NMR Spectroscopy Short Course 2007


Experimental Guide for INOVA-400

NMR Spectrometer

Introduction:                NMR Experiment Procedures

Experiment 1:           1D proton and Basic Operations

Experiment 2:           1D Carbon and Basic Operations

Experiment 3:           Proton 90° Pulse Width Calibration

Experiment 4:           Carbon 90° Pulse Width Calibration

Experiment 5:           APT (Attached Proton Test)

Experiment 6:           DEPT

Experiment 7:           INEPT

Experiment 8:           Presaturation

Experiment 9:           COSY

Experiment 10:         HETCOR

Experiment 11:         1D NOE

Experiment 12:         1D Carbon with Complete Fluorine Decoupling by Using Wave Function Generator

Experiment 13:         Variable Temperature Experiment

 

Introduction:   NMR Experiment Procedures

Step 1. Login the Computer System

All modern NMR spectrometers are controlled by host computers. For the security and administration reason, all research groups using NMR in Emory University are issued their group usernames and passwords for the host computers. All authorized NMR users need to use your own group usernames and passwords to logon to the host computers.

Step 2. Change Sample

A standard CDCl3 or other deuterated sample is always placed in the magnet to keep the instrument in working condition when nobody is using it. Every user needs to replace the standard sample with your own sample at the beginning of each experiment and change back at the end of experiment.

Step 3. Lock and Shim the Magnet

Locking the magnet keeps its field constant and stable during data acquisition. Shimming the magnet makes it uniform and homogenous for a sample.

Step 4. Setup Parameters and acquire Data

Retrieve default parameters that are setup and updated for you by the NMR Center so that you may collect your data efficiently.

Step 5.  Process Data and Plot Spectrum

This step converts data into visible NMR spectra.

Step 6. Logout the Computer System

Your mission is completed and you need to do a few things for next user.

The step 1, 2, 3, and 6 are the same for all experiments. We will describe them in detail in Experiment One. The step 4 and 5 differ from experiment to experiment.

   

Experiment One: 1D Proton and Basic Operations

Sample to be used: 10% Strychnine in CDCl3

 

Step 1. Login the Computer System

1.1       Type in your username at login prompt-------- INOVA400> username

Type in your account password------PASSWD> password

The VNMR software will be loaded in several seconds with the display of four windows. The upper left window is used for entering commands and selecting menu buttons; the middle left for displaying NMR spectra and the lower left for displaying NMR parameters and other text messages. The upper right window is called “ACQUISITION STATUS” window, indicating the working status of the instrument, such as lock level, spin rate, temperature and acquisition time, etc.

Step 2. Change Sample

2.1            2.1.         Click on the Acqi or type acqi in the command line to display the fifth window at lower right, which is called “ACQUISITION” window. All the operations hereafter in Step 2 and Step 3 will be operated. There are CLOSE, LOCK, FID, SHIM and LARGE popup menu buttons, and Sample eject and Sample insert buttons. We will be focusing on the LOCK and the SHIM buttons first.

 
 

2.2            Click on LOCK button to display lock sub-window then click SPIN: off button to turn off sample spinning; click LOCK: off to turn off the lock.

2.3       After a few seconds, click on the SAMPLE: eject button to eject the standard sample onto the top of the magnet; remove the spinner, replace the sample with your own, use sample depth gauge to measure the correct tube position, put the spinner back onto the top of the magnet again. Caution: place the spinner with a sample on top of the magnet only when ejecting air is on.

2.4       Click on the SAMPLE: insert button to put the spinner back down inside the probe. Wait for 3 to 5 seconds, then click on the SPIN: on button to make the sample spin.

 

 

  Lock sub-window

 Step 3. How to Lock and Shim the Magnet

In this step, you need to adjust lock and shim parameters by using the mouse. Each parameter can be increased or decreased by 1, 4, 16 and 64 units as shown by corresponding –1+, -4+, -16+ and –64+ buttons. For example, when you place the cursor on a button, say –4+, if you click the left mouse button once, the corresponding parameter is decreased by 4 units; if you click the right mouse button twice, the parameter is increased by 8 units.

3.1            If you are using CDCl3 solvent, the magnet could be locked almost instantly most of the time. This is indicated by the display of lock signal plateau and a green LOCKED message below it. Then you directly go to step 3.3.

3.2            If you don’t see a plateau signal with a LOCKED message or you are using solvent other than CDCl3, click on LOCK: off button, adjust Z0 to make wave-like lock signal into plateau-like resonance, then click LOCK: on button. A green LOCKED should be shown. Then adjust the lock phase to make the lock level as high as possible. You need to reduce the lock power to 26-28 and lock gain 30-36 if the lock level excesses 100.  You may also check our record sheets on the desk. There are Z0, Lock power, Lock gain, and Lock phase values for different solvents recorded by previous users.

3.3       Now it is shimming time. There are as many ways of shimming as NMR users. We suggest you shim it this way:

                       

 

3.2.1       Click SHIM button to display shim sub-window. The lock level is shown in this window as two color bars and exact lock value is shown underneath the bars. In the middle of the window, there is one triangular button after ‘SHIM:’ which let you switch from shim sub-windows and lock sub-window by just click on it. Now it should show coarse z by the button.

 

3.2.2       Adjust Z1C (Z1 coarse) and Z2C by alternatively changing 1 or 4 units to increase the lock level as high as you can. If the lock level increases to 100, decrease lock gain and then continue to adjust Z1C and Z2C. Higher lock level indicates better homogeneity.

3.2.3       Click on triangular button to switch to fine z sub-window. Adjust Z1, Z2 and Z3 to make the lock level as high as possible.  We do not suggest you to adjust Z4 and Z5 at this stage. If you really want to adjust them, it is better write them down before you change them, so that you are able to go back in the case you get a worse shimming.

3.3            Finally click the CLOSE button to end the shimming procedure.

Tip 1. Reinstall our default standard shimming file.

If you have a very bad shim, type “loadshim” in the command window to reinstall our default shim file for the standard sample.

        

Step 4. Setup Parameters And Run 1D proton Experiment

4.1       Type h1 to load the standard parameter for proton with a default CDCl3 solvent. If your solvent is not CDCl3, you may simply type h1(‘solvent’), where the solvent may be one among acetone, DMSO, D2O, etc. You may also type h1 and then solvent=’DMSO’ etc.

 

Proton standard parameters.

 

4.2       Type nt=16 or 32, according to sample concentration.  

4.3       Type ga (submit to acquisition) to begin acquisition.

4.4            After acquisition is completed, a proton spectrum should be displayed on the screen now. If not, type wft (Weight and Fourier transform) to show it. Then enter aph (automatic phasing) to phase the spectrum. Type vsadj (vertical scale adjustment) to adjust the vertical scale of the spectrum automatically. Type dscale or click on Dscale button to display chemical shift scale under the spectrum.                                                          

Tip 2.  Manual Phasing  

If you need more improvement on the phase, you may manually phase the spectrum by clicking on Phase button first and then clicking on the region of interest and moving up or down the mouse while pressing LMB to adjust its phase. You may continue to click the cursor on the next region and adjust the phase same way. Clicking on the Box or Cursor button will exit the manual phase routine.

                                                           

Tip 3.  Manual Vertical Scale Adjusting  

Place the cursor above the base line and click middle mouse button to increase the vertical scale. Place the cursor below the base line and click middle mouse button to decrease the vertical scale.  

Step 5. Data Processing       

5.1       Zoom operation

Click LMB on the left side of a peak and RMB on the right side, two vertical red cursors appear at the click points. Then click the expend button on the menu bar, the region between the red cursors will be expanded. Just click on full button to get back to the full spectrum after you did expending, or type f then press enter. You may do this zooming operation as many times as you like.                                                

5.2       Set reference

After zooming the region containing the reference peak, click the LMB around the top of the peak, type nl to place the cursor on the top of the peak exactly. Then type rl(x.xxp) to set its chemical shift. For example, rl(4.80p) will set a peak frequency to 4.80ppm.

 

5.3       Integral operation

            Enter vp=20. Type ds cz to clear all the previous integral values in the buffer.

Click on Part Integral button for the full spectrum or its expanded region to display an integral curve. Then click on Resets button. Now click LMB at both sides of each peak or integral region to get separated integral lines of the spectrum.

 

If you know the proton number a specific peak presents, you may set the value for that integral as follows:  placing the cursor on that integral and click on Set Int. The computer will then ask what value to assign that integral. After you put the value for that integral, the computer will recalculate the value of every other integral. You are able to see these values by typing dpir.

                                                         

Tip 3.  Redo integral operation  

If you made a mistake during the integral operation, You can redo the integral operation by click on Full Integral and then No Integral button to clear the integral line. Then type cz, click on Part Integral button and repeat the procedure 5.3.

 

5.4       Peak picking operation

Type ds and click on Th buttons to display a yellow horizontal threshold line. Press and hold LMB to move the yellow cursor to the position you desire, then click on the Th button again. Type command dpf to display peak frequencies above spectrum peaks. 

Tip 4. Back to interactive adjustment

 During above operations, if you are not able to find the button you need or you are not be able to use red cursor, just click on Main manue > Display > Interact

 

5.5       Label your experiment data

Type ctext to clear any text label for the present experimental data.  Type text(‘Your sample name, date\\solvent, e’) to label the present experimental data. Command dtext  will display the text on the top left corner of your spectrum. 

5.6       Print and plot spectrum and related information

            pl---to begin print the present spectrum;

pap---to print all the parameters including the text labels, if any.

ptext---to print text labels without other parameters.

pscale---to print the spectrum scale in ppm or hz by axis=“p” or  axis=“h”.

            ppf---to print all the peak frequencies over the respective peaks.

            pll---to print a list of the frequencies and intensities of all the peaks in the spectrum.

            pirn---to print normalized integral values below the respective peaks.

            pir---to print proton numbers below the respective peaks.

            page ---to expel the paper from printer.

Remember, every printing procedure starts with pl and ends with page. You can put other printing commands selectively between these two commands.

 

5.7       Save your experimental data and reload your experimental data

            You may save your spectrum data.

The directory path is /export/home/nmrusr/Login_ID/ when you login. It is your group directory. You should make a directory of your own files.

Type mkdir(‘yourname’) to make a directory of your own. The computer automatically goes into your directory /export/home/nmrusr/Login_ID/yourname/. If it does not, click on Main manue > File then highlight your directory name and click on Set director. Remember you can go back to your group directory at any time by typing cd.

Type svf(’file_name’) to save your data in the form of FID.

5.8.          To reload your file, go to your directory first as described above then click on Main manue > File  > highlight your file > click on load.

 

Step 6. Logout the Computer System

6.1       Retrieve standard file and replace your sample with the standard.         

Type h1 to set up standard proton file. This is especially important after you have done a carbon-13 experiment. It will turn off the decoupling channel automatically.

            Then see Step 2 for details about replacing NMR sample tubes.

6.2       Lock and shim the magnet

See Step 3 for details. You are required to shim the magnet to the required level: lockpower=32, lockgain=40 with lock level of over 75%!!!

6.3       Exit the VNMR program

Type exit to close the VNMR program. Now you will see a full screen of Varian’s logo.

6.4       Exit UNIX system

Press the RMB to select Exit item in the popped up menu, release RMB. You will be prompted to select one of two choices: Exit or Cancel. Click on Exit to exit UNIX system. The screen will become blank and a prompt appears.

6.5       Logout

            Type logout. Sign off the logbook for the instrument.

  Congratulations!!! You have finished a complete 1D proton NMR experiment.

  

   

Experiment Two: 1D Carbon-13 and Basic Operations

Sample to be used: 30% Strychnine in CDCl3

 

Step 1. Login The Computer System

(See Experiment One)

Step 2. Change Sample

            (See Experiment One)

  Step 3. Lock And Shim The Magnet

            (See Experiment One)

Step 4. Setup Parameters Carbon Experiment

4.1       Standard C-13 spectrum (Fully decoupled with NOE enhancement):

            Type c13 to call up the standard carbon-13 experiment parameters with CDCl3 solvent.           

4.2       Enter nt=256, according to the nature of your sample.

4.3       Type ga to begin acquisition.

4.5            After acquisition is done, a carbon spectrum is displayed. If not, enter wft. Type aph for automatic phasing. Manual phasing is often necessary for a carbon-13 spectrum.

Step 5. Data Processing       

5.1       Zoom operation

  Click LMB on the left side of a peak and RMB on the right side, two vertical red cursors  appear at the click points. Then click the Expand button on the menu bar, the region between the red cursors will be expanded. You may do this zooming operation as many times as you like.

 

5.2       Set  reference

After zooming the region containing the reference peak, click the LMB around the top of         the peak, type nl to place the cursor on the top of the peak exactly. Then type rl(x.xxp) to   set its chemical shift. For example, rl(77p) will set a peak frequency to 77.0ppm.

 

5.3       Peak picking operation

Type ds and click next and th buttons to display an horizontal threshold line. Press and hold LMB to move the yellow cursor to the position you desire, then click on the th button again. Type command dpf to display peak frequencies above spectrum peaks.

           

5.4       Label your experiment data

            Type ctext to clear any text label for the present experimental data.

            Type text(“Your sample name, date\\solvent, etc”) to label the present experimental data.

            Type dtext to display the text label of present data on the upper left of the spectrum.

           

5.5       Print and plot spectrum and related information

            pl----to begin print the present spectrum;

            pap---to print all the parameters including the text labels, if any.

            pscale---to print the spectrum scale in the unit of ppm or hz by axis=“p” or     

            axis=“h”.

            pltext--- to print the label.

ppf---to print all the peak frequencies over the respective peaks.

            page ---to expel the paper from printer.

 

5.6       Save your experiment data

            You have to save your data in /export/home/nmrusr/Login_ID/yourname/

            Type svf(’file_name’) to save your data in the form of FID.

Step 6. Logout the Computer System

6.1       Retrieve standard file and replace your sample with the standard.         

            Type h1 to set up standard proton file. That will turn the decoupler power off too.

            Then see Step 2 for details about replacing NMR sample tubes.

 

6.2       Lock and shim the magnet

See Step 3 for details. Before clicking Disconnect, you are required to shim the magnet to the required level: lockpower=32, lockgain=40 with lock level of over 75%!!!

6.3       Exit the VNMR program

Type exit to close the VNMR program. Now you will see a full screen of Varian’s logo.

6.4       Exit UNIX system

Press the RMB to select Exit item in the popped up menu, release RMB. You will be prompted to select one of two choices: Exit or Cancel. Click on Exit to exit UNIX system. The screen will become blank and a prompt appears.

6.5       Logout

            Type logout. Sign off the logbook for the instrument.

  Congratulations!!! You have finished a complete 1D carbon-13 experiment.

 

Experiment Three: Proton 90° Pulse Width Calibration

Sample to be used: 10% Strychnine in CDCl3

 

Step 1. Login The Computer System

(See Experiment One)

Step 2. Change Sample

            (See Experiment One)

  Step 3. Lock And Shim The Magnet

            (See Experiment One)

 

Step 4. Setup Parameters and Run Experiment

4.1            Type h1 to retrieve standard proton experiment parameters.

4.2            Set d1=1 nt=1

4.3            Type ga to begin acquisition.

4.4            Expend the spectrum between 1ppm away from the left of the left most peak of the spectrum and 1ppm away from the right of the right most peak.

Type movesw to reduce the spectrum width and increase the digital resolution.

4.5            Set d1=20 or a value which enables protons relax back to thermal equilibrium status.

tpwr=58

4.6       Type array and input the following parameter and numbers as computer requests:

parameter to be arrayed:  pw.

enter number of steps in array: 20

enter starting value: 2

enter array increment: 2

You are able to see the whole array parameters in the Text Window on the bottom by typing da                    

4.7       Type ai ga to begin acquisition. The ai means absolute intensity mode.

 

Step 5. Data Processing       

5.1       Display arrayed spectrum

When the acquisition is finished, enter ds(1) for displaying the first spectrum of the array. Type aph for autophasing. Chose a singlet or a doublet peak to expend and then enter dssa for displaying peaks at the same chemical shift range in all of the individual spectrum horizontally.

5.2       Null point spectrum and 90° pulse width

From the arrayed spectrum, find out first null point (the peak intensity is 0), check the pw value corresponding to it. The 90° pulse width is this pw value divided by 2 at given power (tpwr). In this case tpwr=58.

            Type pw90=xx to set up this parameter, where xx was obtained as above.

 

5.3       Label your experiment data

            See Experiment One.

5.4            Print and plot spectrum and related information

Type plww pap page to print spectra in white wash mode (after the first spectrum, each spectrum is blanked out in regions in which it is behind an earlier spectrum).

5.6       Save your experimental data

            See Experiment One

           

Step 6.  Exit And Logout

            See Experiment One

 

Congratulations!!! You have finished a complete Proton 90° Pulse Width Calibration experiment.

 

Experiment Four: 90° Pulse Width Calibration for Carbon Channel

Sample to be used: 40% p-dioxane in benzene-d6 (ASTM)

 

Step 1. Login The Computer System

            See Experiment One

  Step 2. Change Sample

            See Experiment One

  Step 3. Lock And Shim The Magnet

            See Experiment One

  Step 4. Setup Parameters And Run Experiment 

4.1       Type c13(‘benzene’) to load standard carbon parameter set. Check following parameters:

tpwr=60, nt=1, pw=6  gain=‘46’, then enter ga to begin acquisition.

4.2       After the acquisition is finished, phase the spectrum if necessary.

4.3       Set d1=20 or a value which enables protons relax back to thermal equilibrium status.           

4.4       Type array and input the following parameter and numbers as computer requests:

parameter to be arrayed:  pw.

enter number of steps in array: 20

enter starting value: 2

enter array increment: 2

You are able to see the whole array parameters in the Text Window on the bottom by typing da                    

4.5       Type ai ga to begin acquisition. The ai means absolute intensity mode.

 

Step 5. Data Processing       

5.1       Display arrayed spectrum

When the acquisition is finished, enter ds(1) for displaying the first spectrum of the array. Type aph for autophasing. Chose a singlet or a doublet peak to expend and then enter dssa for displaying peaks at the same chemical shift range in all of the individual spectrum horizontally.

5.2       Null point spectrum and 90° pulse width

From the arrayed spectrum, find out first null point (the peak intensity is 0), check the pw value corresponding to it. The 90° pulse width is this pw value divided by 2 at given power (tpwr). In this case, tpwr=60.

            Type pw90=xx to set up this parameter, where xx was obtained as above.

 

5.3       Label your experiment data

            See Experiment One.

  5.4       Print and plot spectrum and related information

            See Experiment Three.

5.5       Save your experiment data

            See Experiment One.

Step 6.  Exit And Logout

            See Experiment One.  

Experiment Five: APT

Sample to be used: 30% 2-butanol in CDCl3

 

Step 1. Login The Computer System

            See Experiment One.

  Step 2. Change Sample

            See Experiment One.

Step 3. Lock And Shim The Magnet

            See Experiment One.

 

Step 4. Setup Parameters and Run Experiment

4.1       type c13 to load standard proton experiment parameters.

4.2       Set nt=8. Type ga to begin acquisition for a common 1D-carbon spectrum.

4.3       After acquisition, type aph for automatic phasing and /or manual phasing if needed.

4.4       Type apt to load standard APT parameters. Double check following parameters:

            pw-----a normal observe pulse. It need not be 90° pulse.

            p1-----180° observe pulse width.

            d2-----the tau delay time. d2=0.007 (7ms)will make CH, CH3 down and C, CH2 up.

            d1-----the pulse delay. It is set to around 10 seconds.

            dm----decouple mode. It is set as dm=‘yny’ for APT.

            nt------scan numbers. It may be set to 32

4.5       Type ga to acquire data.

 

Step 5. Data Processing         

 

When the acquisition is finished, type aptaph macro to phase APT automatically.

Type pl pscale pap page to plot the spectrum.

 

Step 6.  Exit And Logout

            See Experiment One

 

Congratulations!!! You have finished a complete APT experiment.

 

 

Experiment Six.  DEPT

Sample to be used: 30%  2-butanol in CDCl3

 

Step 1. Login The Computer System

See Experiment One

  Step 2. Change Sample

See Experiment One

  Step 3. Lock And Shim The Magnet

See Experiment One

Step 4. Setup Parameters And Run Experiment

4.1       Type command c13 to setup standard parameters for 1D carbon spectrum.

4.2       Set nt=4 or 8 to acquire an 1D carbon spectrum, and set the chemical shift.

4.3       Type dept to setup standard parameters for DEPT experiment.

4.4       Check and/or change the following important parameters:

            ss=4                             steady-state pulse or dummy scans

            pw=15                         90° pulse for carbon-13;

            tpwr=60                      transmitter power of observer channel;

            pp=14.6                       proton 90° pulse from decoupler channel;

            pplvl=60                      proton pulse power from decoupler channel;

            d1=5                            relaxation time

            j=140                           average C-H coupling constant;

            mult=0.5,1,1,1.5         arrayed pulses for CHx, CH, CH2 and CH3;

            nt=32                          number of scans, A multiple of 16 is suggested.

4.5       Type command ga to acquire data.

 

Step 5. Data Processing

5.1       After the acquisition, type ds(1) to display the first spectrum.

5.2       Phase the spectrum and select a proper threshold by using menu button of th.

5.3       Enter adept dssa to analyze and display arrayed spectra of CHx, CH, CH2 and CH3.

5.4       Type pldept to plot the DEPT spectra as displayed by dssa. 

 

Step 6.  Exit And Logout

            See Experiment One

 

Congratulations!!! You have finished a complete DEPT experiment.

   

Experiment Seven.  INEPT

Sample to be used: 30%  2-butanol in CDCl3

 

 Step 1. Login The Computer System

            See Experiment One

  Step 2. Change Sample

            See Experiment One

  Step 3. Lock And Shim The Magnet

            See Experiment One

 

Step 4. Setup Parameters And Run Experiment

4.1       Type command c13 to setup standard parameters for 1D carbon spectrum.

4.2       Set nt=4 or 8 to acquire an 1D carbon spectrum, and set the chemical shift value.

4.3       Type inept to setup standard parameters for DEPT experiment.

4.4       Check the following important parameters:

            ss=4                             steady-state pulse or dummy scans

            pw=15                         90° pulse for carbon-13;

            tpwr=60                      transmitter power of observer channel;

            pp=16.5                       proton 90° pulse from decoupler channel;

            pplvl=60                      proton pulse power from decoupler channel;

            d1=30                          relaxation time

            j=140                           average C-H coupling constant;

            mult=3                                    multiplicity to make CH, CH3 up and CH2 down;

            nt=64                          number of scans, A multiple of 16 is suggested.

            dm=‘nnn’                   setting for coupled spectrum;

            focus=‘y’                    refocusing for coupled spectrum;

            normal=‘y’                 normal multiplets in coupled spectrum

4.5       Type command ga to acquire data.

 

Step 5. Data Processing

5.1       After the acquisition, the first spectrum will be displayed automatically.

5.2       Phase the spectrum, if necessary.

5.3       Type pl  pscale pap page to plot the INEPT spectrum.

Step 6.  Exit And Logout

            See Experiment One

 

Congratulations!!! You have finished a complete INEPT experiment.

   

Experiment Eight: Presaturation

Sample to be used: 10% Strychnine in CDCl3

 

Step 1. Login The Computer System

See Experiment One

  Step 2. Change Sample

See Experiment One

  Step 3. Lock And Shim The Magnet

See Experiment One

   

Step 4. Setup Parameters And Run Experiment

4.1       type h1(‘solvent’)  su, where the solvent may be one among cdcl3, acetone, DMSO, d2o,     etc. The standard proton experiment parameters will be loaded.

4.2       Set nt=1. Type ga to begin acquisition.

4.3       After acquisition, type aph for automatic phasing and manual phasing if necessary.

4.4       Set reference. Then move the cursor close to or on the peak to be removed, type nl  sd commands.

4.5       Type presat to retrieve standard file for presaturation experiment. Check and change             following parameters to: ss=4, nt=8, d1=3 - 5, satdly=2,

            satpwr=10 (Min=1, Max=30 step by 1 unit), spin=0 and finally, su.

4.6       Type ga to acquire data. A spectrum will be displayed when acquisition is finished.

4.7       Check if the peak of interest is efficiently removed in the resultant phased spectrum. If not, increase satpwr by to 4 units, then repeat Step 3.6. If yes, set nt=64 to 128, enter ga to formally acquire data.

           

Step 5. Data Processing

The data processing for Presat experiment is same as that of 1D Proton experiment.

            See Experiment One.

 

Step 6.  Exit And Logout

See Experiment One

 

Congratulations!!! You have finished a complete PRESATUATION experiment.

   

Experiment 9:  COSY on INOVA-400 

 

Step 1. Login The Computer System

See Experiment One

  Step 2. Change Sample

See Experiment One

  Step 3. Lock And Shim The Magnet

See Experiment One

 

Step 4. Setup Parameters And Run Experiment

 

4.1       Run a proton 1D spectrum by using proton standard parameters (See Experiment One)

4.2       Set two cursors in the desired sw; type movesw gain=‘y’;  then ga , check the spectrum again, make sure no peak is fold in. At this time, you may type gain? to display an autoset value then set gain to this value.

4.3       Print a 1D spectrum as a reference. pl pap pscale page

4.4       Look at the top of the screen:

            seq:     s2pul               EXP:1             index:  1

That means you are using  s2pul pulse sequence in experiment 1.

 

4.5       Now use mouse:

main menu ---workspace---create new. A new experiment will be created and  a new button Exp 2 is displayed on the menu bar. If you already have Exp 2 button on the manual bar, omit this step.

 

4.6       Type mp(1,2)  then jexp2 (or simply click Exp 2 button) to open the “exp2”. Look at the top of screen, Exp:1 will change to Exp: 2.  The parameters will move from Exp1 to Exp 2. You will use Exp2 to run COSY experiment. Your proton parameters are still remained in Exp1, in case you need check them again.

 

4.7       Type cosy on the command line. Pulse sequence and some COSY parameters will be loaded for COSY experiment but remain sw, tof  and some others from exp1, so you don’t have to reset reference and spectra width. The following commands and parameters should be checked to setup the experiment:

 

dg                                display the 2D parameters

time                             check the total experiment time

ss=4                             dummy scans for COSY

d1=2                            delay; set to 2 seconds.

tpwr=60                      set up the transmitter power

pw=14.5                      proton 90 degree pulse width

ni=256                        number of FIDs, (128, 256,512).

nt=8                            minimum nt is 8 (16, 32).

fn1=2048                    zero-fill the f1 dimension to 2K data points

gain?                           check the gain value (default value gain=20). Do not use gain='n'.

dp=‘y’                         set double precision.

ga                                Start the experiment

svf(‘cosy_mydata’)     save the date after the acquisition completed.

   

Step 5. Data Processing and Spectrum Printing

5.1.      Recall the data “cosy_mydata.fid”

            Click on Main Menu  and File , then highlight the file name and click on  Load

5.2.          Type wft2d and foldt to show 2-D spectrum and symmetrize the spectrum.

5.3.          Click on Return, Size and Center buttons to place the spectrum in the middle of the screen.

5.4.          Adjust the vertical scale of the 2-D spectrum using the vs+20and vs-20   button to eliminate the unnecessary background color.

5.5.          Click on Proj, Hproj(max)and Plotbuttons to display horizontal projection 1-D spectrum on the top of the 2-D spectrum. Using MMB to adjust the vertical scale of the 1-D projection spectrum.

Click on Vproj(max)and Plotbuttons to display vertical projection 1-D spectrum on the left of the spectrum. Using the MMB to adjust the scale of the 1-D spectrum.

5.6.      Type pcon page to print the 2-D spectrum with 1-D projection spectra.

 

Step 6.  Exit And Logout

See Experiment One

 

Congratulations!!! You have finished a complete COSY experiment.

 

  Experiment 10:  HETCOR on INOVA-400

 Step 1. Login The Computer System

See Experiment One

  Step 2. Change Sample

See Experiment One

  Step 3. Lock And Shim The Magnet

See Experiment One

 

Step 4. Setup Parameters And Run Experiment

4.1.      In exp1, run 1D proton spectrum by using standard parameters. After phasing and setting reference, narrow the spectrum of interest by 1ppm upfield and 1ppm downfield. Type movesw to change the spectral width.

4.2.      Type ga to run 1D proton experiment with a modified sw. Phase the spectrum.

4.3.      Type jexp2 to joint exp2. Run a 1D-carbon experiment. After phasing and setting reference, narrow the spectrum of interest by 4ppm upfield and 4 ppm downfield. Type movesw to change the spectral width.

4.4       Type ga to run 1D carbon experiment with a modified sw. Phase the spectrum.

4.5       Enter jexp3 mv(2,3) to joint exp3 and copy the parameters in exp2 to exp3. Then type hetcor to set up standard HETCOR experiment. Remember your proton spectrum is in exp1.

4.6.      Double-check the following important parametes:

            ss=4                 dummy scans

            tpwr=60          transmitter power in observer channel

            pw=15             90° pulse for carbon

            pp=14.6           90° pulse for proton from decoupler channel

            pplvl=60          proton pulse power from decoupler channel;

            sw=                 sw should equal the modified sw value in exp2 for carbon domain

            sw1=               sw1 should equal the modified sw value in exp1 for proton domain;

            dof=                dof should equal the modified tof in exp1;

            nt=n*16          acquisition scans of multiple of 16;

            ni=256            number of increments in 1st dimension;

            np=2048          number of points to record FID;

            d1=2                relaxation time

4.7.      Type time to check the experiment time. d1 can be adjusted to make up proper time.

 

4.8.          Stop spinning the sample and do a minor shimming. Enter go or ga to acquire data.

 

4.9.      Save the spectrum by typing svf(‘hetcor_mydata’)

 

Step 5. Data Processing and Spectrum Printing

 

5.1.      Recall the data “hetcor_mydata.fid”

            Click on Main Menu  and File , then highlight the file name and click on  Load

5.2.      Type wft2d.

 

5.3       Click on Return, Size and Center buttons to place the spectrum in the middle of the          screen.

5.4.      Adjust the vertical scale of the 2-D spectrum using the vs+20and vs-20   button to

eliminate the unnecessary background color.

 

5.5.      Click on Proj, Hproj(max)and Plotbuttons to display horizontal projection 1-D spectrum 

on the top of the 2-D spectrum. Using MMB to adjust the vertical scale of the 1-D projection spectrum.

5.6.      Click on Vproj(max)and Plotbuttons to display vertical projection 1-D spectrum on the left of the spectrum. Using the MMB to adjust the scale of the 1-D spectrum.

5.7.      Type pcon page to print the 2-D spectrum with 1-D projection spectra.

 

Step 6.  Exit And Logout

See Experiment One

 

Congratulations!!! You have finished a complete HETCOR experiment.

  

Experiment 11: 1D NOE

Sample to be used: 10% Strychnine in CDCl3

 

Step 1. Login The Computer System

See Experiment One

  Step 2. Change Sample

See Experiment One

  Step 3. Lock And Shim The Magnet

See Experiment One

 

Step 4. Setup Parameters And Run Experiment

4.1       type h1 to load standard proton parameters with CDCl3 as solvent.

4.2       Set nt=1. Type ga to begin acquisition.

4.3       After acquisition, type aph for automatic phasing and manual phasing if necessary.

4.4       Set reference by using cusor and commands nl rl(x.xxp). Then move the cursor close to or on the peak to be irradiated, type nl  sd to display its frequency shown as dof=xxx.xx on the top of the screen. Write down this first dof value. Then move the cusor to the no-peak region close to the peak to be irradiated, type sd to display the second dof value and write it down.

4.5       Type presat to retrieve standard file for presaturation experiment. Check and change following parameters to: ss=4, nt=8, d1=5, satdly=2, satfrq=the first dof value

satpwr=2 (Min=1, Max=30 step by 1 unit), and type su. Finally, turn off the spin. Type ga to run a quick spectrum. Check if the irradiated peak is removed from the resultant spectrum. If not,  repeatedly increase satpwr by 2 units and run a spectrum again until the peak is removed.

4.6.      Change the nt to 128 or 256 and  then type ga to acquire data for the first spectrum. After

            the acquisition is finished, save the spectrum.

4.6.1.     Change the parameter satfrq to the second dof value written down, and type ga to get second spectrum and save it.  

Step 5. Data Processing

5.1.      Type jexp5 to go to Exp5 and load the first spectrum by following procedure: Click on Main Manu, File  and highlight the first spectrum file, then click on load. Type lb=5 wft  to show the spectrum.

5.2.          Type jexp1 to go to Exp1. Load and process the second spectrum by the procedure as same as step 5.1.

5.3.          Type spsub to substrate the second spectrum (in Exp1) from the first one (in Exp5) and then type jexp5 and  ds to show the resulting spectrum in the Exp5.

5.4.          Plot the spectrum.

 

Step 6.  Exit And Logout

See Experiment One

 

Congratulations!!! You have finished a complete 1D NOE experiment

 

Experiment Twelve: 1D Carbon with Complete Fluorine Decoupling by Using Wave Function Generator

Sample to be used: 20% Perfluoro-2-methyl-2-pentene

 

Step 1. Login The Computer System

(See Experiment One)  

Step 2. Change Sample

            (See Experiment One) 

Step 3. Lock And Shim The Magnet

            (See Experiment One)

 

Step 4. Setup Parameters And Run Experiment

4.1       type f19 to load standard fluorine parameters with CDCl3 as solvent. Run a F19   spectrum and save.

4.2       Find the value of the value of tof from the dg and write it down. tof  is the center frequency of the observe transmitter.

 

4.3            Find the transmitter frequencies for each peaks as follows:

4.3.1       Put the cursor on a peak and typing movetof, and write down the new tof value in dg.

4.3.2       Load original spectrum again and find the tof value for another peak as in step 4.3.1.

4.3.3       Repeat step 4.3.1 and 4.3.2 until all the frequency values for all peaks are recorded.

 

4.4            Calculate offset value.

4.4.1    Subtract the transmitter frequency value of each peak recorded in step 4.3 from the tof value of the spectrum recorded in step 4.2. to obtain  an offset value for each peak and write them down.

            Remember, always subtract the frequency value of a peak from the tof value, so that when the frequency of a peak is higher than the tof value, you get a positive offset value and when the frequency of a peak is lower than tof¸you get a negtive offset value.

 

4.5       Use Waveform generator to generate a shaped decoupling sequence:

4.5.1       Load the F19 spectrum and type wft

4.5.2       Click Pbox button on the menu bar

Click Het-dec

Click Adiabatic and then type 300 after J in Hz on the top of the screen for C-F coupling.

Click Options

Click Offset and input the offset value of the first (leftmost) peak which should be decoupled.

Click Bandwidth and input 4000------- the area will be decoupled.

Click Return.

Click WURST,  three parameters will be displayed for the decoupling on the top.

4.5.3.     Click Options and repeat the remaining steps in the step 4.5.2. until all the offset value are input.

Click Close.

4.5.4.     Click Name and give a file name for the shaped pulse.

Click Close.

The computer will ask you to

Enter reference 90 degree pulse width (mse): (F19 90 degree pulse width)

Enter reference power level: (power level when you measure the F19 90 degree)

A shaped pulse  will be displayed on the screen.

 

Write down the parameters:

dres=x

dmf=xxxxx

Remember you shaped pulse file name.

 

4.5.5       Load the standard C13 experiment parameters by typing c13

Input following parameters:

dres = x

dmf = xxxxx

dm = ‘nny’

dmm = ‘ccp’

dseq =’shaped pulse file name’

dof = the tof of F19 spectrum  

Type ga to acquire data.

 

Step 5. Data Processing

            See steps in Experiment Two.

 

Step 6.  Exit And Logout

            See Experiment One.

 

Experiment 13: Variable Temperature Experiment 

(25 to 90 oC)

Sample to be used: 10% Strychnine in DMSO

 

Step 1. Login The Computer System

See Experiment One  

Step 2. Change Sample

See Experiment One  

Step 3. Lock And Shim The Magnet

See Experiment One

 

Step 4. Setup Parameters And Run Experiment

4.1       type h1(‘solvent’) to load standard proton parameters and run a trial experiment.

4.2            For a single temperature:       

type temp=xx.x, pad=360, then type su to execute temperature change. Wait until the temperature reading on the remote display reaches the desired value.

For arrayed temperatures:

Type temp=30, 50, 70, pad=360, then type su. Wait until the temperature reading on the remote display reaches the first value of the array.

If the lock level has decreased by 10 or more unites, shim the magnet once again.

4.3.          Run a normal 1D experiment.

 

Step 5. Data Processing

            See Experiment One.

 

Step 6.  Terminated the variable temperature function and exit

6.1      Type temp=’n’ su to terminate variable temperature function. Wait until the reading on       the remote display is below 25 oC.

6.2            Exit and logout.

(See Experiment One)

  

 


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Last updated: May 17, 2006.