for structure elucidation of organic molecules

NMR

Theory

Basic Experiments

The Second and Third Dimensions

Neil K. Garg Stoltz Group Literature Meeting Stoltz Group Conference Room (268 Crellin)

Thursday January 9, 2003 at noon ------------------------------------------------------------------------

A Superconducting Magnet

superinsulation and high vacuum

NMR tube sample lift and spinner

liquid nitrogen

shim assembly (kept at room temp) probe

magnetic coils and liquid helium

Basic Theory:
Magnetic Field and Electromagnetic Radiation Some nuclei have spins No magnetic field
1. apply magnetic field spins are oriented randomly spins align either parallel or antiparallel to the magnetic field

With magnetic field

parallel spin is considered to be slightly lower in energy

2. irradiate with E proper frequency E

The magnetic nuclei are in resonance with the applied radiation!

A spin flip occurs!

------------------------------------------------------------------------

Basic Theory:
Frequency and Nuclei
irradiate with proper frequency

What is the 'proper frequency'???

1. the strength of the magnetic field

This depends on...

2. the identity of the nuclei These give rise to magnetic phenomenon: anything with an ODD number of protons ie. 1H, 2H, 1 4N, 19 F, 3 1P anything with an ODD number of neutrons ie. 13C

E=h These do not rise to magnetic phenomenon: anything with an EVEN number of both protons and neutrons ie. 12C, 16 O, 32S strength of

Basic Theory:
Consider the 1H nuclei Do all 1H nuclei absorb at the same frequency?
effective

applied

local

The moving electrons around a nucleus set up a tiny local magnetic field Thus, the effective magnetic field will be different for each 1 H nuclei
shielded, deshielded, upfield, downfield, etc...

The Chemical Shift

NMR data is reported using the delta scale: 1 = 1 part per million (ppm) of the spectrometer operating frequency ie. 60 MHz instrument ie. 300 MHz instrument 1 1 = 1 ppm of 60 MHz = 60 Hz = 1 ppm of 300 MHz = 300 Hz

or ppm units are constant regardless of the spectrometer operating frequency

------------------------------------------------------------------------

Acquisition
Two Basic Methods
1. keep magnetic field constant, then vary electromagnetic radition 2. keep electromagnetic radiation constant, then vary magnetic field

BUT, acquisition time can be LONG

(especially with 13C NMR, or dilute samples)

Two important developments

1. signal averaging - increased sensitivity background noise will average out to zero nonzero signals will stand out clearly 2. Fourier Transform - increased speed

Fourier Transform
The sample is placed in a magnetic field of constant strength, then irradiated with a short burst / pulse of energy that covers the entire range of useful frequencies.

All 1 H nuclei resonate at once, giving a complex composite signal that must be mathematically manipulated using Fourier Transforms

This is FAST since all resonance signals are collected at once

For the mathemetically inclined...

The Fourier transform, in essence, decomposes or separates a waveform or function into sinusoids of different frequency which sum to the original waveform. It identifies or distinguishes the different frequency sinusoids and their respective amplitudes

------------------------------------------------------------------------

The Pulse Sequence

The basic 1H NMR Experiment:

pw = pulse width controls angle, and thus the strength of the pulse

2D experiments have much more complicated pulse sequences

Spin-Spin Splitting / Coupling

the local magnetic field produced by one nucleus affects the magnetic field felt by neighboring nuclei

consider

CH3 CH2 Br

Some rules to remember:

CH2
app lied

1. chemically equivalent protons do not show spin-spin splitting

fro m CH 3 pr otons

2. the signal of a proton that has n equivalent neighboring protons is split into a multiplet of n+1 peaks with coupling constant J. (usually only 2 carbons away, but secondary interactions can also be observed)

3. two groups of protons coupled to each other have the same coupling constant J. 1 3 3 1

n + 1 rule : protons that have n equivalent neighboring protons show n + 1 peaks

-----------------------------------------------------------------------

Some Typical Coupling Constants

J (Hz) Ha C Ha 0 Hb Ha C C Ha Ha C C Hb 6-8 C 2-30 Ha C C C Hb Hb 12-18 Ha Ha Ha Ha C C Hb 4-10 C Hb Hb Coupling between two adjacent hydrogens is sensitive to the dihedral angle between them 0-1 Hb 2-3 C Hb Ha 0-3 Hb Ha 1-3 0-1 J (Hz) Ha Hb 6-12 J (Hz) Ha Hb 6-8 J (Hz)

For 2 adjacent hydrogens on sp carbons

Sample preparation
How much solvent volume should I use?
To get good resolution you need at least 0.7 mL of solvent in a 5 mm NMR tube. If you have a limited amount of sample you can increase its effective concentration by reducing the solvent volume to 0.4 - 0.5 mL, however you will need to spend more time shimming.

What about a Shigemi Tube?

Can use 0.3 mL of solvent!!!

+

...but pretty expensive

glass the magnetic susceptibility of the glass is adjusted to that of your solvent your average tube

the Shigemi tube

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More on NMR tubes

Cleaning and Drying
Clean NMR tubes by rinsing with solvent. Be careful if scrubbing with a brush since scratches can lead to bad spectra. Air or dessicator drying is recommended. Oven drying can lead to deformities in the NMR tube! These deformities can lead to bad spectra and destroy the probe-coil insert. If you insist on oven drying, place in the oven lying flat for no longer than 1 hour.

Breaking a tube
Notify Dr. Lee, Tom Dunn, or a GLA ASAP! The probe must be removed and cleaned immediately! Leave a note warning others not to use the spectrometer. Prevention: always use the depth gauge (samples centered too low can destroy the probe-coil insert) make sure NMR tubes are good quality and are in good condition

Tuning the Probe

When an instrument sends out a pulse, it comes at the NMR tube at a certain angle (ie. 90 ) If poorly tuned, the probe will reflect a lot of the power of the pulse This gives bad signal to noise! Especially for more advanced experiments

If using a 300 MHz instrument, you do NOT need to tune the probe For higher field strength instruments, tuning should be done on each individual sample Frequency, solvent, sample height, NMR tube quality are all factors that affect tuning If you see no peaks in your spectrum (not even solvent), chances are that the probe is not tuned sufficiently

There are typically two channels to tune: 1." H, F " or "high band channel" 2. "X-channel" or "broadband channel" or "lowband channel"
1 19

On a 300Mhz instrument...
1

H 300.08 N 30.4
31

19

F 282.3
13

15

P 121.5

C 75.5

------------------------------------------------------------------------

Locking
Purpose:
magnetic fields and frequencies are not very stable over long periods of time locking provides a special field and frequency stabilization

What happens?
A separate experiment is always run in parallel to the one you are running By locking on a deuterium signal from the solvent, resonance is held at a set position

How to lock?
1. Make sure the LOCK is turned OFF 2. Increase the LOCKPOWER and LOCKGAIN and look for a sine wave by adjusting Z0 3. Adjust the Z0 until the frequency of the wave becomes zero 4. Reduce the LOCKPOWER and LOCKGAIN so the LOCK LEVEL is not 100 5. Turn ON the LOCK, then repeat step #4 if necessary

LOCKPOWER and LOCKGAIN must be balanced. This varies by solvent. In general, higher gain and less power. If Z0 is set wrong, your deuterated solvent peak will need to be referenced after the acquisition.

Problems Encountered with Locking

"There is no lock signal"
1. are you using a deuterated solvent? 2. sometimes this happens after the instrument is rebooted type rts('standard') load='y' su It may also be necessary to logout, then log in again

"The lock is lost as soon as I turn ON the LOCK"

the LOCKPHASE is probably not maximized this can happen when the instrument is rebooted try to turn OFF the LOCK, then maximize the signal by adjusting LOCKPHASE, then turn the LOCK back ON

"The lock signal is very weak"

this can happen if the shims are way off reload the standard shim file by typing rts('standard'), followed by su

"The lock signal is very noisy or fluctuates"

this usually means that the signal is saturated saturation - more lock power is sent to the sample than it can dissipate To Check: reduce the LOCKPOWER by 6, and the lock level should drop by 50%. If it drops by significantly less that 50%, the lock is saturated If saturated, reduce the LOCKPOWER

------------------------------------------------------------------------

The Art of Shimming

What is it?
the shim system: a set of coils focused around the sample region that cause very specific magnetic field contours

Purpose?
Optimizes magnetic field homogeneity high resolution

Types of Shims
Spinning Shims Z0, Z1, Z2, Z3, Z4, Z5, etc... we typically adjust these when shimming

Non-Spinning Shims all others ie. x, y, zx, zy, xy, etc... typically not adjusted responsible for spinning sidebands

Shimming Procedure

Quick Shimming on 300MHz Instruments

1. reload standard shims, type rts('standard') su 2. Maximize lock level by adjusting Z1C 3. Maximize lock level by adjusting Z2C, then return to Z1C 4. repeat 1 and 2 until no further improvement can be made 5. if changes in shims are large, repeat Lock procedure

Fine Course

------------------------------------------------------------------------

The Art of Shimming

The optimization processes are:

Zero Order - This is a straightforward process of adjusting the control for the best response.

First Order - Adjust one control, then the next control, then the next until all controls in the set have been adjusted. Repeat the process until no further response improvement can be obtained.

Second Order - Note the current response level. Adjust a shim control from the current value a defined amount (a rule of thumb would be enough to change the response to between 50% and 75% of the original value) to a new value. Optimize all other shims in the set with a first order process. If the new response is better than the previous response, note the new response level and adjust the him to a new value in the same direction and repeat the process. If the new response is less than the original response, adjust the shim control a defined amount in the opposite direction and repeat the process. Continue until the best value is clearly determined. This means it is necessary to go too far to make sure no further improvement can be made and then return to the optimum value.

The Art of Shimming

Advanced Technique: First Try

If the NMR spectrometer is in a state of unknown homogeneity or is known to have poor homogeneity, a simple optimization of certain shims is the first step in the shimming sequence. A swept NMR signal is the recommended method of judging the response for this operation: * Spin the sample (20-30 Hz) and adjust the Z1 and the Z2 shims interactively to produce the tallest swept signal response (first-order process). * Stop the spinner and adjust X and Y for the tallest swept signal response (first-order process). * Adjust X and ZX for the tallest swept signal response (second-order process). * Adjust Y and ZY for the tallest swept signal response (second-order process). * Adjust XY and X2-Y2 for the tallest swept signal response (first-order process). * If any large shim changes were observed in the above process then the process should be repeated from Step 1.

After the above procedure, the NMR instrument should be capable of a field/frequency lock.

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The Art of Shimming

Advanced Technique: Spinning Shims
The adjustment procedure for the spinning shims should be conducted with the sample spinning at more than 10 Hz. Care should be taken at all times to avoid a vortex. A vortex leads to a false shim optimum, with Z2 usually being the most misadjusted. If the lock signal is being used for shimming, then ensure that the lock signal is not being partially saturated with rf power and that the lock phase is correctly adjusted. The lock phase should be reexamined each time a large change is made in an even-order Z shim. In the adjustment of Z3, Z4, and Z5 described below (steps 2, 3, and 4), it is best to make a plot of the response level versus the shim under adjustment. If the operator is careful to proceed far enough past the maximum response position for the shim under adjustment, then the plot should reveal a broad curve. The best position for the shim can then be determined even if an interpolation between two sample positions is necessary. With experience, this plotting becomes an automatic mental process. Also, confidence is gained that all the shims were correctly optimized when a broad smooth curve is obtained as a result of this process. * Use the first-order process to optimize Zl and Z2. * Use the second-order process to optimize Z3. Note the position of Z3 and the response. Change Z3 enough to degrade the response by 20-30%. Repeat the process in Step 1. If the new position for Z3 has yielded a better response, then continue in the same direction. If the new response is poorer, then try the other direction for Z3. * Use the second-order process to optimize Z4. Note the position of Z4 and the response. Change Z4 enough to change the response by 30-40%. Repeat the process in Step 1. Adjust Z3 to provide the optimum response. If the Z3 shim changes considerably, then repeat Step 1 again and readjust Z3 again for maximum response. If, after optimizing Z3, Z2, and Zl, the new response is better than the previous response, then continue in the same direction. If the response is worse, then try the other direction.

The Z5 shim is difficult to adjust for two reasons. First, only probes with longer coils give significant response change with Z5 owing to its strong dependence on distance. Second, the Z5 shim often has more Zl, Z2, Z3, and Z4 components than a Z5 component in its correction. The Z5 shim normally needs to be adjusted only with wide-bore magnet systems with large-diameter tubes or with longer coil probes. To adjust Z5, note its position and the response. Change Z5 enough to lower the response by 30-50%. Repeat Step 1. Adjust Z3 for the maximum response. Adjust Z4 for the maximum response. If either Z3 or Z4 changed a considerable amount, repeat Step 1 and reoptimize Z3 and Z4. If the new response obtained after this procedure is better than before, continue in the same direction. If the response is worse, try the other direction with Z5.

The Art of Shimming

Advanced Technique: Nonspinning Shims
The nonspin shim set should be adjusted while the sample is not spinning. Changing the nonspin shims which have Z components causes changes in the spinning shim set. If any of these shims change significantly, then the spinning shim sequence should be repeated after completion of the nonspinning sequence. With all shims involving a second-order process, the technique described under the spinning shim sequence of plotting the result and interpolating the shim position should be followed either on paper or mentally. * Adjust X and Y interactively using the first-order process for maximum response. * Use a second-order process to adjust ZX. Note the position of ZX and the response. Change ZX by enough to lower the response 10% and adjust X for a maximum response. If the new response is better, continue in the same direction with ZX. If the response is less, try the opposite direction with ZX. * Repeat Step 2 but using the Y and ZY shims. * Adjust XY and X2-Y2 interactively using the first-order process for maximum response. If either XY or X2-Y2 changed significantly, then repeat Steps 2 and 3. * Use a second-order process to adjust Z2X. Note the position of Z2X and the response. Change Z2X by enough to decrease the response 30%. Maximize the response with ZX. Maximize the response with X. If the new response is larger than the initial response, then continue with Z2X in the same direction. If the response is less, then try the opposite direction. Repeat Step 5 but using Z2Y, ZY, and Y. * Use a second-order process to adjust ZXY. Note the position of ZXY and the response. Change ZXY enough to decrease the response by 20%. Maximize the response with XY. If the new response is larger than the initial response, continue with ZXY in the same direction. If the response is less, try the other direction. * Repeat Step 7 but using Z(X2-Y2) and X2-Y2. * Adjust X3 and X interactively for maximum response (first-order process). * Adjust Y3 and Y interactively for maximum response (first-order process). If the nonspin shim settings have significantly changed, then the spinning shim sequence should be repeated. If there are significant changes in the spin set after optimization, repeat the nonspin set also.

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Problems with Shimming

"All of the peaks are distorted in the same way"
This means that your shims are off The distortions you see are typically as follows:

Z1

Z2

Z3

Z4

Z5

Sidebands are usually a result of the nonspin shims

"Still won't shim"

Inspect sample tube: scratches? warped? homogeneous sample? sample centered? enough solvent? Report problem in log book in case of reoccurance

Gradient Shimming
A More Recent Development

1. Create a shimmap - an image of the magnetic fields homogeneity 2. Run gradient shimming (an automated process) the computer calculates the required changes for good homogeneity then find the optinum homogeneity (Hg3 and the 600MHz(BI) are currently our only spectrometers that are setup for gradient shimming)

A typical shimmap:

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The Acquisition
Parameters to set:
bs= block size ie. 2 or 4 nt= number of scans ie. 16 d1= relaxation time ie. 1 - 10 (10 to get more accurate integration) pw = pulse width affects the pulse angle, which affects the amount of energy being put into the sample; use default settings sw = sweep width determines the frequency range that your spectra will include autoshim=n autolock=n gain= the sensitivity of the receiver (usually use default setting or n)

What is "ADC OVERFLOW" ?

Signal from the NMR is amplified by the receiver, then digitized by the ADC (analog to digital converter) If the signal is too strong, the "ADC OVERFLOW" message is displayed

The Solution - try these one at a time:

1. check the value gain is set at by typing "gain?", then set the gain to a lower value 2. type gain='n' which turns on the "autogain" 3. reduce the pw value by 50% 4. dilute your sample

Starting, Restarting, and Stopping the Acquisition

Some useful commands
go = starts the acquisition ga = starts the acquisition and does a 'wft' at the end sa = stops the acquisition after finishing the next block ra = resumes the acquisition if the "sa"command was used to stop it aa = stops the acquisition immediately

How do I add scans to an acquisition in progress?

type: sa nt=____ (set this to the total number of scans you desire) ra

How do I add scans to a completed acquisition?

1. Acquire data in exp1 2. type: mp(2) jexp2 nt=extra_nt go 3. Once that experiment is complete, or when you have accumulated enough extra signal, type: clradd jexp1 add jexp2 add jexp5 setvalue('nt',<nt + extra_nt>,'processed') setvalue('ct',<ct + extra_ct>,'processed') wft 4. Your combined file will be created and displayed in exp5

------------------------------------------------------------------------

Dilute Samples
1. Can the sample be made more concentrated? 2. Is there a stronger magnet available? ie 500 or 600MHz? 3. Try line broadening! The good side: signal to noise ratio increases The bad side: line width increases small coupling constants will not be observed

How to line broaden

1. Put a cursor on a peak 2. type dres the digital resolution will appear (ie. 0.293) 3. set lb < 0.293 ie. type lb=0.29 4. type wft

line broadening is also very useful for 13 C spectra you may also want to try playing with the other parameters under the 'process' tab

Decoupling Experiments
Sometimes coupling interactions make spectral assignment difficult You can remove an unwanted coupling interaction with a decoupling experiment! How it works?
While an experiment is running, you heavily irradiate the problematic protons. This causes rapid transitions between spin states for each proton being irradiated. The spin averages to zero and there is no observable coupling.

How to run a decoupling experiment on VNMR

1. Take a normal proton NMR spectrum 2. type: HOMODEC (all caps) 3. type: dm='y' 4. type: homo='y' 5. type: dmm? (make sure dmm=ccc) 6. type: dpwr=28 7. type: ss= -2 8. type: fn=16k 9. type dps to display the pulse sequence 10. return to the original proton spectrum, select the peak, and type: sd 11. acquire the new spectrum, take as many scans as needed for the normal proton 12. watch out! the spin sometimes shuts off

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Advanced 13C Experiments

APT
Attached Proton Test Signals of CH1 and CH3 groups are positive Signals of CH0 and CH2 groups are negative N O S O

Advanced 13C Experiments

DEPT
Distortionless Enhancement by Polarization Transfer DEPT 45: all protonated carbons DEPT 90: CH only DEPT 135: CH3 , CH positive CH2 negative

CH3 carbons

N CH2 carbons O S O

CH carbons

all protonated carbons

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Advanced 1H Experiments
NOE
Nuclear Overhauser Effect Commonly used for stereochemical assignment 1H-1H homonuclear through space correlation

Irradiate one peak and the resonance becomes saturated. This affects the intensity of other signals that are close in space (within 5 A) R which diasteromer? olefin geometry of enal? BC CH3 Ph H OH Ph R H O H O O CH3 BC O O O O

[H] Team Saudin

O O O

which diastereomer?

1D NOE: BC in Action

CH3

H OH

irradiate this proton!

T irradiate

Methyl shows anti-periplanar relationship vinyl H phenyl H

CycleNOE Experiments (I500)

type: jexp1 Take a regular proton NMR type: mf(1,2) type: unlock(2) jexp2 type: wft type: cyclenoe Place cursor in the middle of the peak that you want to irradiate type: sd type: satfrq=dof type: dn='C13' Expand the peak you want to irradiate so that it takes up half of the screen type: pattern=1 (for singlet) or pattern=2 (for doublet), etc. Place the left cursor just to the left of the most downfield portion of the peak Place the right cursor just to the right of the most upfield portion of the peak type: axis='h' type: spacing= (type the number at the bottom right of the window, labelled delta) type: axis='p' type: tau=0.1 type: satpwr=-10 type: nt=(some multiple of) 64 type: jexp1 type: wft place cursor in a region with no peaks type: sd (remember the number displayed at the top of the screen type: jexp2 type: control=(the number displayed after you typed 'sd' in experiment 1) turn spinner off and reshim to maximize lock signal type: time (this gives you approximate time of experiment) type: go Process the same way you process a proton nmr spectrum, except do not use autophase (aph)

NOE experiments using glide

Manually lock and shim on your sample In VNMR, start the glide program, then click SETUP

A window will open. Change the experiment setting to "H1 and selective 1D Expt" by right clicking on the arrow next to "Experiment" Choose your solvent, ignore "Sample" shut off AutoLOCK and AutoSHIM

then click "setup" at the bottom of the window

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NOE experiments using glide

Another window will open click Acquire

Yet another window will open. These are the parameters for your normal proton NMR that will run prior to the NOE experiment specify parameters

click NOESY1D This opens another window!!!

NOE experiments using glide

Specify the number of scans

Choose the mixing time the mixing time is directly related to the distance between protons longer mixing time longer distances

typically a mixing time of 500 ms gives distances of 3-4 angstroms Click OK when finished setting NOE parameters

You will return to the proton NMR parameters window

click Do

Your standard proton NMR spectrum will be acquired!

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NOE experiments using glide

When you HNMR acquisition has completed, your spectra will show up on the screen. Expand the peak that you wish to irradiate and put two cursors around the peak Click select, then Click PROCEED

The NOE experiment will run Exit glide and type ds You will now be able to process your NOE data

NOTE: integration ratios can be correlated to the distance between nuclei

2 Dimensions: 1H-1H Homonuclear Through SPACE Correlations

NOESY - 2D NOE has a dependence on molar mass and viscosity signals can be positive or negative, and can sometimes disappear

ROESY - 2D ROE (Rotating frame Overhauser Enhancement) use for FW=1000-3000 g/mole

NOESY of strychnine

The diagonal consists of the 1D spectrum The cross peaks represent NOE effects between two protons

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2 Dimensions: 1H-1H Homonuclear Through BOND Correlations

COSY
Correlation Spectroscopy commonly used for regiochemical assignment Cross-peaks appear if spin coupling is present Protons that are separated by 2 or 3 bonds are usually detected

DQCOSY
Double Quantum Filtered Correlation Spectroscopy COSY with water supression commonly used for COSY experiments of proteins, peptides, and carbohydrates in water solution

TOCSY or HOHAHA
Total Correlation Spectroscopy or Homonuclear Hartmann Hahn Very similar to COSY EXCEPT: TOCSY can give a total correlation of all protons in the same spin system not just 2 or 3 bonds away! (spin systems are separated by heteroatoms) Very useful for structural assignment of peptides or oligosaccharides

Total Correlation is possible by varying the "mixing time" longer mixing time longer distances

2 Dimensions: COSY of Strychnine

The diagonal consists of the 1D spectrum The cross peaks represent through bond coupling interactions

2 or 3 bonds only

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2 Dimensions: TOCSY of Strychnine

The diagonal consists of the 1D spectrum The cross peaks represent through bond coupling interactions

All protons in the same spin system (mixing time dependant)

2 Dimensions: 1H-13C Heteronuclear Through 1 BOND Correlations

HMQC
Heteronuclear Multiple Quantum Coherence Cross signals are observed for protons and their directly bounded carbons (1 bond only) 1H is the observed nucleus

HSQC
Heteronuclear Single Quantum Coherence 1H is the observed nucleus Very similar to HMQC, can sometimes be superior in the case of a crowded
13

C spectrum

HETCOR
Heteronuclear Correlation Also for protons and their directly bonded carbons (1 bond only) 13C is the observed nucleus Usually 2-4 times slower than HMQC and HSQC

-----------------------------------------------------------------------2 Dimensions: 1H-13C Heteronuclear Through MULTIPLE BOND Correlations

HMBC
Heteronuclear Multiple Bond Correlation Cross signals are observed for protons and their long range coupled carbons (2-3 bonds) 1H is the observed nucleus

1D and 2D NMR for Structure Determination: A Practical Example

O OCH3 O OCH3 N2 0.001M in THF 254-400nm 2 hours H H desired H CH3 C O OCH3

2 products with very similar structural data are isolated What are they? Diastereomers? Regioisomers? Product A = higher Rf Product B = lower Rf

Richmond Sarpong

------------------------------------------------------------------------

A Closer Look at Product A (higher Rf)

10 1. IR shows carbonyl at 1668 cm-1 9 2. Mass spec is consistent with proposed structure 3. HNMR is consistent with proposed structure

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

12 Tentative assignment

CDCl3

2 7

11

10 8 9

A Closer Look at Product B (lower Rf)

10 1. IR shows carbonyl at 1706 cm-1 9 2. Mass spec is consistent with proposed structure 3. HNMR is consistent with proposed structure Maybe some more advanced experiments can help

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

Tentative assignment

12

5 CDCl3 2 8 4 9 10

7 6

11

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Product A, (higher Rf): 13C NMR

10 13C NMR Shows the correct number of protons

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

But this spectrum is still somewhat difficult to assign

Product A, (higher Rf): DEPT

10 With the help of DEPT experiments, we can begin to assign the 13C spectrum This will help w/ heteronuclear 2D NMR 9

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

CH3 carbons

CH2 carbons

CH carbons

all protonated carbons

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Product A, (higher Rf): 13C NMR Another Look

10

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

9 With the hel p of DEPT, a tentative assignment of the 13C spectrum can be made

CDCl3

76 2 3 1

11

12 4 8

9 10

Product A, (higher Rf): COSY

Both the COSY and TOCSY spectra can assist HNMR assignment For example: The Vinyl peaks appear to be correctly assigned Notice that #6 is interacting with 4, 7, and even 2 while #7 is interacting with #6, but no interaction with #4 is present 2 7 6 10 9

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

4 6 7 2 2

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Product A (higher Rf): TOCSY

Both the COSY and TOCSY spectra can assist HNMR assignment Remember: COSY involves 2 or 3 bonds TOCSY can include all interactions within the same spin system (but this depends on the mixing time) 9 10

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

Product A (higher Rf): HMQC

HMQC is very helpful in assigning the 13C spectrum 10 Remember: Cross signals are observed for protons and their directly bounded carbons (1 bond only) Example: The vinyl proton peaks are easily correlated to the appropriate carbon atoms 9

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

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Product A (higher Rf): HMBC

Remember: Cross signals are observed for protons and their long range coupled carbons (2-3 bonds) HMBC is crucial for this exercise! The methyl protons interact with carbons #3, #4, and #6 This proves the regiochemistry assigned is in fact correct! 10 9

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

Product B, (lower Rf): 13C NMR

10 13C NMR ALSO Shows the correct number of protons

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

But this spectrum is still somewhat difficult to assign

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Product B, (lower Rf): DEPT

10 With the help of DEPT experiments, we can begin to assign the 13C spectrum This will help w/ heteronuclear 2D NMR 9

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

CH3 carbons

CH2 carbons

CH carbons

all protonated carbons

Product B (lower Rf): 13C NMR Another Look

10

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

9 With the hel p of DEPT, a tentative assignment of the 13C spectrum can be made

CDCl3

6 72 3 1

11

4 12 8

9 10

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Product B, (lower Rf): COSY

Both the COSY and TOCSY spectra can assist HNMR assignment For example: How can we correctly assign the vinyl protons #6 and #7? Vinyl proton #6 interacts with the methyl, while #7 does not. Rather, #7 seems to be interacting with #8 6 7 10 9

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

5 8

Product B (lower Rf): TOCSY

Remember: COSY involves 2 or 3 bonds TOCSY can include all interactions within the same spin system (but this depends on the mixing time) The TOCSY, in this case, picks up many more interactions than the COSY. For instance, #11 interacting with #2, #8, and #10 11 10 9

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

10 8

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Product B (lower Rf): HMQC

HMQC is very helpful in assigning the 13C spectrum 10 Remember: Cross signals are observed for protons and their directly bounded carbons (1 bond only) Example: The vinyl proton peaks are easily correlated to the appropriate carbon. 9

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

Product B (lower Rf): HMBC

For product A, we observed an interactions between the protons of #5 and carbons 3, 4, and 6 For product B, the #5 protons are interacting with both carbon #6 and carbon #7 But there is no interaction between the #5 protons, and carbons #3 and #4 There's something funny going on here... 10 9

12 OCH3 11 1 8 H 7 ??? 6 2 3 4 CH3 5 O

6 3

7 4

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What is Product B?
All of the spectral data supports that product A is the desired product The data for product B suggest that it is a regioisomer of the desired product mass spec, IR, HNMR, 13CNMR, and some 2D techniques are OK HMBC, in particular, does not seem to make sense 10 9

12 OCH3 11 1 8 H 7 6 2 3 4 CH3 5 O

If HMBC is supposed to reach only 2-3 carbons away, how are the methyl protons interacting so strongly with both carbons #6 and #7? #6 is 3 bonds away and #7 is 4 bonds away!

Instead of an allylic methyl, consider a vinyl methyl. R #6 #7 H2 C #5 H

Now, the #6 carbon is 2 bonds and the #7 carbon is 3 bonds away from the methyl protons. This makes more sense.

Looking back at the COSY and TOCSY, there was no interaction between the supposed #4 and #5 protons. This supports that the allylic methyl substitution pattern is incorrect.

What is Product B?
10 9 R H2 C H

12 OCH3 11 1 8 H 7 6 2 3 4 CH3 5 O

this seems to be part of the structure

All of the spectral data indicate that the following fragment is also present:

OCH3 O R

R H

------------------------------------------------------------------------

What is Product B?
There are not too many options left at this point: OCH3 This structure agrees with all spectral data and could potentially arise from a Norrish Type I Fragmentation of Product A 10 9

12 OCH3 11 1 8 H 7 6 2 3 4 CH3 5 O

O H

Product A

CH3

In fact, re-exposure of Product A to the initial reaction conditions results in the formation of "Product B"

OCH3 OCH3 O 0.001M in THF CH3 H Product A 254-400nm 2 hours H CH3 Product B CH3 OCH3 O O H

3 Dimensions
OR ... 2 2D Experiments
HMQC-COSY A 3D spectra containing the HMQC C-H correlations and the COSY H-H correlations Thus the 3 axes are 1H, 1H, and 13C

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3 Dimensions
OR ... 2 2D Experiments
HMQC-TOCSY A 3D spectra containing the HMQC C-H correlations and the TOCSY H-H correlations Thus the 3 axes are 1H, 1H, and 13C

HMQC-NOESY A 3D spectra containing the HMQC C-H correlations and the NOESY H-H correlations Thus the 3 axes are 1H, 1H, and 13C

3 Dimensions
HMQC-TOCSY
Since 2 of the 3 axes are the same (2 1 H axes), varian software avoids the cube representation Instead, the HMQC correlations are shown in blue and the TOCSY correlations are shown red

1 2

red

1 H3 C

2 NH NO2

blue NO 2

Example: The HMQC correlation between proton #2 and carbon #2 is shown in blue Since proton #1 is in the same spin system as proton #2, carbon #2 shows a correlation to proton #1 which is red

-----------------------------------------------------------------------Summary of Key Advanced Experiments

NOESY and ROESY HMQC-COSY or HSQC-COSY

COSY HMQC-TOCSY or HSQC-TOCSY

TOCSY HMQC-NOESY or HSQC-NOESY

HMQC, HSQC, HETCOR DEPT 13C Experiments

DEPT 45: all protonated carbons

HMBC

DEPT 90: CH only DEPT 135: CH 3 , CH positive CH2 negative DEPT Full Edit

How Do I Run 2D and 3D Experiments?

For the average organic chemist, the easiest way is ALWAYS to let VNMR run the experiment for you! 2 options: Glide (all instruments) and Walkup (Hg3 only)

choose your experiments

then customize the experiment parameters

These pre-programmed experiments set a number of default parameters that are correct for most organic molecules. However, there will be exceptions. You should see the administrator if these experiments do not work on your sample.

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Software Upgrades Are Nice

email your spectrum

There are separate queues for Daytime and Nightime

Lots of experiments! Including solvent suppresion techniques & gradient experiments

Gradient Experiments
Varian Software often has options for gradient experiments These experiments are typically faster than then non-gradient analogs

Most of the 2D experiments will ask you to specify both "Scans per inc" and "number of inc"

If you are running non-gradient experiments, you should choose 'scans per inc' > 4 For gradient experiments, 'scans per inc' can be minimal ie. 2,4 The 'number of inc' will vary depending on sample concentration

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Saving Spectra as Picture Files 1. JDesign

After processing your spectrum, type jdesign at the VNMR command line Wait a few seconds, as it takes a few seconds to launch this program

Click 'Preview' Then 'All' Your spectrum will be displayed

Functions add / edit text resize spectrum save as picture file jpeg, gif, bmp, pcx, ps, tiff, etc.

Saving Spectra as Picture Files 2. Snapshot

With this program you can take pictures of any window, and thus of spectra Right click on the solaris desktop Highlight 'Applications' then 'Snapshot' Click Snap when you are ready to take a picture You will be asked to pick the window you want to snap

The image you select will show up in the image viewer Click 'File', choose 'save as' You can save the picture as a tiff file

-----------------------------------------------------------------------

Saving Spectra as Picture Files 3. Postscript Files

Type your normal print commands, except include the filename as shown below: ex. pl pscale ppf page('filename.ps') your file will be saved as a postscript file in the currect working directory

There is a catch!
You must have a postscript printer selected first. On any spectrometer: click 'main menu', click 'more', click 'configure' click 'Show Output Devices' Find the PS plotter name Click 'Select Plotter' until that PS plotter name has been selected Now print to a postscript file as described above Reset the plotter to laserjet instead of PS by following the instructions described above, but be sure that the laserjet printer is chosen when you 'select plotter' On Hg3: type 'ps' to select the postscript plotter type 'lj' to reselect the laserjet plotter

References
Books (and references therein)
150 and More Basic NMR Experiments by Braun, Kalinowski, and Berger Basic One and Two Dimensional NMR Spectroscopy by Friebolin Organic Chemistry by Jones Organic Chemistry by McMurray NMR Spectroscopy : Basic Principles, Concepts, and Applications in Chemistry by Harald Gnther Fundamentals of Nuclear Magnetic Resonance by Hennel and Klinowski ABCs of FT-NMR by Roberts Structure Elucidation by Modern NMR: A Workbook by Duddeck and Dietrich

Websites (and references therein)

http://web.chem.queensu.ca/FACILITIES/NMR/ http://fbr1.usask.ca/guide/eNMR/w34b.html http://nmrsg1.chem.indiana.edu/NMRguide/ http://biomol.uchsc.edu/researchFacilities/nmr/ http://nmr.chem.ualberta.ca/AOWWW/index.htm http://web.mit.edu/speclab/www/nmr_links.html#four http://www.varianinc.com

A Few Other Resources:

Dr. Lee Chris Brandow Scott Ross Uttam Tambar (NOE) Jeremy May (NOE) Jen Love (NOE,Kinetics, etc...) Sarah Tully (decoupling) Jarrett Farias (Varian) Varian