Quadrature detectionUse two detectors 90o out of phase.FT is designed to handle two orthogonal input functions called the real and imaginary componentDetector along X-axis(real component of FT)Detector along Y-axis(imaginary component of FT)Phase of Peaks are different allows differentiation of frequencies relative to carrier
80 Phase Correction of the NMR Spectrum Depending on when the FID data collection begins a phase shift in the data may occur.Phase ShiftPhase correction of the NMR spectrum compensates for this phase shift.
81 Phase Correction of the NMR Spectrum Phase shift depends on the frequency of the signalPhase Shift
82 Phase Correction of the NMR Spectrum Phase ShiftPhase CorrectManually adjust zero-order (PO) and first-order (P1) parameters to properly phase spectra.
83 Phase Correction of the NMR Spectrum What is happening mathematically during manual phasing of an NMR spectrumFourier transformed data contains a real part that is an absorption Lorentzian and an imaginary part which is a dispersion Lorentzianwe want to maintain the real absorption mode line-shapedone by applying a phase factor (exp(iQ)) to set F to zerowe are effectively discarding the imaginary component of the spectrum
84 Phase Correction of the NMR Spectrum If you “over-phase” the spectrum, you get baseline “roll”
85 Phase Correction of the NMR Spectrum Power or Magnitude spectrumobtain a pure absorption NMR spectrum without manual phasingresults in broader spectrum that can not be integratednot a typical or preferred approach to processing an NMR spectrum
86 Zero Filling of the NMR Spectrum Improve digital resolution by adding zero data points at end of FIDessential for n-Dimensional NMR datareal gain in resolution is limited to zero-filling to 2AQ ( in theory) or ~ 4AQ in practice8K data8K zero-fill8K FID16K FIDNo zero-filling8K zero-filling
87 Zero Filling of the NMR Spectra Better example of the resolution gain and benefits of zero-filling NMR spectraNo zero-filling4AQ zero-filling
88 Applying a Window Function to NMR data Emphasize the signal and decrease the noise by applying a mathematical function to the FID.Can also increase resolution at the expense of sensitivityApplied to the FID before FT and zero-fillingGood stuffMostly noiseSensitivityResolution
89 = Applying a Window Function to NMR data X Simply Multiple FID with a Mathematical FunctionF(t) = e - ( LB * t )X=
90 Can either increase S/N Applying a Window Function to NMR dataFTLB = -1.0 HzLB = 5.0 HzIncrease SensitivityIncrease ResolutionCan either increase S/NorResolutionNot Both!
91 Applying a Window Function to NMR data A Variety of Different Apodization or Window functions
92 Applying a Window Function to NMR data A main goal in applying a window function for a nD NMR spectra is to remove the truncation by forcing the FID to zero.Truncated FID with spectra “wiggles”Apodized FID removes truncation and wiggles
93 Spline baseline correction Baseline Correction of NMR SpectrumIt is not uncommon to occasionally encounter baseline distortions in the NMR spectraThe baseline can be corrected by applying a linear fit, polynomial fit, spline fit or other function to the NMR spectrum.Spline baseline correction
94 polynomial baseline correction Baseline Correction of NMR SpectrumA number of factors lead to baseline distortions:Intense solvent or buffer peaksPhasing problemsErrors in first data points of FIDShort recycle tinesShort acquisition timesReceiver gainpolynomial baseline correctionXi & Roche BMC Bioinformatics (2008) 9:234
95 NMR Peak Description LW1/2 Peak height – intensity of the peak relative to the baseline (average noise)Peak width – width (in hertz) at half the intensity of the peakLine-shape – NMR peaks generally resemble a Lorentzian functionA – amplitude or peak height(LW1/2) – peak width at half height (Hz)Xo – peak position (Hz)LW1/2
96 NMR Peak Integration or Peak Area The relative peak intensity or peak area is proportional to the number of protons associated with the observed peak.Means to determine relative concentrations of multiple species present in an NMR sample.Relative peak areas = Number of protonsHO-CH2-CH3123Integral trace
97 NMR Peak Integration or Peak Area Means to determine relative concentrations of multiple species present in an NMR sample.Need to verify complete or uniform relaxationUnknown Xylene Mixturefrom peak heights17.7%57.9%24.4%orthoMethyl Region of NMR Spectrummeta (21.3 ppm)metapara (20.9 ppm)ortho (19.6 ppm)paraimpuritiesimpurities
98 NMR Peak Integration or Peak Area NMR titration experiments are routinely used to monitor the progress of a reaction or interactionBy monitoring changes in the area or intensity of an NMR peak
99 Peak Picking NMR Spectra One of the basic steps in analyzing NMR spectra is obtaining a list of observed chemical shiftsUsually refereed to as peak pickingMost programs have similar functionality, choice is based on personal preferencedisplay the data (zoom, traces, step through multiple spectra, etc)Peak-picking – identify the X,Y or X,Y,Z or X,Y,Z,A chemical shift coordinate positions for each peak in the nD NMR spectraPeak Picking List
100 Peak Picking NMR Spectra Critical for obtaining accurate NMR assignmentsEspecially for software for automated assignmentsOnly provide primary sequence and peak-pick tablesTwo General Approaches to Peak PickingManualtime consumingcan evaluate crowded regions more effectivelyAutomatedpick peaks above noise thresholdORpick peaks above threshold withcharacteristic peak shapeonly about 70-80% efficientcrowded overlap regions and noiseregions (solvent, T2 ridges) cause problemsnoise peaks and missing real peaks causeproblems in automated assignment softwareJ. OF MAG. RES. 135, 288–297 (1998)