KJM4250 - Organic NMR Spectroscopy

Laboratory
Experiments - part 3
Heteronuclear 13C detected 2D Spectroscopy.
XHCORR and COLOC
(Xwinnmr on PC, version 3.5)

on

DPX 200

and
 
DPX 300

Department of Chemistry
University of Oslo
April 2005.

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

XHCORR

			Heteronuclear (X, H) shift CORRelation spectroscopy is a 2D technique which is used to determine which 1H's of a molecule that are bonded to which 13C atom (or another X nuclei) in the molecule.  Those off you that are used to Varian terminology are probably used to the term HETCOR for this experiment.  Before you can perform the XHCORR experiment you must acquire a 1D 1H NMR reference spectrum of your sample and a 1D 13C spectrum or a DEPT 45 spectrum as reference spectra.  More information is found in chapter 10 in the black Bruker Avance manual (pages 115-124).  

			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.   

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 signal almost covers the whole window, but leave at least 1 ppm on each side.  Click on the Sw-sfo1 button 2 times.  Write down the values for sw and o1 which are shown at the bottom of the window the second time.  You will need these values later.  Click on return.  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.  Save. Check that the o1 value corresponds to the middle of the spectrum.  (Type o1¿ and look at the value).   Change the scale between Hz and ppm with the button Hz/ppm and compare.  

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 keep procno to 1.  Click on save. 

11.	Type rpar¿ and select a carbon parameter set with proton decoupling (C13CPD ) and click on copy all.  Alternatively select a DEPT45 experiment.  DEPT gives higher S/N and is faster but does not give the quartenary carbons.  Do you think you need them in this experiment?  

12.	Type getprosol¿

13.	Type o2¿ and change the value to the value for o1 found in 8.  o2 is the decoupling position for the proton decoupler while acquiring the carbon spectrum.  

14.	Type ii¿ Initialize interface.  The computer checks that all parts of the spectrometer is on line.  Type rga¿ Receiver gain adjustment.  Type zg¿  Zero and go.  Type efp¿ and apk¿  when efp is finished.  Exponential multiplication, Fourier transforming and automatic phasing.  Plot if you want a hard copy.

15.	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 13C signals almost cover the whole window, but leave at least 10 ppm on each side.  Click on the Sw-sfo1 button.  Write down the values for sw and o1, which are shown at the bottom of the screen, you will need them later on.  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.  Check that o2 is still having the o1 value from 7.  

16.	Type edc¿  Change Expno to 4 and keep Procno as 1.  Click on save. 

17.	Type rga¿ acquire with zg¿ and process with efp¿ and apk¿ to get the spectrum with reduced width.  

18.	Type edc¿ and change the Expno to 5 and keep Procno as 1. 

19.	Type eda¿  	Change PARMODE to 2D.  Click on Save. Answer  OK to delete fid files and meta.ext files.  The message "New 2D DATA SET" comes up on the screen.  You have now started the creation process of the XHCORR parameter set on top of a carbon data set.  This is the way all 2D experiments are created - on top of a data set for the nuclei that is giving the signals to the receiver.  

20.	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 (13C)and F1 is the second dimension related to the variable delay in the pulse sequence.

21.	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		hxcoqf
		TD					1024
	NS					16				This could also be 4, 8, 32 or 64 depending on the sample consentration.  
		DS					16
D0	3 ms.		Be aware that the D´s and CNST´s are hidden under sub menus in the eda window.  (Click on DONE with the left mouse button to exit these menues.)  
		D1					2 s
		D2					3.45 ms
		D3					2.30 ms
		D11					30 ms
		D12					20 ms
		CNST2				145 Hz
		CNST11			3
		CPDPRG2			waltz16
		
Check that the sw value is the same as the sw for the "reduced" 13C spectrum.  If not change it to the correct value.  

22.	Look around under the F1 parameters and change/check the following parameters in the order given, 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					128
ND0					2 
FnMODE			QF
SW					SW of the optimized proton spectrum, look at the value in the reduced 1H spectrum.

Click on save.

23.	Type edasp¿ and watch the radio sender set up.  Be aware that the radio senders are also denoted with F1 and F2 and that there is necessarily no logical connection to the F1 and F2 acquisition parameter and F1 and F2 processing parameters.  In this experiment the F1 radio sender is for 13C but in the setup menus this is denoted F2.!!!  See that the upper sender is 13C and the lower one is 1H.  

24.	Type rga¿ and wait until the receiver adjustment is performed, then type zg¿ which starts the experiment.   The acquisition will take several hours.  Check the time by typing acqu¿ which brings you into the FID window and watch a new small window giving time information.  Wait 15 minutes before continuing.  Some parameters are brought over into the edit processing parameter file (edp) but this does not happen at once. 

25.	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.  

26.	Look around under the F2 processing parameters and change/check the following parameters:
		SI					2048
		SF				This is 50.31 MHz on Ingolf and 75.47 MHz on Lucy.
	Offset			This is the ppm or Hz value of the left side of the 13C spectrum.  If you don't have it, get the 13C on the screen by typing re 4 1¿ which takes you back to the reduced 13C spectrum.  Put the arrow on the ppm scale and move it to the leftmost side and read the value.  Type re 5 1¿ to get back the XHCORR dataset and type edp¿ to get the processing parameters on screen again.  Type in the Offset value.  
		WDW			QSINE
		SSB				2
		PH_mod		no
		PKNL			TRUE
		LB				3 Hz
	BC_mod		no			This parameter is given a wrong value in the Bruker Avance manual on page 136.

27.	Look around under the F1 processing parameters and change/check the following parameters:
		SI					256
		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 5 1¿ to get back the XHCORR dataset and type edp¿ to get the processing parameters on screen again.  Type in the Offset value.  
		WDW 				QSINE
		SSB					2
		PH_mod			mc
		LB 					1 Hz
		BC_mod			no
		MC2					QF

28.	When the acquisition is finished type xfb¿ to Fourier transform the data in both dimensions.  Click on +/- button until only positive peaks are seen.  If you do get strange numbers on the F1 axis on the 2D spectrum you must re enter the edp menu and put the correct value of sw in once more.  Perform a new xfb¿.

29.	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.

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.  

31.	After defining the correct intensities click on defplot to ensure that the chosen intensities are stored.

32.	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 GR1HET, 2, 1).  Click on save.  Do the corresponding procedure for EDPROJ2 (For instance GR1HET, 4, 1) and click on save. 

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

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

35.	Type plot¿ if you are satisfied.  

36.	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.

COLOC

			COrrelation spectroscopy via LOng-range Coupling is a 2D technique which is used to determine which 1H's of a molecule that are bonded to which 13C atoms (or another X nuclei) in the molecule.  In XHCORR the large one bond coupling constants are used to see direct coupling.  In COLOC the experiment is optimized for small coupling constants so that 2 and 3 bond couplings are observed as well as the one bond coupling.  It is therefor not possible to only run a COLOC spectrum - you must also have the regular XHCORR spectrum to identify the one bond couplings in the COLOC.  Those off you that are used to Varian terminology are probably used to the term long range HETCOR for this experiment.  Before you can perform the COLOC experiment you must acquire a 1D 1H NMR reference spectrum of your sample and a 1D 13C spectrum. Why can you not use a DEPT 45 spectrum as the reference spectrum in this experiment?  More information is found in chapter 10 in the black Bruker Avance manual (pages 115-124).  

			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 signal almost covers the whole window, but leave at least 1 ppm on each side.  Click on the Sw-sfo1 button 2 times.  Write down the values for sw and o1 which are shown at the bottom of the window the second time.  You will need these values later.  Click on return.  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.  Save. Check that the o1 value corresponds to the middle of the spectrum.  (Type o1¿ and look at the value).   Change the scale between Hz and ppm with the button Hz/ppm and compare.  

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 keep procno to 1.  Click on save. 

11.	Type rpar¿ and select a carbon parameter set with proton decoupling (C13CPD ) and click on copy all. Alternatively select a DEPT45 experiment. DEPT gives higher S/N and is faster but does not give the quartenary carbons.  Do you think you need them in this experiment?  

12.	Type getprosol¿

13.	Type o2¿ and change the value to the value for o1 found in 8.  o2 is the decoupling position for the proton decoupler while acquiring the carbon spectrum.  

14.	Type ii¿ Initialize interface.  The computer checks that all parts of the spectrometer is on line.  Type rga¿ Receiver gain adjustment.  Type zg¿  Zero and go.  Type efp¿ and apk¿  when efp is finished.  Exponential multiplication, Fourier transforming and automatic phasing.  Plot if you want a hard copy.

15.	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 13C signals almost covers the whole window, but leave at least 10 ppm on each side.  Click on the Sw-sfo1 button.  Write down the values for sw and o1, which are shown at the bottom of the screen,  you will need them later on.  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.  Check that o2 is still having the o1 value from 8.  

16.	Type edc¿  Change Expno to 4 and keep Procno as 1.  Click on save. 

17.	Type rga¿ acquire with zg¿ and process with efp¿ and apk¿ to get the spectrum with reduced width.  

18.	Type edc¿ and change the Expno to 5 and keep Procno as 1.

19.	Type eda¿  	Change PARMODE to 2D.  Click on Save. Answer  OK to delete fid files and meta.ext files. The message "New 2D DATA SET" comes up on the screen.  You have now started the creation process of the COLOC parameter set on top of a carbon data set.  This is the way all 2D experiments are created - on top of a data set for the nuclei that is giving the signals to the receiver.  

20.	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 (13C) and F1 is the second dimension related to the variable delay in the pulse sequence.

21.	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		colocqf
		TD					1024
		NS					64
		DS					16
D0	3 ms.			Be aware that the D´s and CNST´s are hidden under “arrray" sub menus in the eda window.  Look for them.  Click on DONE with the left mouse button to get out of an array.
		D1					2 s
		D2					0.00345 s
		D3					0.0023 s
		D6					80 ms
		D11					30 ms
		D12					20 ms
		D18					33.3 ms		This corresponds to 10 Hz coupling.  
		CNST2				145 Hz
		CPDPRG2			waltz16
	
		Check the value for sw - this should correspond to the value for the "reduced" 13C spectrum.  

22.	Look around under the F1 parameters and change/check the following parameters in the order given, 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							128
ND0						2
FnMODE				QF
SW 						Sw of the optimized 1H spectrum.  

Click on SAVE with the left mouse button.  

23.	Type edasp¿ and watch the radio sender set up.  Be aware that the radio senders are also denoted with F1 and F2 and that there is necessarily no logical connection to the F1 and F2 acquisition parameter and F1 and F2 processing parameters.  In this experiment the F1 radio sender is for 13C but in the setup menus this is denoted F2.!!!  See that the upper sender is 13C and the lower one is 1H.  Click on save.  

24.	Type rga¿ and wait until the receiver adjustment is done.  Type zg¿ which starts the experiment.   The acquisition will take many hours.  Go into the acqusition window by typing acqu¿ and watch for a small window telling you about the remaining time.  Take a break of at least 15 minutes before continuing, some data must be transferred from the eda file to the edp file, this does not happen immediately.  

25.	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.  

26.	Look around under the F2 processing parameters and change/check the following parameters:
		SI					2048
		SF				This is 50.31 MHz on Ingolf and 75.47 MHz on Lucy.  
		Offset			This is the ppm or Hz value of the left side of the 13C spectrum.  
If you don't have it, get the 13C on the screen by typing re 4 1¿ which takes you back to the reduced 13C spectrum.  Put the arrow on the ppm scale and move it to the leftmost side and read the value.  Type re 5 1¿ to get back the XHCORR dataset and type edp¿ to get the processing parameters on screen again.  Type in the Offset value.  
		WDW			QSINE
		SSB				2 or 3
		PH_mod		no
		PKNL			TRUE
		LB				3 Hz
	BC_mod		no			This parameter is given a wrong value in the Bruker Avance manual on page 136.

27.	Look around under the F1 processing parameters and change/check the following parameters:
			SI					256. 
			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 5 1¿ to get back the XHCORR dataset and type edp¿ to get the processing parameters on screen again.  Type in the Offset value.  

			WDW 				SINE or QSINE
			SSB					2 or 3
			MC2					QF
			LB 					0.3 Hz

			Click on SAVE.

28.	When the acquisition is finished type xfb¿ to Fourier transform the data in both dimensions.  Click on +/- button until only positive peaks are seen.  If you do get strange numbers on the F1 axis on the 2D spectrum you must re enter the edp menu and put the correct value of sw in once more.  Perform a new xfb¿.

29.	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.

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.  

31.	After defining the correct intensities click on defplot to ensure that the chosen intensities are stored.

32.	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 GR1HET, 2, 1).  Click on save.  Do the corresponding procedure for EDPROJ2 (For instance GR1HET, 4, 1) and click on save. 

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

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

35.	Type plot¿ if you are satisfied.  

36.	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.