2D NMR Spectroscopy Overview BRUKER
last edit 2/21/12
‘Essential’ 1H-detected 2D Pulse Sequences: COSY – COrrelation SpectroscopY, good for determining basic connectivity via Jcouplings (through-bond). TOCSY – TOtal Correlation SpectroscopY, same as COSY, but also able to generate cross peaks via intermediate spins (mix). Uses a spin lock that produces rf heating of the sample and hence requires many steady state scans (ds). (see separate training guide for TOCSY. NOESY – Nuclear Overhauser Effect SpectroscopY, allows one to see through-space effects, useful for investigating conformation and for determining proximity of adjacent spin systems. Not so useful for MWs in the 1 kDa range due to problems arising from the NMR correlation time. ROESY – Rotational Overhauser Effect SpectroscopY, same as NOESY, but works for all molecular weights. Has the disadvantage of producing more rf heating, hence it requires more steady state scans. HMQC – Heteronuclear Multiple Quantum Correlation, allows one to pair NH or CH resonances. Often uses X-nucleus decoupling and hence gives rise to rf heating, requires reasonably well calibrated pulses and many steady state scans. HSQC – Heteronuclear Single Quantum Correlation, provides the same information as HMQC, but gives narrower resonances for 1H-13C correlations. Also requires Xdecoupling and hence a large number of steady state scans and is also more sensitive to pulse imperfections. HMBC – Heteronuclear Multiple Bond Correlation, a variant of the HMQC pulse sequence that allows one to correlate X-nucleus shifts that are typically 2-4 bonds away from a proton. Information of individual 2D pulse sequences can be found in the TopSpin Menu [Help]→[NMR GUIDE] Experiment gCOSY gNOESY ROESY gHMQC gHSQC gHMBC
Parameter Set 2D-gCOSY-dcif 2D-gNOESY-dcif 2D-ROESY-dcif 2D-gHMQC-dcif 2D-gHSQCdcif 2D-gHMBC-dcif
Pulse Program cosygpqf noesygpph roesyetgp hmqcgpqf hsqcgpph hmbcgplpndqf
Minimum NS 1 2 8 1 1 1
PLEASE NOTE: If any of the dcif parameter sets have been accidentally deleted, you can load the original Bruker parameters sets by typing rpar. Just make sure the correct pulse program is loaded in the Acqu Pars tab.
Bruker Pulse Program Abbreviations qf gp ph et si nd pr
absolute value gradient pulse phase sensitive phase sensitive (Echo/Anti-Echo TPPI) sensitivity improved no decoupling presat
Summary of Methodology In other words, what you need to do. 1.
Set up variable temperature control (if needed).
2.
Lock, tune, and shim.
3.
Acquire 1D 1H spectra, set: reference, sweep width, and transmitter frequency.
4.
Reacquire 1D 1H spectra with reduced sweep width, and then determine the number of scans required. Record parameter values.
5.
Repeat for 1D 13C spectra to run HSQC or HMBC (if needed).
6.
Calibrate the 90 degree pulse for 1H (if needed).
7.
Load 2D parameter set.
8.
Check 2D pulse program.
9.
Load prosol parameters and setup the reference, sweep width, transmitter frequency, number of scans, and the number of points.
10.
Set receiver gain, acquire.
11.
Transform 2D data, phase and load projections.
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1. Regulate the temperature (if desired). Make sure you have had Bruker VT training BEFORE adjusting the temperature. Open temperature controller: edte a. Select the Carrier Gas: Compressed Air (10-40 C) or Nitrogen. i. Turn off the compressed air (may keep 401 magnet legs on compressed air). The valve is closed when the handle is perpendicular to the pipe. ii. Turn on the nitrogen (see handout on wall by valves). b. Select: [ Corrections] and verify that no correction is applied. c. Select: [Ramp] enter a ramp rate of 2 degrees/min, enable ramp. d. Normal Conditions: [Main Display] i. Sample Temp= 20 C Thermocouple located below tube. ii. Target Temp= 20 C iii. Heater= OFF (Set Max = 10% ) iv. Gas Flow= 270 L/h v. Cooling= Empty e. Increase Gas Flow i. 270 L/h normal, 800 for high/low temp [+/-] a. Extreme temperatures will need a higher flow rate ii. Turn the heater [on] iii. Check the maximum heater power [Set Max] 10%. Increase the heater power if unable to obtain the desired temperature. f. Set temperature at 25 C. The liquid nitrogen dewar is not required for 25 C g. Within the edte window open [Monitoring] i. Use auto scale for both y-axis’: a. Left: Temperature b. Right: Heater Power ii. Let sample equilibrate for 5 to 15 minutes h. Open [Self tune], run Self-tune program
2. Lock, tune and shim. a. Check that the spinning is shut off. b. Shim the magnet: X, Y, Z1-Z5 c. Tune each channel in use during the 2D experiment i. 1H ONLY: COSY, NOESY, TOCSY ii. 1H and 13C: HSQC and HMBC
3. Collect a good 1D spectra Experiment 1 (EXPNO) a. Proton: Acquire a 1D and reference. Zoom in and display all proton signals leaving 0.5 ppm of baseline on each side. Type setsw (or click on the icon) to set the transmitter offset (o1p) and sweep width (sw).
b. Reacquire “reduced-sweep width spectra” with the number of scans (ns) needed to get good signal to noise and phase. This dataset will become the 1H projection. Write down the following values: o1p:______________ sw:______________ sr:______________ ns:______________ **These values will be used in F2 (direct) dimension.** Type the parameter in the command line, hit enter, and TopSpin will display the value for you. Write these parameters to experiment 10 - wrpa 10 (for pulse calibration). Experiment 2 c. X Nucleus (only if acquiring HMQC, HSQC, or HMBC), Acquire a 1D and reference. Zoom in and display all X signals leaving 0.5 ppm of baseline side. Type .setsw to set the transmitter offset (o1p) and sweep width (sw). d. Reacquire “reduced-sweep width spectra”. This dataset will become the X projection. Write down the following values: o1p:______________ sw:______________ sr:______________ **These values will be used in F1 (indirect) dimension.**
How to calibrate the 1H 90o Pulse on the Bruker NMRs Background Information: For many NMR experiments such as DEPT, TOSCY, NOESY, and HMBC, the pulse sequence requires that many specific pulses or a series of pulses (90o, 45o, 180o, etc.) be applied. Without properly calibrated pulses, many of these experiments will yield meaningless results, or most likely, fail outright. Since each compound (and each nucleus) has a different chemical environment, each had a distinct 90o pulse width (p1). The 90o pulse is defined as the duration, in microseconds, that the rf signal must irradiate your sample in order to tilt the magnetizations into the XY-plane, 90o away from the Z-axis of the NMR’s magnetic field. Another way to think of it is how long you must pulse in order to tip all the spins into the XY plane. This pulse is often referred to as the pi/2 pulse. The 90o pulse width for proton NMR experiments is about 10-20 microseconds on most modern spectrometers. The exact value of the 90o pulse width depends on the sample (nucleus, solvent, etc.) as well as the instrument (probe, transmitter power, etc.). It may be 5 microseconds long, 17 microseconds, or 35 microseconds, or some other number determined experimentally. For this reason, it is necessary to measure the 90o pulse for every sample you need to perform 2D experiments on. Lucky for us, the proton 90o pulse is typically quite similar for all the protons in your sample. Measuring the 90o pulse width is simple enough. Remember that the 90o pulse tilts the sample magnetization into the XY plane, which contains the detector. A simple pulse sequence of irradiate-observe should show a maximum for the pulse duration corresponding to a 90o pulse. Because it is difficult to discern maximum signal intensities by comparing similarly intense peaks (i.e. comparing an 89o, a 90o, and a 91o pulse.), we look at the 180o or the 360o pulse.
The 360o pulse corresponds to a ‘null’ – no signal is observed at this irradiation. Searching for this null is easier to determine and has the added advantage of minimizing the time required between pulses due to relaxation issues.
4. Calibrate 90 o pulse (if needed!). Experiment 10 (or any other new experiment) The Bruker nitty gritty:  re 10 and obtain a well-shimmed 1H spectrum.  Type p1, hit enter and notice the current value for the 90o pulse. Record p1 and pl1  Type pulprog zg. Typically, Bruker uses a 30o pulse (zg30) for a proton 1D. This resets this to a 90o pulse.  Change parameters (ns 1; ds 0; d1 60), reacquire, and phase. The value for d1 should be 5xs T1, hence using a value of 60 here is an estimate. If you have a slow relaxer or know your value for T1, you might want to set d1 to a larger value.  Fourier transform (ft) and phase (apk). Type dpl1 to set the display regions. Type phmod pk to use the same phase values for all spectra  Start the acquisition by executing the AU popt program. 1. Check Optimize button 2. Enter parameter to modify: p1 3. Choose optimum value: zero 4. Enter startval value: 8 5. Enter endval value: 64 6. Enter the number of experiments (nexp) 8 7. Enter the increment variation mode (varmod) lin(ear) 8. Enter parameter increment (inc) 8 9. Click [start optimize]
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In PROCNO 999, the finished array will be displayed, similar to Figure 1. On the screen, you should see a series of spectra that start positive, pass through a null at 180o, become negative, and pass through a second null at 360o. Estimate the point where the signal goes from negative values through zero then become positive. This is the location of your 360° pulse. (If you do not see a clear null at 360o, you may need to run popt again, adjusting the entered values.) Run your array again, to determine the 360 o pulse width  0.5 s (i.e. array 60 to 63 with an increment of 0.5) Calculate the 90o pulse by dividing the p1 value of the null by 4. Use this number for your p1 in your subsequent experiments on this sample. p1:______________ 90 o pulse pl1:______________ power level for p1
5. Load 2D parameter set. Experiment 100 (or any other new experiment) rpar Experiment gCOSY gNOESY ROESY gHMQC gHSQC gHMBC
Parameter Set 2D-gCOSY-dcif 2D-gNOESY-dcif 2D-ROESY-dcif 2D-gHMQC-dcif 2D-gHSQCdcif 2D-gHMBC-dcif
Pulse Program cosygpqf noesygpph roesyetgp hmqcgpqf hsqcetgpsi2 hmbcgplpndqf
Minimum NS 1 2 8 1 1 1
6. Check pulse program (in AcqPar tab) and make sure the correct one has be loaded (see above table). 7. Load the prosol parameters by typing getprosol. 8. Edit the basic parameters based on the information from the 1D experiments (the values you recorded in step #3). Homonuclear Experiments  ns Number of Scans  p1 Pulse width (us) (90 degree Pulse)  o1p F2 Transmitter frequency (ppm)  sw Sweepwidth (ppm). Enter the value for F2 and F1 dimensions.  sr Reference (Hz). Enter the value for F2 and F1 dimensions. Inverse experiments ( 1H vs 13C) require additional parameters  p2 Pulse width (us)  o2p F1 Transmitter frequency (ppm)  1 sw F1 Sweepwidth (ppm)  1 sr F1 Reference(Hz) Check these parameters, loaded during getprosol. Adjust as needed (eg. if you did measure the 90 o pulse).  pl1 Power Level p1  pl2 Power Level for p2 9. Check Experiment Specific Parameters (listed below) and adjust as needed. 10. Optional parameter changes  2 td F2 Number of points  1 td F1 Number of points 11. Set receiver gain rga and acquire zg. Use multizg to start multiple incremental experiments (if desired).
12. Data Processing a. Fourier Transform the data by typing xfb b. Phase the data. i. Type ph ii. Choose manual phasing iii. RMB on three peaks that span your spectrum iv. LBM on the “R” button to start phasing the rows 1. Click and drag on the “0” and “1” to adjust the zero and first order phasing 2. When finished, click save and return v. LBM on the “C” button to start phasing the columns 1. Click and drag on the “0” and “1” to adjust the zero and first order phasing 2. When finished, click save and return vi. Click return to go back to the spectra c. Homonuclear experiments ONLY may be symmetrized to reduce the noise. Type sym to symmetrize. 13. Load the 1D projections a. Type edc i. Fill out the name, EXPNO and PROCNO information for both F2 and F1. 14. When finished, remember to ro off, lock off, and eject (ej) your sample.
Experiment Specific Parameters (step #9) COSY  p1  pl1
90 degree pulse power level for p1 pulse
NOESY:  d8
mixing time, 400-500 ms for small molecules, 100-200 ms for larger
ROESY:  d?  ds
mixing time for dipolar or through-space interactions dummy scans to establish thermal equilibrium
HSQC  d1  CNST2
Delay time (average value for 1J(XH) will affect d24 1/(8 * 1J(XH))
See the NMR Guide in TopSpin for more details.