KJM5250 - Organic NMR Spectroscopy

Laboratory 
Experiments - part 2

Homonuclear 2D NMR Spectroscopy
COSY, TOCSY, NOESY, ROESY
(Xwinnmr on PC with W2K, version 3.5)

on

DPX 200

and

DPX 300

Department of Chemistry
University of Oslo
March 2005

2005  © B. J. Wik, F. Rise and D. Petersen.

COSY 45

			COrrelation SpectroscopY is a homonuclear 2D technique which is used to correlate the chemical shifts of 1H nuclei which are J-coupled to each other.  The information obtained is in principle the same as you get from 1D homodecoupling experiments.  The 1D decoupling experiment is faster for small molecules but the COSY experiment is easier to set up and run.  Before you can perform the COSY you must acquire a 1D 1H NMR reference spectrum of your sample.  More information is found in chapter 12 in the black Bruker Avance manual (pages 133-148).  

			A prerequisite for using this manual is that you know how to: Login, enter password and start the xwinnmr, remove the old sample, insert your sample, lock the sample on the actual solvent and shim the magnetic field with Z1 (Z on Lucy), Z2, Z3 and Z4, if needed, stop and start the spinning of the sample and start and stop the automatic shimming.  
			
After doing all this continue with:

1.	Type edc¿ 	Type in the name of the experiment.  Either the name of your synthesis product (FR7653) or for instance GROUP1PW.  Expno is 1 and procno is 1.  Select disk unit M: or D:.  D: is the local disk and M: is your home directory.  

2.	Type rpar PROTON¿  	Choose Copy all.

3.	Type getprosol¿

4.	Type ii¿		Initialize interface.  The computer checks that all parts of the spectrometer is on line.  

5.	Type rga¿ 		Receiver gain adjustment.

6.	Type zg¿			Zero and go, starts the experiment.   

7.	Type efp¿ and apk¿  when efp is finished. Exponential multiplication, Fourier trans-forming and automatic phasing.  Plot if you want a hard copy.

8.	Expand the area around the solvent peak, click on calibrate, place the arrow on top of the central peak, click the middle mouse button and enter the exact chemical shift value.  Reduce the spectral width so that the 1H signals almost covers the whole window.  Click on the sw-sfo1 button.  You have now reduced the spectral width to what was selected on the screen and set the radio sender to the middle of the new spectral window.  If you know how to do it, you might now adjust the acquisition aq time and the si to appropriate values.

9.	Type edc¿  Change Expno to 2.  Keep Procno at 1. Click on save.  Type rga¿ acquire with zg¿ and process with efp¿ and apk¿ to get the spectrum with reduced width. 

9a.	Type edc¿  Change Expno to 3 and keep Procno as 1.  Click on save.  You can now create a new dataset "on top" of the reduced one.  

9b.	Type eda¿  	Change PARMODE to 2D.  Click on Save. Answer OK to delete fid files and meta.ext files and update of a file name.  (Not all of these messages might show up).  The message "New 2D DATA SET" comes up on the screen.  

10.	Type eda¿ 	A new form of the eda window comes up.  You shall now check/change the F2 and F1 acquisition parameters.  F2 is the parameter list for the acquisition channel and F1 is the second dimension related to the variable delay in the pulse sequence.

11.	Look around under the F2 parameters and change/check the following parameters:
Change  PROSOL from false to true.  THIS POINT IS VERY IMPORTANT, the pulse lengths and pulse power levels will be wrong if this is not done.    
			PULPROG		cosyqf45
			TD					1024
			NS					8				This should be 16, 32 or 64 for a weak sample.  
			DS					16
			D0					3 ms		The delays can be found under a submenu in the eda window.
			D1					3 s
			FnMODE			QF

Click on Done whit left mouse button when you are finished with the parameter changes.  

12.	Look around under the F1 parameters and change/check the following parameters:
		Look for:
		Edit nuclei		Click and select 1H.  THIS POINT IS VERY IMPORTANT.  The ppm 									scale one side of the spectrum will be wrong if this is not done.  
		TD					256
		ND0					1
SW		SW of the optimized proton spectrum, look at the value in the 1H spectrum or	at the value for SW in F2 dimension, SW(F1)=SW(F2).  

		Click on Save.  

13.	Type rga¿ and wait until the automatic receiver gain adjustment is finished, then type zg¿ Starts the experiment.   The acquisition will take about 1 hour and 52 minutes with the chosen parameters.  Wait about 10 minutes before starting on point 14.  Some parameters are transferred from the acquisition parameters found in eda to the processing parameters found in edp when the experiment is run for some minutes.  

14.	While the instrument is acquiring data you can set up the correct processing parameters in the edp menu.  Type edp¿ 	Edit processing parameters menu comes up.  

15.	Look around under the F2 processing parameters and change/check the following parameters:
			SI						512
			SF						This is 200.13 MHz on Ingolf and 300.13 MHz on Lucy.
			Offset				This is the ppm or Hz value of the left side of the 1H spectrum.  
If you don't have it, get theon the screen by typing re 2 1¿ which takes you back to the reduced 1H spectrum.  Put the arrow on the ppm scale and move it to the leftmost side and read the value.  Type re 3 1¿ to get back the COSY dataset and type edp¿ to get the processing parameters on screen again.  Type in the Offset value.  
			WDW				SINE 
			SSB					0
			PH_mod			no
			PKNL				TRUE
BC_mod		no			This parameter is given a wrong value in the Bruker Avance manual on page 136.

16.	Look around under the F1 processing parameters and change/check the following parameters:
			SI						512
			SF					This is 200.13 MHz on Ingolf and 300.13 MHz on Lucy.
Offset				This is the ppm or Hz value of the left side of the 1H spectrum.  If you don't have it get the 1H on the screen by typing re 2 1¿ which takes you back to the reduced 1H spectrum.  Put the arrow on the ppm scale and move it to the leftmost side and read the value.  Type re 3 1¿ to get back the COSY dataset and type edp¿ to get the processing parameters on screen again.  Type in the Offset value.  
			WDW 				SINE
			SSB					0
			PH_mod			mc
			BC_mod			no
			MC2					QF

17.	When the acquisition is finished type xfb¿ to Fourier transform the data in both dimensions.  Click on +/- button until only positive peaks are seen.  

18.	Press calibrate place the pointer at the solvent diagonal peak and press the middle mouse button. Type in the shift value for the solvent and press ¿, you will do this two times.

19.	The threshold value of the peaks on the screen can be changed by using the *2 and /2 buttons or the à button together with the mouse.  The spectrum can be displayed in three modes on the screen.  Intensities, contours and oblique.  Try them. 

20.	After defining the correct intensities click on defplot to ensure that the chosen intensities are stored.  Press ¿ for all three questions you are asked and do not change anything.  

21.	Type edg¿   The graphic parameter menu comes up. Click on EDPROJ1.  Remember in which file the 1D Proton spectrum is located and fill in the corresponding PF1 Name, PF1 Experiment no. and PF1 Processno. (For instance GR1COSY, 2, 1).  Click on save.  Do the same for EDPROJ2 and click on save. 

22.	Type setti¿ and type a title for the plot. Save and exit.

23.	Type view¿ to get a preview on the screen of what is coming on the paper.  Press quit.  

24.	Type plot¿ if you are satisfied.  

25.	If you want expansions of the 2D spectra, enclose the area you want to expand with the help of the square button located on the left hand side of the panel with the help of the mouse.  Click with the left mouse button on the upper left corner of the area to be expanded, keep the button depressed and drag the mouse down and to the right and let go of the button when the area is correct.  Click with the right mouse button and the selected area is expanded.  If you want the whole spectrum back - click on the exp button and then the all button.  Ask for help if you don't understand this.  After expanding and increasing or decreasing the threshold (by the *2, /2 or the à button and hold left mouse button pressed down and move the mouse up and down to increase or decrease the intensity of the peaks) click on defplot and answer the questions and type plot¿ to get the spectrum out.

TOCSY.

			TOtal Correlation SpectroscopY is a homonuclear 2D technique which is used to identify 1H spin systems.  In principle all members of a J coupled spin system couples with all the other members regardless of how many bonds there are between the protons as long as there are no breaks in the chain of CH, CH2 and CH3 groups.  In practice you will only see coupling over a distance of five groups of protons.  The longer the so-called spin-lock period (p6) the more couplings are seen.  More information is found in chapter 12 in the Bruker Avance manual (pages 167-174).  

			The experiment can be set up in two different ways, either using another dataset for a homonuclear 2D spectrum run previously on the same sample (for instance COSY) or set up the experiment from scratch.  This manual tells you how to do it from scratch.  If you are doing this experiment as a part of the KJM5250 course you can also start from the COSY spectrum you just acquired.  In this case use the re XX X¿ command (where XX an X are the correct expno and procno) or the open button under file on the left hand side of the window to find the correct parameter set and then use edc to change to a new expno and change the appropriate parameters (see points 10-12 below).  
			A prerequisite for using this manual is that you know how to: Login, enter password and start the xwinnmr, remove the old sample, insert your sample and shim the magnetic field with Z1 (Z on Lucy), Z2, Z3 and Z4, if needed, stop and start the spinning of the sample and start and stop the automatic shimming and giving the experiment a file name with edc.  

1.	Type edc¿ 		Type in the name of the experiment.  Either the name of your synthesis product (FR7653) or for instance GROUP1TOCSY.  Expno is 1 and procno is 1.  Select disk unit M: or D:.  D: is the local disk and M: is home directory.  

2.	Type rpar PROTON¿  	Copy all.

3.	Type getprosol.

4.	Type ii¿			Initialize interface.

5.	Type rga¿ 		Receiver gain adjustment.

6.	Type zg¿			Zero and go.   

7.	Type efp¿ and apk¿  		Exponential multiplication, Fourier transforming and automatic phasing.  Plot if you want a hard copy.

8.	Expand the area around the solvent peak, click on calibrate, place the arrow on top of the central peak, click the middle mouse button and enter the exact chemical shift value.  Reduce the spectral width so that the 1H signal almost covers the whole window.  Click on the Sw-sfo1 button.  You have now reduced the spectral width to what was on the screen and set the radio sender to the middle of the new spectral window.  

9.	Type edc¿  Change expno to 2.  Click on save.  Type rga¿ acquire with zg¿ and process with efp¿ and apk¿ to get the spectrum with reduced width. 

10.	Type edc¿  Change Expno to 3 and Procno as 1.  Click on save.  You can now create a new dataset "on top" of the reduced one.  

11.	Type eda¿  	Change PARMODE to 2D.  Click on Save. Answer OK to delete meta.ext files and to update.  The message "New 2D DATA SET" comes up on the screen.  

12.	Type eda¿ 	The 2D form of the eda window comes up.  You shall now check/change the F2 and F1 acquisition parameters.  F2 is the parameter list for the acquisition channel and F1 is the second dimension related to the variable delay in the pulse sequence.

13.	Look around under the F2 parameters and change/check the following parameters:
		Change PROSOL from false to true.  THIS POINT IS VERY IMPORTANT, the 						pulse lengths and pulse power levels will be wrong if this is not done.    
		PULPROG		mlevph
		TD					1024
		NS					8				This should be 16, 32 or 64 for a weak sample.  
		DS					16
		P17					2.5ms	P’s, D’s and L’s can be found under sub menu in the eda window.  
		D0					3 ms
		D1					2 s
		D9					200ms
		D12					20 ms
		D13					3 ms
		L1						30

Click on DONE with the left mouse button.  

14.	Look around under the F1 parameters and change/check the following parameters:
		Look for :
		Edit nuclei		Click and select 1H.  THIS POINT IS VERY IMPORTANT.  
		TD					512			
		ND0					1
		FnMODE			TPPI
	SW					SW of the optimized proton spectrum, look at the value in the 1H spectrum or at the value for SW in F2 dimension and use this value.
Click on SAVE with the left mouse button.

15.	Type rga¿ and wait until the receiver gain adjustment is done, then type zg¿ which starts the experiment.   The acquisition will take about 2 hours and 50 minutes with the chosen parameters.  

16.	While the instrument is acquiring data you can set up the correct processing parameters in the edp menu.  Type edp¿ 	Edit processing parameters.  

17.	Look around under the F2 processing parameters and change/check the following parameters:
		SI						512
		SF						This is 200.13 MHz on Ingolf and 300.13 MHz on Lucy.
	Offset				This is the ppm or Hz value of the left side of the 1H spectrum.  If you don't have it get the 1H on the screen by typing re 2 1¿, this takes you back to the reduced 1H spectrum.  Put the arrow on the ppm scale and move it to the leftmost side and read the value.  Type re 3 1¿ to get back the TOCSY dataset and type edp¿ to get the processing parameters on screen again. Type in the Offset value.  
		WDW				SINE
		SSB					2
		PH_mod			pk
		PKNL				TRUE
	BC_mod			no		This parameter is given a wrong value in the Bruker Avance manual on page 136.

18.	Look around under the F1 processing parameters and change/check the following parameters:
		SI						512 
		SF					This is 200.13 MHz on Ingolf and 300.13 MHz on Lucy. 
	Offset				This is the ppm or Hz value of the left side of the 1H spectrum with reduced SW.  If you don't have it get the 1H on the screen by typing re 2 1¿ which takes you back to the reduced 1H spectrum.  Put the arrow on the ppm scale and move it to the leftmost side and read the value.  Type re 3 1¿ to get back the TOCSY dataset and type edp¿ to get the processing parameters on screen again.  Type in the Offset value.  

		WDW 				SINE
		SSB					2
		PH_mod			no
		BC_mod			no
		MC2					TPPI

Click on Save with left mouse button.  

19.	When the acquisition is finished type xfb¿ to Fourier transform in both dimensions.  When this is completed click on +/- button and watch the difference between positive, negative and both positive and negative peaks.  If all signals are displayed in only one color, click on colors and move the mouse upwards. Red colors represent positive values and violet represent negative values.  The spectrum probably looks terrible in all modes.  

20.	The threshold value of the peaks on the screen can be changed by using the *2 and /2 buttons or the à button together with the mouse.  The spectrum can be displayed in three modes on the screen.  Intensities, contours and oblique.  Try them and end up with intensities.  

21.	The TOCSY spectrum must be phased.  Phasing of 2D spectra is complicated and you must be careful and somewhat intelligent to succeed.   Type rser 2 this means read serial file 2.  A 1D FID comes up and is stored temporarily in ~TEMP 1 1.
(It is only for TOCSY and ROESY spectra that you use the serial file 2 for phasing, for all other 2Ds to be phased the rser 1¿ command shall be used to get the serial file 1.)  Read more about this in the Avance manual page 171.  

22.	Type sinm¿ to enter a sine bell window function.  
What is a sine bell window function? This is a good examination question.  

23.	Type ft¿  To Fourier transform the spectrum.

24.	Manually phase the 1D spectrum.  Click on Phase, Cursor, and tie the cursor on the spectrum by clicking on biggest.  Click on PH0 and use the mouse to phase the spectrum.  Then click on PH1 to phase the rest of the spectrum.  Click on return, and also on Save as 2D & return.  In this way the phase corrections you have made are stored in the TOCSY 2D data file.

25.	Click on 2D in left lower corner and the last 2D comes back (The TOCSY presumably). Type xfb¿ to Fourier transform once more.  The phasing along the F2 axis should now be better.  

26.	You must now perform phase correction in the F1 dimension.  This is done in a different way.  Click on the phase button.  A new window with several small sub windows comes up.  

27.	Click on col for columns, a new moveable cross comes up.  The cross shall be used to define which columns are to be moved into the three windows on the right.  The three columns moved shall then be used to get correct phase corrections in the other dimension.

28.	Select a col to the right of the small spectrum by moving the mouse to a position, (where there are peaks outside the diagonal) click with the middle button to define the column.  Click then with the left mouse button to get out of this submenu.  Then click on move: 1 to move the spectrum into the upper of the three windows.  

29.	Repeat this for a column in the middle of the spectrum with peaks outside the diagonal and move that to window 2.  Continue with a column in the left side with crosspeaks outside the diagonal and move it to window 3.  When all are moved scale them to a correct size with the *2, /2 buttons at lower left corner.  You must click on 1, 2 or 3 in the green windows to get the scaling to work. 

30.	Click then on window 1.  Use PH0 to phase the spectrum right side of the upper spectrum manually. Click then on window 3 and use PH1 to phase the left side of the lower spectrum. Watch that all three spectra are phased at the same time and select the phasing corrections that give best overall result.  All peaks shall be positive or as positive as possible.  Click on return.  Save and return.  Confirm xf1p by clicking OK. The computer Fourier tansforms in one dimension.  The resulting spectrum should look better.  If it not looking better try to move three rows into the green windows and phase them manually.  Confirm xf2p.  Then try xfb¿ to phase in both dimensions.  If still not OK go back to columns and then to rows.  If you are phasing while still acquiring you must most probably phase again when the whole data set is acquired.  

31.	Press calibrate place the pointer at the solvent diagonal peak and press the middle mouse button. Type in the shift value for the solvent and press ¿, you will do this two times.

32.	The threshold value of the peaks on the screen can be changed by using the *2 and /2 buttons or the à button together with the mouse.  The spectrum can be displayed in three modes on the screen.  Intensities, contours and oblique.  Try them. 

33.	After defining the correct intensities with *2, /2 buttons or the à button, click on defplot to ensure that the chosen intensities are stored.  Press ¿ for all of the three questions you are asked and change nothing.

34.	Type edg¿   The graphic parameter menu comes up. Click on EDPROJ1.  Remember in which file the 1D Proton spectrum is located and fill in the corresponding PF1 Name, PF1 Experiment no. and PF1 Processno. (For instance GR1COSY, 2, 1).  Click on save.  Do the same for EDPROJ2 and click on save. 

35.	Type setti¿, and write a title for your spectrum in the notepad that appears. Select file-save and file-exit to save and exit.

36.	Type view¿ to get a preview on the screen of what is coming on the paper.  Press quit.  

37.	Type plot¿ if you are satisfied.  

38.	If you want expansions of the 2D spectra, enclose the area you want to expand with the help of the square button located on the left hand side of the panel with the help of the mouse.  Click with the left mouse button on the upper left corner of the area to be expanded, keep the button depressed and drag the mouse down and to the right and let go of the button when the area is correct.  Click with the right mouse button and the selected area is expanded.  If you want the whole spectrum back - click on the exp button and then the all button.  Ask for help if you don't understand this.  After expanding and increasing or decreasing the threshold (by the *2, /2 or the à button and hold left mouse button pressed down and move the mouse up and down to increase or decrease the intensity of the peaks) click on defplot and answer the questions and type plot¿ to get the spectrum out.

NOESY

			Nuclear Overhauser Effect SpectroscopY identifies spin pairs undergoing cross-relaxation. the most common cross-relaxation "method" is magnetic dipolar coupling.  Magnetic dipolar coupling denoted (The coupling constant denoted D) goes through space and is extremely dependent upon distance.  Noesy therefore gives information about which nuclei are close in space.  The same information is obtainable from the 1D technique NOEDIFF.  Noediff is faster for small molecules, and NOESY is used for large molecules with hundreds and thousands of correlations.  The Bruker program Aurelia which is available over the network from NLH can be used in the interpretation of NOESY spectra.  For the regular user NOEDIFF is better but for a person wanting just one or two spectra NOESY is simpler to set up and run since the Bruker NOEDIFF experiment is not easy to perform.  More information is found in chapter 13 in the Bruker Avance manual (pages 149-157), and in the curriculum of KJM5250.

			The experiment can be set up in two different ways, either using another homonuclear 2D run previously on the same sample (for instance TOCSY) or set up from scratch.  This manual tells you how to do it from scratch.  If you are doing this experiment as a part of the KJM5250 course you can also start from the TOCSY or COSY spectrum you just acquired.  In this case use the re NAME XX X¿ command (where XX an X are the correct expno and procno) or the open button on the left hand side of the window to find the correct dataset and use edc to change to a new expno and change the appropriate parameters (see points 10-12 below).  It is recommended to start from scratch. 
			A prerequisite for using this manual is that you know how to: Login, enter password and start xwinnmr, remove the old sample, insert your sample and shim the magnetic field with Z1 (Z on Lucy), Z2, Z3 and Z4, if needed, stop and start the spinning of the sample and start and stop the automatic shimming and giving the experiment a file name with edc.

1.	Type edc¿ 		Type in the name of the experiment.  Either the name of your synthesis product (FR7653) or for instance GROUP1NOE.  Expno is 1 and procno is 1.  Select disk unit D: or M:.  D: is the local disk and M: is your home directory.  

2.	Type rpar PROTON¿  	Copy all.  

3.	Type getprosol¿

4.	Type ii¿			Initialize interface.

5.	Type rga¿ 		Receiver gain adjustment.

6.	Type zg¿			Zero and go.   

7.	Type efp¿ and apk¿  		Exponential multiplication, Fourier transforming and automatic phasing.  Plot if you want a hard copy.

8.	Expand the area around the solvent peak, click on calibrate, place the arrow on top of the central peak, click the middle mouse button and enter the exact chemical shift value.  Reduce the spectral width so that the 1H signals almost covers the whole window.  Press the utilities button and the press sw-sfo1 button.  Click on return.  You have now reduced the spectral width to what was on the screen and set the radio sender to the middle of the new spectral window.  

9.	Type edc¿  Change Expno to 2 and keep procno as 1.   Click on save.  Type rga¿ acquire with zg¿ and process with efp¿ and apk¿ to get the spectrum with reduced width. 

10.	Type edc¿  Change Expno to 3 and Procno to 1.  Click on save.  You can now create a new dataset "on top" of the reduced one.  

11.	Type eda¿  	Change PARMODE to 2D.  Click on Save. Answer  OK to delete meta.ext files and to update.  The message "New 2D DATA SET" comes up on the screen.  By creating the NOESY dataset from the 1H dataset most of the parameters are set correctly.  

12.	Type eda¿ 	A new form of the eda window comes up.  You shall now check/change the F2 and F1 parameters acquisition parameters.  F2 is the parameters for the acquisition channel and F1 is second dimension related to the variable delay in the pulsesequence.

13.	Look around under the F2 parameters and change/check the following parameters:
Change  PROSOL from false to true. THIS POINT IS VERY IMPORTANT, the pulse lengths and pulse power levels will be wrong if this is not done.    
		PULPROG		noesyph
		TD					1024
		NS					32				This should be 48, 52 or 64 for a weak sample.  
		DS					16
		D0					3 ms.			The D’s are found in a submenu in the eda window.  						D1					2 s
D8		350 ms		Will be optimized to a different value later with the go setup (gs¿) procedure.  

		Click on DONE and not on SAVE when you have changed the values.  

14.	Look around under the F1 parameters and change/check the following parameters:
		Look for:
		Edit nuclei		Click and select 1H.  THIS POINT IS VERY IMPORTANT.  
		TD						256			This should be 512 or 1024 if higher resolution is required.
		ND0						1
		FnMODE				TPPI
SW	SW of the optimized proton spectrum, look at the value in the 1H spectrum or at the value for SW in F2 dimension which was automatically set correctly from the 1D spectrum.

		Click on SAVE.  

15.	Optimization of d8.  d8 is the so called mixing period when the NOE is building up.  This time is around one T1.  d8 is optimized in a 1D experiment below.  Type edc¿ and change Expno to 4 and Procno to 1.  Click on save.

16.	Type eda¿ and change PARMODE to 1D and click on save and OK to delete some files.  

17.	Type eda¿ again and the new 1D eda window shows up.  Change PULPROG to zg, NS to 1 and DS to 0 and TD to 16k (if you do not change this last parameter the 1D spectra will have to low resolution.).  The pulse program is changed to zg, the number of scans to one and the dummy scans to zero.  Click on save. 

18.	Type rga¿ and wait until the adjustment of the receiver is finished, followed by typing zg¿ and Fourier transform the FID with ef¿.  Phase the spectrum manually (Phase, Biggest, PH0, PH1) and save the phasing corrections.  Be aware that this phasing must be performed correctly since this phasing is to be used later on.  Return¿ followed by Save and return¿.

19.	Type eda¿ again and the new 1D eda window shows up.  Change PULPROG to t1ir1d which is a T1 inversion recovery pulse program.  Change d7 (D7) to 500 ms.  The d7 value here corresponds to the d8 value used in the 2D experiment later on.  Click on done, then on save.  

20.	Click on Acquire and observe fid window and type gs¿ .  The go setup routine is started, in which a whole lot of parameters can be optimized interactively.  A new frame for a window comes up - click on it to fix it on the screen.  Select the correct button for optimizing delays (d7).    

21.	Click on Display then Show frequency domain then Display once more, Frequency domain phasing and finally Pk phasing.  You are watching the Fourier transformed data instantly during the gs routine. 

22.	While monitoring the Fourier transformed data on screen change d7 by typing d7¿ and change the value in the window that is showing up.  With short d7’s all peaks are negative.  With long d7’s all peaks are positive.  Why?  Find the value where the signals just have passed zero and are slightly positive.  Type stop¿ to get out of the go setup routine.  Or use the command kill¿ and select the gs process.  

23.	The d7 value just found shall be used as d8 in the NOESY experiment.  Type re 3 1¿, the NOESY experiment setup returns.  Type d8¿ and enter the chosen value (for instance 750ms) followed by ¿.  

24.	Press the SPIN ON/OFF button on the shim console to stop spinning the sample.

25.	Type rga¿ to allow the instrument to adjust the receiver for the chosen d8 value.  Then type zg¿  which starts the experiment.  Type expt¿ to see how long time it will take.  The acquisition will take several hours depending on how long d8 is.  Take a 15 minuets break before continuing with the next point.

26.	While the instrument is acquiring data you can set up the correct processing parameters in the edp menu.  Type edp¿ 	and the edit processing parameters window comes up.  

27.	Look around under the F2 processing parameters and change/check the following parameters:
			SI						1024	
			SF						200.13 or 300.13 MHz depending upon which instrument you are on. 
			Offset				This is the ppm or Hz value of the left side of the 1H spectrum.  
If you don't have it - get the 1H on the screen by typing re 2 1¿ which takes you back to the reduced 1H spectrum.  Put the arrow on the ppm scale and move it to the leftmost side and read the value.  Type re 3 1¿ to get back the NOESY dataset and type edp¿ to get the processing parameters on screen again.  Type in the correct OFFSET value.  
			WDW				SINE or QSINE  Chose either sine or qsine here and in point 28.
			SSB					2 or 3					Chose either 2 or 3 here and in point 28.
			PH_mod			pk
			PKNL				TRUE
BC_mod	no		This parameter is given a wrong value in the Bruker Avance manual on page 153.

28.	Look around under the F1 processing parameters and change/check the following parameters:
			SI						512 	
			SF						200.13 or 300.13 MHz depending upon which instrument you are on. 
			Offset				This is the ppm or Hz value of the left side of the 1H spectrum.  
If you don't have it get the 1H on the screen by typing re 2 1¿ which takes you back to the reduced 1H spectrum.  Put the arrow on the ppm scale and move it to the leftmost side and read the value.  Type re 3 1¿ to get back the NOESY dataset and type edp¿ to get the processing parameters on screen again.  Type in the SF value.  
			WDW 				SINE or QSINE  Chose either sine or qsine here and in point 27.
			SSB					2 or 3					Chose either 2 or 3 here and in point 27.
			PH_mod			no
			BC_mod			no
			MC2					TPPI

		Click on SAVE.  

29.	When the acquisition is finished type xfb¿ to Fourier transform in both dimensions.  

30.	The threshold value of the peaks on the screen can be changed by using the *2 and /2 buttons or the à button together with the mouse.  The spectrum can be displayed in three modes on the screen.  Intensities, contours and oblique.  Try them.  When you are in the Intensities mode click on the +/- button to become used to see negative peaks, positive peaks, both positive and negative peaks and none.  A common beginner’s error for people working with small molecules is to phase and plot the NOESY with only negative or only positive peaks, getting a completely meaningless result plotted.  Select both positive and negative values before going on.  

31.	The NOESY spectrum must be phased.  Phasing of 2D spectra are complicated and you must be careful and somewhat intelligent to succeed.   Type rser 1 (It is only for TOCSY and ROESY spectra that you use the serial file 2 for phasing, for all other 2Ds to be phased the rser 1¿ command shall be used to get the serial file 1.)  Read more about this in the Avance manual page 171.  

32.	Type sinm¿ to enter a sine bell window function.  	
What is a sine bell window function?  This is a good examination question.  

33.	Type ft¿  To Fourier transform the spectrum.

34.	Manually phase the 1D spectrum.  Click on Phase, Biggest, and phase around the biggest peak by  clicking  PH0 and use the mouse to phase the spectrum.  Then click on PH1 to phase the rest of the spectrum.  Click on return, Save as 2D & return.  In this way the phase corrections you have made are stored in the NOESY 2D data file.

35.	Click on 2D in left lower corner and the last 2D comes back (The NOESY presumably). Type xfb¿ to Fourier transform once more.  The phasing along the F2 axis should now be better.  

36.	You must now perform phase correction in the F1 dimension.  This is done in a different way.  Click on the phase button.  A new window with several small sub windows comes up.  

37.	Click on col for columns, a new cross comes up.  The cross shall be used to define which columns are to be moved into the three windows on the right.  The three columns moved shall then be used to get correct phase corrections in the other dimension.

38.	Select a col to the right of the small spectrum by moving the mouse to a position, (containing a diagonal peak) click with the middle button to define the column.  Click then with the left mouse button to get out of this submenu.  Then click on 1 to move the spectrum into the upper of the three windows.  

39.	Repeat this for a column in the middle of the spectrum with a diagonal peak and move that to window 2.  Continue with a column in the left side containing diagonal peaks and move it to window 3.  

40.	Click then on window 1 (the upper one).  Click on Biggest then click on PH0 and phase the spectrum manually.  Click then on PH1 and phase the rest of the spectrum, especially the part which is farthest away from the diagonal in window 1, that is the diagonal peak in window 3.  Watch that all three spectra are phased at the same time and select the phasing corrections that gives best overall result.  Click on Save and return.  Confirm  xf1p by clicking OK.  

If you are not satisfied please phase the rows manually just as you phased the columns.

If you are still not satisfied read on pages 154 - 155 in the Avance manual for instructions about phasing rows. (F2 dimension- xf2p).  Be aware that the Avance manual is written for an earlier version of the software and some symbols might have different names.  When NOESY is phased correctly all diagonal peaks will all be either negative or all positive.  Crosspeaks can be both negative and positive. Why?  There can also be COSY artifacts which is seen as anti phase peaks.  

41.	After defining the correct intensities with *2, /2 buttons or the à button, click on defplot to ensure that the chosen intensities are stored.  Press ¿ for all the questions you are asked and do not change anything.  At this stage it is time to stop for a moment and think, were you asked about both positive and negative levels and the number of contours?  If not you must have done something wrong.  Both positive and negative contours must be defined - and for instance 15 different contour level chosen for positive peaks and 3 for negative to ensure easy selection between them when plotted on a black and white plotter/printer.  Click on the +/- button followed by defplot, repeat this two step procedure until both negative and positive contours are defined.  

42.	Type edg¿   The graphic parameter menu comes up.  Click on EDPROJ1.  Remember in which file the 1D Proton spectrum is located and fill in the corresponding PF1 Name, PF1 Experiment no. and PF1 Processno. (For instance GR1NOESY, 2, 1).  Click on save.  Do the same for EDPROJ2 and click on save. 

43.	Type setti¿, and write a title for your spectrum in the notepad that appears. Select file-save and file-exit to save and exit.

44.	Type view¿ to get a preview on the screen of what is coming on the paper.  Press quit.  

45.	Type plot¿ if you are satisfied.  

46.	If you want expansions of the 2D spectrum, enclose the area you want to expand with the help of the square button located on the left hand side of the panel with the help of the mouse.  Click with the left mouse button on the upper left corner of the area to be expanded, keep the button depressed and drag the mouse down and to the right and let go of the button when the area is correct.  Click with the right mouse button and the selected area is expanded.  If you want the whole spectrum back - click on the exp button and then the all button.  Ask for help if you don't understand this.  After expanding and increasing or decreasing the threshold (by the *2, /2 or the à button and hold left mouse button pressed down and move the mouse up and down to increase or decrease the intensity of the peaks) click on defplot and answer the questions and type plot¿ to get the spectrum out.  

ROESY

			Rotating-frame Overhauser Effect SpectroscopY measures the homonuclear NOE effects under "spin-lock" conditions.  ROESY is especially valuable since ROESY crosspeaks are always positive and there is no risk of getting crosspeaks of zero intensities.  In NOESY there exist the possibility that w*tc is about 1 and if this condition is fulfilled the NOESY crosspeaks are zero even if the protons are close in space.  Some care is needed when intepretating the results.  Both COSY and TOCSY peaks can sometimes bee seen and if you don't realize it you might be in trouble.  COSY peaks in the ROESY experiment have anti phase appearance, that is positive and negative parts of the same peak.  ROESY have only positive.  Peaks impossible to phase (se several pages later) are therefore probably COSY peaks.  TOCSY cross peaks are identified most easily since they have the same phase as the diagonal peaks which are negative relatively to the ROESY cross peaks.  More information is found in chapter 14 in the Bruker Avance manual (pages 159-166).  
			The experiment can be set up in two different ways, either using another homonuclear 2D run previously on the same sample (for instance COSY) or set up from scratch.  This manual tells you how to do it from scratch.  If you are doing this experiment as a part of the KJM5250 course you can also start from the COSY spectrum you just acquired.  In this case use the re XX X¿ command (where XX an X are the correct expno and procno) or the open button on the left hand side of the widow to find the correct dataset to get the correct parameter set and use edc to change to a new expno and change the appropriate parameters (see points 10-12 below).  
			A prerequisite for using this manual is that you know how to: Login, enter password and start xwinnmr, remove the old sample, insert your sample and shim the magnetic field with Z1 (Z on Lucy), Z2, Z3 and Z4, if needed, stop and start the spinning of the sample and start and stop the automatic shimming and giving the experiment a file name with edc.

1.	Type edc¿ 		Type in the name of the experiment.  Either the name of your synthesis product (FR8764) or for instance GR1ROESY.  Expno and procno are both 1.  

2.	Type rpar PROTON¿  	Copy all.  

3.	Type getprosol¿

4.	Type ii¿			Initialize interface.

5.	Type rga¿ 		Receiver gain adjustment.

6.	Type zg¿			Zero and go.   

7.	Type efp¿ and apk¿  		Exponential multiplication, Fourier transforming and automatic phasing.  Plot if you want a hard copy.

8.	Expand the area around the solvent peak, click on calibrate, place the arrow on top of the central peak, click the middle mouse button and enter the exact chemical shift value.  Reduce the spectral width so that the 1H signal almost covers the whole window.  Click on the sw-sfo1 button.  You have now reduced the spectral width to what was on the screen and set the radio sender to the middle of the new spectral window.  This change will be brought over into the next experiment in 9.  

9.	Type edc¿  Change Expno to 2 and keep Procno as 1.  Click on save.  Type rga¿ acquire with zg¿ and process with efp¿ and apk¿ to get the spectrum with reduced width. 

10.	Type edc¿  Change Expno to 3 and Procno to 1.  Click on save.  You can now create a new dataset "on top" of the reduced one.  By creating the ROESY dataset from the 1H dataset most of the parameters are set correctly.   

11.	Type eda¿  	Change PARMODE to 2D.  Click on Save. Answer OK to delete some files and to update.  The message "New 2D DATA SET" comes up on the screen.  

12.	Type eda¿ 	A new form of the eda window comes up.  You shall now check/change the F2 and F1 acquisition parameters.  F2 is the parameter list for the acquisition channel and F1 is second dimension related to the variable delay in the pulsesequence.

13.	Look around under the F2 parameters and change/check the following parameters:
		Change  PROSOL from false to true.    THIS POINT IS VERY IMPORTANT, the 						pulse lengths, delays and pulse power levels will be wrong if this is not done.   
		PULPROG		roesyph
		TD					1024
		NS					32
		DS					16
	P15					200 ms.  Should be of the same length as d8 in the NOESY experiment.  If this experiment is run on a sample where you have not acquired a NOESY spectrum please look at the NOESY manual to see how you shall determine the d8 value.  
		D0					3 ms.		The delays are found in a sub menu in the eda window. 
		D1					2 s
		D12					20 ms

Click on DONE.

14.	Look around under the F1 parameters and change/check the following parameters:
		Look for:
		Edit nuclei		Click and select 1H.  THIS POINT IS VERY IMPORTANT.  
		TD					256			This should be 512 or 1024 is higher resolution is required.
		ND0					1
		FnMODE			TPPI
SW	SW of the optimized proton spectrum, look at the value in the 1H spectrum or at the value for SW in F2 dimension.

Click on SAVE.  

15.	Type rga¿ and wait until the receiver is adjusted for the selected parameter then type zg¿ 	which starts the experiment.   The acquisition will take several hours depending especially upon the length of P15 and the values for NS and TD.  Take a break for at least 15 minutes before continuing with 15.  

16.	While the instrument is acquiring data you can set up the correct processing parameters in the edp menu.  Type edp¿ 	 and the edit processing parameters window shows up.  

17.	Look around under the F2 processing parameters and change/check the following parameters:
		SI						512
		SF						200.13 or 300.13 MHz.
	Offset				This is the ppm or Hz value of the left side of the 1H spectrum.  Should already be correct since you are building on top of a correct 1D spectrum.  If you don't have it get the 1H on the screen by typing re 2 1¿ which takes you back to the reduced 1H spectrum.  Put the arrow on the ppm scale and move it to the leftmost side and read the value.  Type re 3 1¿ to get back the ROESY dataset and type edp¿ to get the processing parameters on screen again.  Type in the OFFSET value.  
		WDW				SINE or QSINE		Chose either sine or qsine here and in point 18.
		SSB					2 or 3					Chose either 2 or 3 here and in point 18.
		PH_mod			pk
		PKNL				TRUE
BC_mod	no		This parameter is given a wrong value in the Bruker Avance manual on page 162.

18.	Look around under the F1 processing parameters and change/check the following parameters:
		SI						512 		
		SF						200.13 or 300.13 MHz.

		Offset				This is the ppm or Hz value of the left side of the 1H spectrum.  
If you don't have it get the 1H on the screen by typing re 2 1¿ which takes you back to the reduced 1H spectrum.  Put the arrow on the ppm scale and move it to the leftmost side and read the value.  Type re 3 1¿ to get back the ROESY dataset and type edp¿ to get the processing parameters on the screen again.  Type in the SF value.  
		WDW 				SINE or QSINE		Chose either sine or qsine here and in point 17.
		SSB					2 or 3					Chose either 2 or 3 here and in point 17.
		PH_mod			no
		BC_mod			no
		MC2					TPPI

Click on SAVE.  

19.	When the acquisition is finished type xfb¿ to Fourier transform in both dimensions.  

20.	The threshold value of the peaks on the screen can be changed by using the *2 and /2 buttons or the à button together with the mouse.  The spectrum can be displayed in three modes on the screen.  Intensities, contours and oblique, the last option gives you the possibility to look at the spectrum from many sides by changing the Q and F angles.  Try them.  Click on the +/- button until both positive and negative peaks are displayed.  This must be done since the diagonal peaks have opposite sign of the crosspeaks.  Go back to intensities, both positive and negative contours must be defined to ensure good phasing below.  

21.	The ROESY spectrum must be phased.  Phasing of 2D spectra are complicated and you must be careful and somewhat intelligent to succeed.   Type rser 2¿  (It is only for TOCSY and ROESY spectra that you use the serial file 2 for phasing, for all other 2Ds to be phased the rser 1¿ command shall be used to get the serial file 1.)  Read more about this in the Avance manual page 171.  

22.	Type sinm¿ to enter a sine bell window function.  
What is a sine bell window function?  This is a good examination question.  

23.	Type ft¿  To Fourier transform the spectrum.

24.	Manually phase the 1D spectrum.  Click on Phase, Biggest, and phase the biggest peak.  by clicking on PH0 and use the mouse to phase the spectrum.  Then click on PH1 to phase the rest of the spectrum.  Click on return, Save as 2D & return.  In this way the phase corrections you have made are stored in the ROESY 2D data file.

25.	Click on 2D in left lower corner and the last 2D comes back (The ROESY presumably). Type xfb¿ to Fourier transform once more.  The phasing along the F2 axis should now be better.  

26.	You must now perform phase correction in the F1 dimension.  This is done in a different way.  Click on the phase button.  A new window with several small sub windows comes up.  

27.	Click on col for columns, a new cross comes up.  The cross shall be used to define which columns are to be moved into the three windows on the right.  The three columns moved shall then be used to get correct phase corrections in the other dimension.

28.	Select a col to the right of the small spectrum by moving the mouse to a position, containing a diagonal peak) click with the middle button to define the column.  Click then with the left mouse button to get out of this submenu.  Then click on 1 to move the spectrum into the upper of the three windows.  

29.	Repeat this for a column in the middle of the spectrum with diagonal peak(s) and move that to window 2.  Continue with a column in the left side with diagonal peak(s) and move it to window 3.  

30.	Click then on window 1.  Click on PH0 and phase the spectrum manually.  The diagonal peak in window one shall be phased correctly.  Click then on PH1 and phase the rest of the spectrum, the diagonal peak in window 3 farthest from the diagonal shall be phased correctly.  Watch that all three spectra are phased at the same time and select the phasing corrections that give best overall result.  Click on return in the lower left corner and Save and return in the little window that comes up.  Confirm xf1p by clicking OK.  If you are not satisfied read on page 164 in the Avance manual about how to continue phasing of the rows. 

31.	Type edg¿   The graphic parameter menu comes up. Click on EDPROJ1.  Remember in which file the 1D Proton spectrum is located and fill in the corresponding PF1 Name, PF1 Experiment no. and PF1 Processno. (For instance GR1COSY, 2, 1).  Click on save.   Do the same for EDPROJ2 and click on save. 

32.	Type setti¿, and write a title for your spectrum in the notepad that appears. Select file-save and file-exit to save and exit.

33.	Type view¿ to get a preview on the screen of what is coming on the paper.  Press quit.  

34.	Type plot¿ if you are satisfied.  

35.	If you want expansions of the 2D spectrum, enclose the area you want to expand with the help of the square button located on the left hand side of the panel with the help of the mouse.  Click with the left mouse button on the upper left corner of the area to be expanded, keep the button depressed and drag the mouse down and to the right and let go of the button when the area is correct.  Click with the right mouse button and the selected area is expanded.  If you want the whole spectrum back - click on the exp button and then the all button.  Ask for help if you don't understand this.  After expanding and increasing or decreasing the threshold (by the *2, /2 or the à button and hold left mouse button pressed down and move the mouse up and down to increase or decrease the intensity of the peaks) click on defplot and answer the questions and type plot¿ to get the spectrum out.