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NMR Training for Advanced Users
NMR Training for Advanced Users
Overview
Overview
A schematic Diagram for NMR
A schematic Diagram for NMR
Insider scoops: a systematic approach for NMR quantitation
Insider scoops: a systematic approach for NMR quantitation
Common Misconceptions
Common Misconceptions
Outlines for this talk
Outlines for this talk
Sample
Sample
Samples of smaller volumes
Samples of smaller volumes
Sensitivity for smaller volumes
Sensitivity for smaller volumes
Tune and match the probe
Tune and match the probe
Significance of tuning/matching
Significance of tuning/matching
Recognizing Bruker probe types
Recognizing Bruker probe types
Lock
Lock
Shims
Shims
Lock: lock gain
Lock: lock gain
Lock: avoid high lock power
Lock: avoid high lock power
Evaluate shims
Evaluate shims
Shim by line-shape
Shim by line-shape
Understanding NMR
Understanding NMR
RF pulses
RF pulses
RF pulse calibration
RF pulse calibration
Proton pulse calibration
Proton pulse calibration
NMR observables
NMR observables
Chemical shifts
Chemical shifts
Chemical shifts of solvents/impurities
Chemical shifts of solvents/impurities
Example: aliasing
Example: aliasing
Spectral aliasing (cont’d)
Spectral aliasing (cont’d)
Scalar coupling (J)
Scalar coupling (J)
Scalar coupling: simulation helps
Scalar coupling: simulation helps
Example: satellites and spinning side-bands
Example: satellites and spinning side-bands
Relaxation
Relaxation
Line-shape
Line-shape
Lorentzian peak Integration
Lorentzian peak Integration
Multiple chemical environments: chemical or conformational exchange
Multiple chemical environments: chemical or conformational exchange
(N)H line-shape: influence of relaxation and scalar coupling
(N)H line-shape: influence of relaxation and scalar coupling
Examples of (N)H resonance
Examples of (N)H resonance
Direct observe: 31P, 13C or 15N
Direct observe: 31P, 13C or 15N
Direct observe: 31P, 13C or 15N
Direct observe: 31P, 13C or 15N
Dipolar coupling: NOE
Dipolar coupling: NOE
NOE implication in Quantification
NOE implication in Quantification
Improving sensitivity: receiver gain
Improving sensitivity: receiver gain
Improving sensitivity: Ernst angle
Improving sensitivity: Ernst angle
Missing a carbonyl carbon presumably due to insufficient relaxation
Missing a carbonyl carbon presumably due to insufficient relaxation
Solution: Use H2O
Solution: Use H2O
1D acquisition for very long hours
1D acquisition for very long hours
Chemical shift referencing
Chemical shift referencing
From 1D to 2D
From 1D to 2D
2D NMR
2D NMR
2D NMR
2D NMR
2D NMR essentials: acquisition
2D NMR essentials: acquisition
2D processing
2D processing
HSQC: a Block Diagram
HSQC: a Block Diagram
HMQC or HSQC
HMQC or HSQC
HMQC and HSQC comparison
HMQC and HSQC comparison
Data Presentation
Data Presentation
Pulse sequence: the heart and soul of NMR
Pulse sequence: the heart and soul of NMR
Where Things are: Bruker File Structure
Where Things are: Bruker File Structure
Gradients
Gradients
Simulations
Simulations
Shaped Pulse: What and Why
Shaped Pulse: What and Why
How is Shaped Pulse Different
How is Shaped Pulse Different
Shaped Pulse Examples
Shaped Pulse Examples
Choosing Shaped Pulses
Choosing Shaped Pulses
Shaped Pulse Calculation
Shaped Pulse Calculation
Example: Setting up a Sinc Pulse
Example: Setting up a Sinc Pulse
Example: a Sinc Pulse (cont’d)
Example: a Sinc Pulse (cont’d)
Pulse Simulation
Pulse Simulation
Pulse Simulation (cont’d)
Pulse Simulation (cont’d)
Demo
Demo
Backup slides
Backup slides
Safety
Safety
Xwinnmr: Spectra addition/subtraction
Xwinnmr: Spectra addition/subtraction

Презентация: «Про автора джон харвуд». Автор: huaping mo. Файл: «Про автора джон харвуд.ppt». Размер zip-архива: 2247 КБ.

Про автора джон харвуд

содержание презентации «Про автора джон харвуд.ppt»
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1 NMR Training for Advanced Users

NMR Training for Advanced Users

Huaping Mo Summer, 2009 http://www.pinmrf.purdue.edu http://people.pharmacy.purdue.edu/~hmo/index.htm

2 Overview

Overview

Our facility PINMRF: Purdue Interdepartmental Facility (http://www.pinmrf.purdue.edu) Staff include John Harwood (director, D.Sc), Huaping Mo (associate director, Ph.D.), Jerry Hirschinger (engineer) and other members. Our capabilities 800MHz, 600MHz, 500 MHz (2), 400 MHz(2) and 300MHz (4) Observe almost all NMR active nuclei Detect as low as 1 mM to as high as 100 M proton concentrations VT from -80°C to above 100°C* Many problems can be converted to and addressed by NMR observables! Our expertise Various 1D, 2D and 3D experiments Novel pulse sequence development and simulations Structural determinations Quantitative analysis NMR hardware trouble shooting and repair Our track record Mo, Harwood et al. J. Magn. Reson., in press; Ye, Mo et al. Anal. Chem. 2009; Mo & Raftery Anal. Chem. 2008; Bai, Mo, Shapiro, Bioorg Med Chem, 2008; Mo & Raftery J. Bio. NMR, 2008; Mo & Raftery J. Magn. Reson. 2008 Acknowledged in a number of publications (Thank you!) Determined the structures for a series of novel natural products / metabolites

3 A schematic Diagram for NMR

A schematic Diagram for NMR

R

g(RG)

receiver gain function

receiving efficiency

NMR signal: A = A0 * h * c * V *R *sin(q)*I(q)*g(RG)

excitation

t90

Mz

transmitter

probe tuning network

q

receiver

observation

pulse sequence

pulse calib/angle q

RF properties; I(q)

spectral analysis

spectral processing

Shims; chemical shift; coupling; line-shape; relaxation; exchange; h, c & V

© Huaping Mo, 2009

raw FID

FT

FID

ADC

4 Insider scoops: a systematic approach for NMR quantitation

Insider scoops: a systematic approach for NMR quantitation

Receiving efficiency (Mo et al. J. Magn. Reson. in press) conceptually, it is similar to extinction coefficient in UV spectroscopy in characterizing how efficient a unit magnetization can be detected 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 Receiver gain function (to be submitted) how much gain is actually achieved by the receiver Solvent signal offers a universal and robust concentration internal standard (Mo & Raftery Anal. Chem. 2008) Normalized NMR signal size is strictly proportional to the concentration for a given sample, regardless how concentrated or dilute the sample is Unit magnetization generates the same amount of total NMR response in the RF coil, which is indifferent to chemical shift or line-shape No need to make additional internal or external standard

5 Common Misconceptions

Common Misconceptions

You need to prepare either an internal or external concentration reference for quantitative NMR You need a chemical shift referencing compound in a hetero-nuclear spectrum A compound has to be in a deuterated solvent to be observed by NMR You need to separate the compound to find out if it is right or how much is there

6 Outlines for this talk

Outlines for this talk

Basic preparations for NMR: safety, sample, lock, shim and tune Understanding NMR: excitation and observation RF pulse calibration Data acquisition: sweep width, carrier freq. and # of scans etc. NMR observables: Chemical shift, scalar couplings, NOE and relaxations Chemical shift referencing Introduction to basic 2D's Simulations for spin systems, pulses and sequences Basic operation demonstrations

7 Sample

Sample

RF coil

Basic requirements: Proton observe: 1 uM or more Cabron observe: 1 mg or more Volume: 300 ul or more Solvent: most solvents will do;10% deuterated solvent is sufficient for locking Spectrometers can be run without lock (deuterium). 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!

~18mm

Coil Center

? 20mm

8 Samples of smaller volumes

Samples of smaller volumes

Follow rule # 1 and then rule #2 Shimming might be challenging due to air/glass and air/solution interfaces Bubbles should be avoided Consider Shigemi tubes Be careful with spinning Non-spinning is recommended for volume ~ 300 ul or less 500 ul is sufficient for a regular tube

300ul

400ul

500ul

9 Sensitivity for smaller volumes

Sensitivity for smaller volumes

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 For a regular tube, larger volume helps shims/line-shape, but not sensitivity directly

10 Tune and match the probe

Tune and match the probe

Why do we need tune and match (wobb)? For best pulses and sensitivity Only higher fields (500, 600 and 800 HMz) in our facility need tuning Lower field probes have been tuned! Most of the time only proton requires tuning Drx500-2 with BBO needs special attention Proton always needs tuning BB (used for 13C or 31P etc) channel needs tuning, by first setting the numbers to the pre-set values

RF reflection

tune

match

Frequency

Carrier frequency

11 Significance of tuning/matching

Significance of tuning/matching

Shorter 90° pulse More efficient use of RF power Protects transmitter More uniform excitation in high power Better sensitivity Reciprocity: if excitation is efficient, then detection is equally efficient Potentially quantitative: NMR signal size is about inversely proportional to the 90° pulse length

12 Recognizing Bruker probe types

Recognizing Bruker probe types

magnet

BBO probe on drx500-2

TXI probe

Side-view

Side-view

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

13 Lock

Lock

Lock depends on shims: bad shims make bad lock Initialize shims 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 auto-lock if multiple deuterium signals exist)

14 Shims

Shims

The goal of shimming 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) + lock field Shimming can be done either manually or by gradient, which can be very efficient and consistent if done properly Sample spinning may improve shims However, spinning-side bands appear Recommendations: 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

15 Lock: lock gain

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

16 Lock: avoid high lock power

Lock: avoid high lock power

Bad lock

Good lock

Lock power okay

Lock power too high

unstable and lower lock signal

17 Evaluate shims

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 Small (<1%) or no spinning side-bands

18 Shim by line-shape

Shim by line-shape

make z4 smaller first

z4 too small

z4 too big

Plot made by G. Pearson, U. Iowa, 1991

19 Understanding NMR

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 xy- plane magnetization

20 RF pulses

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

21 RF pulse calibration

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

22 Proton pulse calibration

Proton pulse calibration

Most hard (highest power) 90° pulses are typically from 5 us to 20 us. High power pulse for proton (or other heteronuclei if sensitivity is sufficient) is directly calibrated 360° method (not quite sensitive to radiation damping or relaxation) 180° method

90?

180?

q

270?

360?

90?

360?

450?

180?

270?

First pulse with 2 us; 2 us increment

23 NMR observables

NMR observables

Chemical shifts Expressed in ppm; reflects chemical environment Scalar couplings Expressed in Hz; causing splitting / broadenings in 1D 2D or nD bond correlations NOEs / relaxation / line-shapes Reveals distance/conformation information Peak size Potentially useful in quantitative analysis

24 Chemical shifts

Chemical shifts

Reflects chemical environment: Ring current effect Outside: high ppm Inside: low ppm Effect of electron withdrawing groups Donating: low ppm Withdrawing: high ppm

25 Chemical shifts of solvents/impurities

Chemical shifts of solvents/impurities

Gottlieb et al. JOC 1997

26 Example: aliasing

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

27 Spectral aliasing (cont’d)

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

28 Scalar coupling (J)

Scalar coupling (J)

Scalar coupling: proportional to gyromagnetic ratio through bond/electrons split into 2nI + 1 lines.

29 Scalar coupling: simulation helps

Scalar coupling: simulation helps

pro-chiral!

a

8Hz

12Hz

b

8Hz

These are not impurities!

ssb

Ha and Hb are not exactly equivalent, with chemical shift difference of 0.025ppm

simulated

Observed at 300 MHz

30 Example: satellites and spinning side-bands

Example: satellites and spinning side-bands

6.6 Hz; 29Si satellites; 2.3% each

ssb: 20 Hz or multiple of 20Hz from center

~120 Hz; 13C satellites; 0.55% each

TMS

31 Relaxation

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-width offers an estimate of T2

32 Line-shape

Line-shape

FWHM (2?)

Lorentzian: A(w)= ? / (?2 + (?-?0)2) 2?=1/(pT2*)

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 1H: T1 ~ T2 in the order of seconds 13C: seconds to tens of seconds; even longer if no proton attached (CO and quaternary) Large molecules 1H: T1 ~ T2 hundreds of mini-seconds or shorter 13C: seconds or sub-seconds

33 Lorentzian peak Integration

Lorentzian peak Integration

integration

n

n (2l)

-n (2l)

0

34 Multiple chemical environments: chemical or conformational exchange

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)

35 (N)H line-shape: influence of relaxation and scalar coupling

(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

Slow 14N relaxation (compared to JNH)

medium14N relxation

Fast relaxation

JNH ~65 Hz

this might be the very reason why CHCl3 proton appears as a singlet though JH-35Cl and JH-37Cl exist

36 Examples of (N)H resonance

Examples of (N)H resonance

800MHz

500MHz

300MHz

14N decoupling

no 14N decoupling

Hz

NH4Cl in DMSO. Triplet is due to 14N coupling (52 Hz)

Urea in water (6% D2O)

37 Direct observe: 31P, 13C or 15N

Direct observe: 31P, 13C or 15N

19F, 31P and 13C can be observed directly on all PINMRF 300 and 400 MHz instruments (please follow local PINMRF instructions) 13C 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 pre-amplifier BBO tuning is needed for both proton and observed nucleus Double check filters if re-cabled

38 Direct observe: 31P, 13C or 15N

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!

39 Dipolar coupling: NOE

Dipolar coupling: NOE

NOE depends on correlation time (molecule size) and resonance frequency NOE does not always enhance the observed signal

13C

31P

1H

15N

Molecule size

Temperature

40 NOE implication in Quantification

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

41 Improving sensitivity: receiver gain

Improving sensitivity: receiver gain

Receiver gain needs to be maximized which frequently requires good water suppression Avoiding excessive large receiver gain (for signal clipping) Excessive acquisition time may end up with spending time collecting noise and down-grade signal-to-noise ratio

42 Improving sensitivity: Ernst angle

Improving sensitivity: Ernst angle

Acquire more scans in a given amount of time Use Ernst angle a for excitation: cos a = exp(-Tc/T1) Increase concentration and lower the solvent / salt amount

Tc/T1

sensitivity

Pulse angle (degrees)

43 Missing a carbonyl carbon presumably due to insufficient relaxation

Missing a carbonyl carbon presumably due to insufficient relaxation

About 5 mg in CD3OD. 2800 scans (~4 hrs)

?

Missing a carbonyl

44 Solution: Use H2O

Solution: Use H2O

Why it works: 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).

45 1D acquisition for very long hours

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): multizg 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

46 Chemical shift referencing

Chemical shift referencing

1H chemical shift can be readily referenced by the solvent signal or TSP/TMS Heteronuclei can be indirectly referenced, by PROTON chemical shift! No need to have a separate internal or external reference

47 From 1D to 2D

From 1D to 2D

FT

1D

t2

w1

time domain

frequency domain

FT(t2)

FT(t1)

2D

w1

t1

t1

t2

w2

w2

frequency domains

time domains

48 2D NMR

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

49 2D NMR

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

50 2D NMR essentials: acquisition

2D NMR essentials: acquisition

Proton tuning and matching Calibration of proton (90 degree) pulse length 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 Modest receiver gain 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) 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) Larger td1 improves resolution in the indirect dimension Rarely exceeds 512 (except occasionally in COSY Detection method in the indirect dimension 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

51 2D processing

2D processing

Window functions Allow FID to approach zero at the end of the acquisition time Sine bell functions with some shifts are recommended most of the time Zero filling Typically double data points in each dimension Phasing Indirect dimension 0th and 1st order corrections are recommended in the pulse program. If not, use 0 for both to start with. First data point is typically scaled by 1 or 0.5, depending on the pulseprogram Direct dimension’s 1st order phase is rarely more than 50 degrees. 0th order can be anywhere from 0 to 360 degrees Phase in the 2D mode for best appearance Referencing Can be done by picking a known resonance in the spectrum by (external) protons

52 HSQC: a Block Diagram

HSQC: a Block Diagram

Magnetization transfer pathway: F1(H) -> F2(X) -> F2(X,t1) -> F1(H) -> F1(H,t2)

90

180

H

90?

180

X

dec

acq

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.

1/4J

1/4J

1/4J

1/4J

t1/2

t1/2

53 HMQC or HSQC

HMQC or HSQC

Magnitude HMQC (9 mins) Easy set up and slightly higher sensitivity

Phase sensitive HSQC (18 mins) Better resolution

codeine

adapted from acornnmr.com

54 HMQC and HSQC comparison

HMQC and HSQC comparison

HMQC Fewer pulses More tolerant to pulse mis-calibrations Allows homonuclear (proton) coupling in the indirect dimension

HSQC More pulses Less tolerant to pulse mis-calibrations No homonuclear (proton) coupling in the indirect dimension

55 Data Presentation

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

56 Pulse sequence: the heart and soul of NMR

Pulse sequence: the heart and soul of NMR

90x

180x

1H

90-x

90-x

G

On-res: dephased by two gradients Off-res: refocused by two gradients

Delay only; be very careful with critical command in a labeled line

label

Delay

define f1 power level

90° pulse on f1

Gradient pulse

Shaped 90° pulse

Acq. and go to label 2

Write to disc. And go to label 2

Phases

;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…

57 Where Things are: Bruker File Structure

Where Things are: Bruker File Structure

User NMR data /u/data/username/nmr Pulse programs /u/exp/stan/nmr/lists/pp Gradient programs /u/exp/stan/nmr/lists/gp Shaped pulses /u/exp/stan/nmr/lists/wave decoupling /u/exp/stan/nmr/lists/cpd Frequency(f1) lists /u/exp/stan/nmr/lists/f1 Parameter sets /u/exp/stan/nmr/par Shim sets /u/exp/stan/nmr/lists/bsms Macros /u/exp/stan/nmr/mac

58 Gradients

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

59 Simulations

Simulations

Can be easily performed for pulses, spin-systems or pulse sequences Save experimental time Enhance our understanding of NMR Most frequently used for shaped pulses

60 Shaped Pulse: What and Why

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

61 How is Shaped Pulse Different

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)

62 Shaped Pulse Examples

Shaped Pulse Examples

Square pulse: simplest shaped pulse; good for simple hard excitation Gaussian and Sinc: good selectivity; for proton Gaussian cascade: G4, G3, Q5 and Q3; for carbon G4 for excitation G3 for inversion Q5 for 90° Q3 for 180°

Gauss

Sinc1

G4

G4: four Gaussian lobes

63 Choosing Shaped Pulses

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

64 Shaped Pulse Calculation

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

65 Example: Setting up a Sinc Pulse

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

66 Example: a Sinc Pulse (cont’d)

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

67 Pulse Simulation

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

68 Pulse Simulation (cont’d)

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”.

69 Demo

Demo

Sample preparation; Shigemi tube Lock and shim Tune and match Calibration of 90° pulse Water suppression Calculation / simulation of pulses Set up 1D and 2D’s: mutizg; COSY and HSQC Data processing: addition and subtraction Data presentation: xwinplot

70 Backup slides

Backup slides

71 Safety

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 and be conservative Probe limits: avoid excessive long decoupling, hard pulses or their equivalents Double check pulse program and parameters for any non-standard new experiment. Pay special attention to power switch statements in the pulseprogram 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

72 Xwinnmr: Spectra addition/subtraction

Xwinnmr: Spectra addition/subtraction

Operations on processed data (spectra) have no impact on raw data edc2: define 2nd dataset (to be compared) and 3rd dataset (to save results into) dual: allow comparison

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