NMR Training for Advanced Users Huaping Aug 18, 2008 Two Insider Scoops • Receiving efficiency – conceptually, it is similar to extinction coefficient in UV spectrospcopy; it characterizes how efficient a unit magnetization can produce a signal by a given NMR receiver – NMR signal size is proportional to receiving efficiency – Receiving efficiency can be pre-calibrated as a function of 90° degree pulse length – receiving efficiency is the same for all nuclei of the same type (indifferent to chemical shifts) in the same sample • Solvent signal offers a universal and robust concentration internal standard – Normalized NMR signal size is strictly proportional to concentration for a given sample, regardless how concentrated or dilute the sample is – Unit magnetization generates the same amount of total NMR response, which is indifferent to chemical shift or line-shape Outlines • Basic preparations for NMR: safety, sample, lock, shim and tune • Understanding NMR: excitation and observation • RF pulse calibration • NMR observables: – Chemical shift, scalar couplings, NOE and relaxations • Introduction to basic 2D's • Simulations for spin systems, pulses and sequences Safety • Personal safety – Cryogens: do not lean on or push magnets – Cryoprobes: avoid contact with transfer line – Magnetic and RF hazards • Instrument safety – Know the limits of instruments – Probe limits: avoid excessive long decoupling and long hard pulses or their equivalents – Be conservative – Double check pulse program and parameters for any non-standard new experiment • Data Safety – Back up data promptly and regularly – Data processing or manipulation has no impact on the raw (FID) data – Do not change parameters after data are acquired Sample • Rule #1: for Bruker NMR spectrometers, the NMR tube insert cannot exceed max depth (19mm or 20mm) from the center of the RF coil – Longer insert than recommended may present problems for the probe, as well as cause frictions during spinning – Varian is more flexible in allowing longer insert • Rule #2: center of NMR sample should be as close as possible to the center of RF coil. – Normal sample needs to about 500 ul or slight more – Too much solvent is a waste! – Too little solvent may make shim difficult, but it does work! • 10% deuterated solvent is sufficient for locking RF coil 18mm Coil Center 20mm Samples of smaller volumes • Follow rule # 1 and then rule #2 • Shimming might be challenging due to air/glass and air/solution interfaces • Consider Shigemi tubes 300ul • Be careful with spinning – Non-spinning is recommended for volume ~ 300 ul or less 400ul 500ul Sensitivity for smaller volumes 1.1 1 0.9 relative senstivity • Volume less than 300 ul may not offer additionally sensitivity improvement over that achieved by 300 ul, if the total amount of analyte is constant 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0 200 400 volume (ul) 600 800 1000 • Only higher fields (500, 600 and 800 HMz) in our facility need tuning • Most of the time only proton channel requires tuning RF reflection Tune and match the probe Carrier frequency tune match • Drx500-2 with BBO needs special attention – Proton always needs tuning – BB (used for 13C or 31P etc) channel needs tuning, by first dialing the numbers to the pre-set values Frequency Significance of tuning/matching • Shorter 90 degree pulse – More efficient use of RF power 1.1 • Protects transmitter • Better sensitivity – Reciprocity: if excitation is inefficient, then detection is equally inefficient NMR signal size – More uniform excitation in high power 0.9 0.7 • Potentially quantitative: – The product of NMR signal size is inversely proportional to the 90 degree pulse length 0.5 8 10 12 90 degree pulse length (ms) 14 Recognizing Bruker probe types side view side view magnet 1H tuning/matching rods are labeled as yellow bottom view Do not touch those! Dials for broadband (BB) tuning/matching Tabulated values for BB tuning/matching BB Dialing stick BBO probe on drx500-2 TXI probe Lock • Lock depends on shim: bad shim makes bad lock – Initialize shim by reading a set of good shims (i.e. rsh shims.txi) – Inheriting a shim set from previous users may present difficulties – Unusual samples (esp. small volumes) may need significant z1/z2 adjustments • Use “lock_solvent” or “lock” command – The default (bruker) chemical shift may appear as dramatically changed if the spectrometer assumes another solvent • Avoid excessive lock power – Lock signal may go up and down if lock power is too high due to saturation of deuterium signal – Apply sufficient lock power and gain so that lock does not drift to another resonance (this may happen by autolock if multiple deuterium signals exist) Shim • The goal of shim is to make the total magnetic field within the active volume homogeneous (preferably <1Hz). Total magnetic field = static field (superconductor) + cryoshim (factory set) + RT shim (user adjust) • Shim can be done either manually or by gradient, which can be very efficient and consistent if done properly • Sample spinning may improve shim – However, spinning-side band appear • Recommendation: – Start from a known good shim set (by rsh on bruker or rts on varian). – Do not inherit shims from other users unless you know they’re good – Non-spinning and higher order (spinning) shims should not change dramatically from sample to sample for most applications Lock: lock gain recommended not recommended higher lock gain Lower lock level due to lower lock gain may easily lose lock; change in lock level (during shimming) is less visible Lock: avoid high lock power Bad lock Good lock Lock power okay Lock power too high unstable lock and lower level Evaluate shims • Look for a sharp peak – No clear distortion – Full width at half height should be about 1 Hz or less for small molecules – Small (1% or smaller) or free of spinning side-bands • Check if peak distortions are individual or universal • Make sure that phasing is not causing peak distortions • Maximize the lock level – Higher lock level => better shim • Lock level does not drop significantly when spinning is turned off Shim by line-shape Plot made by G. Pearson, U. Iowa, 1991 z4 too small z4 too big make z4 smaller first Understanding NMR • Modern NMR spectrum is an emission spectrum • Equilibrium state – Magnetization is along +z axis – It is desired to have the largest +z magnetization prior to excitation • Excitation by a RF pulse – A projection of magnetization is made on xy plane – It is desired to have the largest xy plane project for observation • Observation – Precession of the projected xyplane magnetization RF pulses • RF pulse manipulates spins – Important in excitation and decoupling – Defined by length, power and shape • RF power is expressed in decibels – Bruker • Power range: typically 0db (high power) to 120db (low power) – Varian: • Coarse power: typically 60db (high) to 0db (low); 1 db increment; absolute • Fine power: 4095 (high) to 0 (low); default is 4095; relative – e.g. 54.5db can be roughly achieved through setting coarse power to 55 and fine power to 3854 in out coarse attenuator fine attenuator RF pulse calibration • Hard pulse (high power pulse) can be calibrated directly or indirectly • For best calibrations, pulses need to be on resonance (know the chemical shift or resonance frequency!) • Soft or shaped pulsed can be first calculated and then fine-tuned to optimum – Shapetool (by Bruker) or Pbox (by Varian) can be used for calculation and simulation – Be aware of possible minute phase shift (several degrees for soft pulses), which can be critical in water flip back or watergate Proton pulse calibration • Most hard (highest power) 90° pulses are typically from 5 us to 20 us. • Direct observation for high power proton pulse calibration (or even for heteronuclei if sensitivity is sufficient) – 360° method (not quite sensitive to radiation damping or relaxation) – 180° method 90º First pulse with 2 us; 2 us increment 90º 180º 360º 450º 270º 360º 180º 270º NMR observables • Chemical shifts – expressed in ppm • Scalar couplings – expressed in Hz – 2D or nD bond correlations • NOEs / relaxation / line-shapes • Peak size – Potentially useful in quantitative analysis Chemical shifts • Reflects chemical environment: – Ring current effect • Outside of ring: high ppm • Inside: low ppm – Effect of electron withdrawing groups • Donating: low ppm • Withdrawing: high ppm Chemical shifts of solvents/impurities Gottlieb et al. JOC 1997 Example: aliasing okay sw=16ppm aliased (from arx300) aliased from 0 ppm with phase distortion, because the peak is out of the “detection window” • Oversampled proton spectrum on higher fields (500 – 800 MHz) does not have the aliasing issue: peaks outside of sw will disappear Spectral aliasing (cont’d) • In direct observe dimension, spectral aliasing is generally avoided by either increasing spectral width (sw) or moving center frequency (sfo1) • Sometimes the indirect detection dimension (in nD spectrum) may intentionally adopt aliasing to improve resolution in that dimension Scalar coupling 1 1:1 1:2:1 1:3:3:1 AB system “roofing” JAB JAB dA dB Scalar coupling: simulation helps! pro-chiral! Ha 8Hz O O R P O 12Hz Hb 8Hz These are not impurities! O Ar Ha and Hb are not exactly equivalent, with chemical shift difference of 0.025ppm Observed (300 MHz) simulated Example: satellites and spinning side-bands 6.6 Hz; 29Si satellites; 2.3% each spinning sideband; 20 Hz from center 120 Hz; 13C satellites; 0.55% each TMS Dipolar coupling: NOE • NOE depends on correlation time (molecule size) and resonance frequency NOE + 1 • NOE does not always enhance the observed signal 3 2 13C 1 31P 1H 0 -1 0 2 4 6 8 10 15N -2 H P -3 C N -4 w htc Molecule size Temperature NOE implication in Quantification • The observed nucleus should be free of interference from other nuclei • Pre-saturation in aqueous samples may not be appropriate for accurate quantification – Small molecules tend to gain signal size due to positive NOE from saturated water – Large molecules tend to lose signal size due to spin diffusion Relaxation • T1 relaxation allows magnetization to recover back to +z axis – Nuclei with larger gyromagnetic ratios (resonance frequencies) tend to relax faster • 1H: 0.1 – 10 s (proteins have short T1’s) • 13C, 15N, 31P: much longer than 1H – Nuclei in a proton rich environment tend to relax faster • T2 relaxation contributes to the observed resonance line-shape – T2~T1 for small molecules – Line-shape offers an estimate of T2 Line-shape • Full Width at Half Maximum is 1/(pT2*) Hz, with T2* as apparent spin lattice relaxation time • Magnetic inhomogeneity (shim) can increase FWHM (2l) or distort the line-shape (reduce T2*) • T1 > T2 > T2* • Small molecules – – • FWHM (2l) 1H: T1 ~ T2 in the order of seconds seconds to tens of seconds; even longer if no proton attached (CO and quaternary) 13C: -20 Large molecules – – -15 -10 -5 0 5 10 15 20 of f se t ( H z ) 1H: T1 ~ T2 hundreds of mini-seconds or shorter 13C: seconds or sub-seconds Lorentzian: A(w)= l / (l2 + (w-w0)2) 2l=1/(pT2*) multiple chemical environments: chemical or conformational exchange • Fundamentally, chemical shift reflects chemical environment surrounding a nucleus’ • Multiple chemical environments may alter chemical shift or even cause significant peak broadening Fast exchange slow exchange Jin, Phy. Chem. Chem. Phys. (1999) (N)H line-shape: influence of relaxation and scalar coupling In addition to chemical exchange, (N)H proton line-shape is also influenced by the coupled nucleus 14N JNH ~65 Hz Slow 14N relaxation (compared to JNH) medium14N relxation Fast relaxation this might be the very reason why CHCl3 proton appears as a singlet though JH-35Cl and JH-37Cl exist Improving Sensitivity • More scans in a given amount of time Tc/T1 sensitivity • Use Ernst angle a for excitation: cos a = exp(-Tc/T1) • Increase concentration with less solvent / salt Pulse angle (degrees) Improving sensitivity • Receiver gain needs to be maximized which requires good water suppression • Excessive acquisition time end up with spending time collecting noise and downgrade signal to noise ratio S/N • Avoiding excessive large receiver gain (for signal clipping) sensitivity vs receiver gain (arx300; chloform signal) 0 100 200 300 receiver gain 400 500 Missing a carbonyl carbon presumably due to insufficient relaxation R' O OR About 5 mg in CD3OD. 2800 scans (~4 hrs) OH NH OH O ? Missing a carbonyl Solution: Use H2O Why it works: R' O OR OH NH OH O • Carbonyl 13C is reduced due to presence of a proton rich environment in H2O. • Potential intra-molecular hydrogen bond is weakened or broken, and decoupled from ring movement In H2O:D2O (1:1). 1400 scans (~2 hrs). Direct observe: 31P, 13C or 15N • 19F, 31P • 13C and 13C can be observed directly on all PINMRF 300 and 400 MHz instruments (please follow local PINMRF instructions) can be observed on higher fields (500 MHz and above), without any cable change • Drx500-2 with BBO probe offers higher sensitivity for 31P, 13C, 15N and most other heteronuclei (19F excluded) – Observed nucleus needs to be cabled to x-broadband preamplifier – BBO tuning is needed for both proton and observed nucleus – Double check filters if re-cabled Direct observe: 31P, 13C or 15N • Satellite peaks can frequently be indirectly observed in proton spectrum (so that we know the less sensitive heteronuclei are there to be observed directly!) • Decoupling of proton may improve signal by – Sharper peaks – NOE – Proton channel has to be tuned! 1D acquisition for very long hours • helpful – – – • Split long experiments into smaller blocks and save data regularly (multiple data can always be summed if needed) Dissolve the compound in water (H2O) might be helpful (shorter relaxation time) Lower sample temperature may help Not helpful – – Save several days’ data into one single FID Use 300 ul or less volatile solvent From 1D to 2D FT 1D w1 t2 time domain frequency domain FT(t2) 2D FT(t1) w1 t1 t1 t2 time domains w2 w2 frequency domains 2D NMR • Correlate resonances through bond or space – COSY: coupling • Magnitude mode recommended. • 1 mg or less will do • Minutes to a couple of hours – TOCSY: coupling network • ~ 70 ms mixing time • 1 mg or less will do • An hour or longer – NOESY / ROESY: distance / NOE • Mixing time ranging from less than 100 ms (proteins) to 500 ms (small molecules) • 1 mg or more • Hours or longer – HSQC/HMQC: proton correlation to X, typically through one-bond scalar couplings (two or three bond correlation possible) • 1mg or less will do • An hour or longer – HMBC: proton correlation to X, through multiple bond scalar couplings • 1 mg or more • Hours or longer 2D NMR • Resolve overlapping peaks – Resolution is provided largely through the indirect dimension – No need to have highest resolution in the direct detected dimension • Limit direct acquisition time to 100ms or less if heteronuclear decoupling is turned on • Lower decoupling power if longer acquisition time is needed – Change in experimental conditions may help 2D NMR essentials: acquisition • • Proton tuning and matching Calibration of proton (90 degree) pulse length – – • Modest receiver gain – • • • Larger td1 improves resolution in the indirect dimension Rarely exceeds 512 (except occasionally in COSY Detection method in the indirect dimension – – • • The pulse program recommends NS (a integer times 1, 2, 4, 8 or 16) Needs some dummy scans, especially with decoupling / tocsy Number of increments in the indirect dimension (td1) – – • rg about half of what rga gives or less Carrier frequency (center of spectrum in Hz) and SW (sweep width) in both dimensions (avoid aliasing unless intended to) Number of scans (NS) – – • Standard pulse lengths can be used if the solution is not highly ionic (< 50 mM NaCl equivalent) All proton pulses are likely getting longer if the solution is ionic and/or the probe is not tuned Determined by the pulse program Typically is either states (and/or TPPI) or echo-antiecho Acquisition time (aq) less than 100 ms with decoupling Modest gradients (cannot be more than the full power of 100% and typically less than 2 ms in duration) Go through the pulse program if you really care 2D processing • Window functions – – • Zero filling – • Typically double data points in each dimension Phasing – – – • Allow FID approach zero at the end of the acquisition time Sine bell functions with some shifts are recommended most of the time Indirect dimension zeroth and 1st order corrections are recommended in the pulprogram. If not, use 0 for both Direct dimension first order phase is rarely more than 50 degrees. Zeroth order can be anywhere from 0 to 360 degrees Phase in the 2D mode to best appearance Referencing – – Can be done by picking a known resonance in the spectrum Or referenced by (external) protons HSQC: a Block Diagram • Magnetization transfer pathway: F1(H) -> F2(X) -> F2(X,t1) -> F1(H) -> F1(H,t2) 90 H 180 1/4J 1/4J 1/4J 1/4J acq 180 90 t1/2 X t1/2 dec INEPT States: =x and =y are acquired for same t1 and treated as a complex pair in Fourier transform. No need to change receiver phase TPPI: =x, y, -x and –y are acquired sequentially in t1, and receiver phase is incremented too. Real Fourier transform. HMQC or HSQC codeine Magnitude HMQC (9 mins) Phase sensitive HSQC (18 mins) Easy set up and slightly higher sensitivity Better resolution adapted from acornnmr.com HMQC and HSQC comparison • HMQC • HSQC – Fewer pulses – More tolerant to pulse mis-calibrations – More pulses – Less tolerant to pulse mis-calibrations – Allows homonuclear (proton) coupling in the indirect dimension – No homonuclear (proton) coupling in the indirect dimension Data Presentation • Processed data can be readily viewed, manipulated and printed by xwinplot (wysiwyg) • Xwinplot can readily output .png, .jpg or .pdf files for publications or presentations • Files can be transferred through secure ftp Pulse sequence: the heart and soul of NMR label ;zggpwg ;this is a bruker sequence prosol relations=<triple> #include <Avance.incl> #include <Grad.incl> "d12=20u" 1 ze 2 30m d1 10u pl1:f1 p1 ph1 50u UNBLKGRAD p16:gp1 d16 pl0:f1 (p11:sp1 ph2:r):f1 4u d12 pl1:f1 (p2 ph3) 4u d12 pl0:f1 (p11:sp1 ph2:r):f1 46u p16:gp1 d16 4u BLKGRAD go=2 ph31 30m mc #0 to 2 F0(zd) exit ph1=0 2 ph2=0 0 1 1 2 2 3 3 ph3=2 2 3 3 0 0 1 1 ph31=0 2 2 0 ;comments for parameters… Delay only; be very careful with critical command in a labeled line Delay 90x 1H 180x 90-x 90-x define f1 power level 90° pulse on f1 G Gradient pulse Shaped 90° pulse Acq. and go to label 2 Write to disc. And go to label 2 Phases On-res: dephased by two gradients Off-res: refocused by two gradients Where Things are: Bruker File Structure • • • • • • • • • User NMR data Pulse programs Gradient programs Shaped pulses decoupling Frequency(f1) lists Parameter sets Shim sets Macros /u/data/username/nmr /u/exp/stan/nmr/lists/pp /u/exp/stan/nmr/lists/gp /u/exp/stan/nmr/lists/wave /u/exp/stan/nmr/lists/cpd /u/exp/stan/nmr/lists/f1 /u/exp/stan/nmr/par /u/exp/stan/nmr/lists/bsms /u/exp/stan/nmr/mac Gradients • Homospoil gradients – Size of duration may not matter much – Stronger ones tend to clean up unwanted magnetization better • Gradient echoes: – Exact ratios between multiple gradients must follow – Diffusion loss must be considered for small molecules, especially during long echoes • Log of signal size is proportional to -g2g2d2D Simulations • Can be easily performed for pulses, spinsystems or pulse sequences • Save experimental time • Enhance our understanding of NMR • Most frequently used for shaped pulses Shaped Pulse: What and Why • What – Narrow sense: amplitude modulation only, while phase is constant – Broad sense: amplitude and phase modulation • Why – To achieve perturbation over a certain frequency range (uniform and selective) • Narrow bandwidth: shaped pulse. e.g. Gaussian • Wide bandwidth: adiabatic pulse How is Shaped Pulse Different • Composite pulse is typically a block of square pulses with constant phases – Pulse integration does not correlate with pulse angle – Pulse calibration come from individual component • Adiabatic pulse sweeps frequency (phase has strong time dependence) – Pulse integration does not correlate with pulse angle – Pulse calibration depends on sweep range, and somewhat on adiabaticity too • Simple shaped pulse can be calibrated by integration – Caveat: a 180° pulse is not necessarily twice of a 90° pulse – Some shaped pulses are good for 180° inversions (z -> -z) while others are good for 90° excitations (z -> x/y) Shaped Pulse Examples • Square pulse: simplest shaped pulse; good for simple hard excitation • Gaussian and Sinc: good selectivity; for proton Gauss • Gaussian cascade: G4, G3, Q5 and Q3; for carbon – – – – G4 for excitation G3 for inversion Q5 for 90° Q3 for 180° Sinc1 G4 G4: four Gaussian lobes Choosing Shaped Pulses • Define the goal – excitation, inversion or refocusing – length or power level • Rule of thumb: bandwidth is ~ 1/P360 or RF strength (for square pulses) – shape • Power requirement – peak power may not exceed certain level • Length requirement – Be aware of probe limit on length in case of high power – While longer pulses tend to have better selectivity, relaxation / scalar coupling may limit pulse length • Run pulse simulation and calculation – Bandwidth needs to be first satisfied – Simulated frequency profile is to have top-hat behavior – Phase needs to be linear in the region of interest Shaped Pulse Calculation • Rule of thumb: – 6db change in power results two fold change in pulse length DdB = 20 log (P90/P90ref) – e.g. 10us @0db => 20us @6db for the sample pulse angle • For a shaped pulse with a imperfect linear amplifier, DdB = 20 log (P90*shape_integ/P90hard*comp_ratio) Modern spectrometers have comp_ratio close to 1 • Adiabatic pulses require different treatments Example: Setting up a Sinc Pulse • Within xwinnmr, launch shape tool by typing “stdisp” or from menu • Within shape tool, choose shapes -> sinc. Change lobe number to 1 and click “OK” • On the left is the amplitude profile (sinc shape) and (constant) phase is shown on the right 1 means one sinc lobe Example: a Sinc Pulse (cont’d) • • • • • Within shape tool, choose analyze -> integrate pulse. Make necessary updates. In this particular case, we assume the reference is 9.5 us @1.5db and you wish to calculate for 1000us 90 degree pulse. Then click OK The power level is calculated as 35.8db compared with the reference. Click “seen” If satisfied, you can save this shaped pulse under /u/exp/nmr/stan/lists/wave/. Go back to xwinnmr->ased, and update the sinc1 shaped pulse as pulse length of 1ms, and power level to be 35.8 + 1.5 (since reference 9.5us is @ 1.5db) = 37.3db If needed, the shaped pulse power can be fine tuned by gs, or a careful calibration Pulse Simulation • Within shape tool, choose analyze -> simulate. Update the length as 1000us and rotation angle as 90 (for sinc1 we just set up). Click “OK”. • A new Bloch module will show default (x,y) profile for excitation. Click on z to view z profile. z Pulse Simulation (cont’d) • If you decide that the starting magnetization is x, you can click (in Bloch module) “calculate”>”excitation profile”. Change initial Mx to 1 and Mz to 0. Click “OK” and then the excitation profile will be updated. • If you wish to examine trajectory (how a magnetization at a given frequency responds to the sinc1 pulse), you can click “time evolution”, and update initial values etc (may not allow too many steps). Click “OK”. Advanced NMR Training 10-12 noon, 8/18 (Monday) BROWN 3106 Contact Huaping Mo for details Demo • • • • • • • Sample preparation; Shigemi tube Lock and shim Tune and match Calibration of 90 degree pulse Calculation / simulation of pulses Set up 2D: COSY and HSQC Processing and present data (xwinplot)
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