Index  (Very much under construction revised 10-24-2001)
 

Signing up for time on the Bruker 500

Saving and Archiving Data

Heteronuclear Setups:

General procedures for observing heteroatoms

13C with 1H decoupling

13C APT

15N with and without 1H decoupling

17O

31P with and without 1H decoupling

1H with 31P decoupling

2H with 1H decoupling

Observe 19F

Observe 13C with 19F decoupling

DPFGSE-1D-NOESY (gradient selected 1D-NOESY with selective excitation)

gs-SELINCOR  (gradient selected 1D-HSQC with selective excitation- gives 1 bond CH connectivites)(under construction)

SELINQUATE (1D-INADEQUATE- gives C-toC connectivities)(under construction)

Water suppression

Variable Temperature Setup working but now directions yet (under construction)

Kinetics Array

¹H - ³¹P HMBC

gCOSY

gHMQC

gHMBC (under construction)

gNOESY

gROESY (under construction)

gTCOSY (under construction)

Phasing 2D Spectra (under construction)

Manipulating and Plotting 2D Spectra(under construction)

Probe Changes (under construction)

Pule and Delay Appendix (under construction)

Selective Excitation Pulse Lengths and Powers

---

Signing up for time on the Bruker 500

• Go the online sign up.  Contact the Lab Manager (336-758-5981) for the User ID and Password.

---


Saving and Archiving Data
• You should be logged in and running xwinnmr under your user id.
• All data is saved to x/data/"user id"/nmr/
• I archive all data to CD on the first of each month.
• You can FTP data from the SGI to the nmr PC and burn to CD.  See me and I can help you do this.

---


General Procedures for observing nuclei other than ¹H
 
• Insert sample and cable to the following diagram for the BBO (When using the TBI change the cable from ¹³C to BB on the probe head):
• Before inserting the probe tune and match to the rough values on the card (except when observing ²H, the X channel needs to be turned away from ²H to start).




• Read a standard parameter file for the nuclei you want to observe:
 

Nucleus to observe	Parameter set	Comments
13C without 1H coupling	C13CPD	sensitivity limited
13C attached proton test	C13APT	nice experiment
17O	O17	sensitivity limited
15N	N15	sensitivity limited
15N with 1H decoupling	N15	better sensitivity.  For 1H decoupling change the pulse program to zgdc and make sure in edsp the F2 is set to 1H.
31P	P31	sensitivity limited
31P with 1H decoupling	P31CPD	set pl12 and pl13 to 20 , better sensitivity
1H with 31P decoupling	PRO31PDEC	optimization is questionable
2H with and without 1H decoupling	DEUTERIUM	Tune, match, lock and shim for a normal 1H spectra.  For acquisition recall the parameter set.  Change locknuc to 2H and change your solvent then turn locknuc off.  On the BSMS; no sweep or lock, lock power to -60, lock gain to 75, no filters on X channel.  For 1H decoupling change the pulse program to zgdc and make sure in edsp the X nuc is going to the 2H preamp and F2 is set to 1H.

• Read shims for your solvent.
• Lock on your solvent.
• Tune and match with the sliders (and knobs if also interested in ¹H)
• Run your experiment




---


Observe ¹³C and decouple ¹⁹F
 
• Using the BBO probe obtain a normal ¹³C spectrum and set the SW and O1 to region of interest
• Next, you must detune the ¹H channel to ¹⁹F
• Remove the 1H cable from the probe head
• Remove the BB cable from the probe head and connect it to 1H input of the probe head.
• See figure below for cables:




• Next you will collect the ¹⁹F spectrum.  Type rpar F19 all then type ii
• Type a and then wobb
• Change the sweep width to 20MHz by typing wbsh and then entering 20MHz.
• Tune and match the signal on the 1H cannel.  Tune first then match.  You may have to open the sweep width more to see the dip.
• Type p1 5
• Type pl1 0
• Type  pulprog and confirm the pulse program is zgflqn.
• Type eda, change the solvent and prosol to true.
• Type edsp and make sure the connections are as shown below:




• Type rga then zg. Process and calibrate the ¹⁹F spectrum.
• Display the region of interest on the screen and click on the sw-sfo1 button. Write down the SW and O1 frequency.
• Now you will recable and collect the ¹⁹F decoupled ¹³C spectrum.
• Type iexpno to start a new experiment and type rpar C13CPD all.
• Type  eda , set the solvent and prosol to true.
• Click on NUCLEI edit and change NUC2 to 19F and make the following connections:

• Check 19F as the preferred output for 19F.
• Click on SAVE and OK the message " Warning: connection to preamplifier is missing"
• In the eda window, change O2 to the value of O1 you found in the 19F spectrum.
• Change CPDPRG2 to the following depending upon the SW to be decoupled in the 19F spectrum:

 

SW in 19F Spectrum	CPDPRG2
Less than 10,000 Hz	waltz16
Between 10,000 and 30,000 Hz	garp
More than 30,000 Hz	p5m4


 
• Use the following parameters in the PCPD and PL arrays for the particular CPDPRG2 used:

 

CPDPRG2 used	PCPD (2)	PL (12)	PL (13)
waltz16	90 usec	20 db	20 db
garp	64 usec	14 db	14 db


 
• You may have to optimize PL (12) for the best results.
• Click on SAVE and then recable according to the following diagram:

• Type rga and then zg.
• When finished recable as show below:




---


DPFGSE -1D NOESY(This experiment provides nOe information on a peak by peak basis.  Each 1D scans takes about 3 to 6 minutes depending upon number of scans.  This is much faster than a 7 to 12 hr 2D-NOESY experiment and should be considered for small molecules.)
 
• Take a reference proton spectra without spinning.  Use the minimum number of points (td) as you can tolerate.  Set o1 to the middle of the spectra and narrow the sw to encompass all the peaks.  Note your value of o1 since all experiment will need this reference value. Write down the Hz value for each peak you want to investigate.
• Increment the experiment with iexpno and rpar sel-1D-noe all
• Type eda and change your solvent and then change prosol to true.
• Verify the following:

Parameter	Comment
o1	same as reference proton
sw	same as reference proton
td	same as reference proton
si	same as reference proton
ns	64 to 128 is ok
ds	4

• Type ased and make sure the following parameters are correct:

Parameter	Value
spoff1	(Hz value of peak - o1 value)  can be negative
sp1	70 db
p12	100 msec (the 180 deg shaped pulse, the 90 is 50 ms @ 70 db)
d8	mixing time (700 ms for small molecules)
d20	d8/2 -1.5 msec

• Type zg, when finished type ef and phase so the peak of interest is negative.
• Use xwinplot to do a stacked plot.

---


Water Suppression Parameter Sets
 
 

Parameter Sets	Comments
jump-return-drug-dna	must set the o1p to water
dpfg-water-supression	must set the o1p to water (works the best)
watergate.mww	must set the o1p to water

---


Kinetics Array
 
• Set up experiment (use temperature control) and narrow sweep width to the peak(s) of interest.
• Define the plot region.
• Find the amount of time it takes for 1 experiment by typing expt.
• Set the time between experiments by calculating calculate d1: (d1 = Desired time increment - time for one experiment determined from expt command)
• Type paropt and enter.
• Make d1 the parameter to change, initial value is the value you calculated two steps above, increment will be 0 and the number of experiments should span 4 to 5 half lives.
• The spectra are displayed horizontally and can be integrated.
• If you get a message that the file size is too big try decreasing td.  You will also need to make si = td/2.  Sometimes making a new file with the edc command will allow the experiment to run.




---


¹H - ³¹P HMBC
• Run you 1H spectra on the region of interest and record the sw, o1p and file name.
• Run the 31P spectra with 1H decoupling and record the sw, o1p and file name.
• Run the 31P spectra without 1H decoupling.
• The HMBC parameter set is called H_P_HMBC
• Change the following parameters in the order that are encountered below:

Parameter	Value
prosol	true
solvent	your solvent


 

---


Gradient Selected COSY (gCOSY)
• Obtain a normal ¹H NMR spectrum (see previous section)
• Expand your spectrum to the region of interest
• Click on DP1 (lower left) to define the plot region and then click on SF-SFO1 to define the sweep width (SW) and O1 (off set)
• Type sw and write down the value. Press enter to go back to the command line and type o1 and write down its value.
• Type rga then zg to Collect and process a proton spectrum to display on the F2 and F1 dimensions (write down the file name and spectrum number)
• Stop the sample from spinning and shim.
• Type iexpno to increment the experiment number.
• Type rpar wfu.4min.gCOSY all to copy all the gCOSY parameters
• Type eda and change the existing values to those listed below:

Parameter	Value	F2	F1	Notes
TD		2048	128	Use 4k and 2k for better resolution of crowded spectra
NS	1
DS	8
SW		determined value	determined value	From 1H spectra
O1		determined value	determined value	From 1H spectra
Solvent	Choose your solvent
PROSOL	True			Must be set when a new parameter set is recalled

• Click on Save
• Type expt to see how much time the experiment will take.  You can change the values of TD to alter the desired S/N or resolution.  Those listed are the minimum and will only take 4 to 5 minutes.  When TD is set to 4096 and 2048 it will take about 1 hr and 20 minutes and gives better resolution.
• Type rga to reset the gain
• Type zg to start the experiment
• Type edg to edit the graphics menus (this can be done while the experiment is running)
• Click on EDPROJ1 and make these changes:

Parameter	Value
PF1USER	your login name
PF1NAME	exact name of the 1H NMR spectrum you initially ran
PF1EXP	experiment number of the 1H NMR spectrum you initially ran

• Click on Save
• Click on EDPROJ2 and make these changes

Parameter	Value
PF2USER	your login name
PF2NAME	exact name of the 1H NMR spectrum you initially ran
PF2EXP	experiment number of the 1H NMR spectrum you initially ran

• repeat the previous step and save all changes. (all the parameters names with a 1 will now have a 2 in the name)
• Type xfb when the experiment is done.
• See the section on 2D Spectra Manipulation to plot and expand regions of your 2D spectrum.

---


Gradient HMQC (g-HMQC)
• Collect and process a proton spectrum of the region in interest (write down the file name, spectrum number, SW and O1)
• Collect and process a proton decoupled carbon spectra (write down the file name, spectrum number, SW and O1)
• Stop the sample from spinning and shim.
• Type iexpno to increment the experiment number.
• Type rpar wfu.birdHMQC all
• Type eda and change the existing values to those listed below:

Parameter	Value	F2	F1	Notes
PULPROG	invbst
TD		1k	128
NS	8
DS	16
NDO			2
NUCLEI		EDIT and change NUC2 from off to 13C	EDIT and choose 13C
SW		value from 1H spectra	value from 13C spectra or use 230 if no carbon has been run
O1	value from 1H spectra
O2P	value from 13C spectra or use 1/2 of SW for F1 if no carbon has been run
SOLVENT	Chose your solvent
PROSOL	True

• Click on Save
• Verify the following values by typing in the parameter and pressing enter.  Press enter again and then go to next parameter.

Parameter	Value
PL1	Pulse and Delay Appendix
PL2	Pulse and Delay Appendix
P1	Pulse and Delay Appendix
P2	Pulse and Delay Appendix
P3	Pulse and Delay Appendix
P4	Pulse and Delay Appendix
D1	1.5* average T1 (1.5 s should be OK)
CNST	145
D2	0.00345 s
CPDPRG2	garp
D7	see section on Optimizing D7

• Optimizing D7:  Type D7 and enter 0.400 s.  Type a to see the acquisition window.  Type gs and you should see the FID.  Change D7 to 0.1, wait a few seconds and note the amplitude of the FID.  Increase D7 by 0.1 increments.  Look for the smallest FID amplitude and note the value of D7.
• Type expt to see how much time the experiment will take. These parameters will take about 1.5 hours.
• Type rga to reset the gain
• Type zg to start the experiment
• Type edg to edit the graphics menus (this can be done while the experiment is running)
• Click on EDPROJ1 make these changes:
Parameters when there is no stored ¹³C spectrum:

Parameter	Value
PF1EXT	Click and choose Positive

Parameters when there is a stored ¹³C spectrum:

Parameter	Value
PF1USER	your login name
PF1NAME	exact name of the 13C NMR spectrum you initially ran
PF1EXP	experiment number of the 13C NMR spectrum you initially ran

• Click on Save
• Click on EDPROJ2 make these changes:

Parameter	Value
PF2USER	your login name
PF2NAME	exact name of the 1H NMR spectrum you initially ran
PF2EXP	experiment number of the 1H NMR spectrum you initially ran

• Type xfb when the experiment is done
• Type prow to phase the first row of data. Phase the spectra manually and click on Return and choose "Save as 2D and return"
• Type xau calcphinv then enter xfb.
• Set the contour level with the *2 and /2 buttons.
• See the section on Phasing 2D Spectra if phasing is needed.
• See the section on Manipulating and Plotting 2D Spectra your 2D to plot regions of interest.

---


Gradient NOESY (g-NOESY)
• Obtain a normal ¹H NMR spectrum (see previous section)
• Expand your spectrum to the region of interest
• Click on DP1 (lower left) to define the plot region and then click on SF-SFO1 to define the sweep width (SW) and O1 (off set)
• Type sw and write down the value. Press enter to go back to the command line and type o1 and write down its value.
• Type rga then zg to Collect and process a proton spectrum to display on the F2 and F1 dimensions (write down the file name and spectrum number)
• Stop the sample from spinning and shim.
• Type iexpno to increment the experiment number.
• Type rpar wfu.gNOESY all.
• Type eda and change or verify the existing values to those listed below: (these can entered through the command line using small case letters)

 

Parameter	Value	F2	F1	Notes
PULPROG	noesygpst
PL1	Pulse and Delay Appendix
DO	3 usec
D8	250 msec			just a starting value
D16	100 usec
TD2		2k
SW2		from previous 1H experiment
O1			from previous 1H experiment
NS	2
P1	Pulse and Delay Appendix
P2	Pulse and Delay Appendix
P16	1 msec
D1	2 sec
D11	30 msec
D20	D8*0.5 - P16 -D16
ND0	1
TD1	256
SW1			from previous 1H experiment
L3	use default
DS	16
gpnam1	SINE.1000
gpnam2	SINE.1000
INO	1/(SW1)
gpz1	40%
gpz2	-40%


 
• Type rga then zg
• Type edp to change the processing parameters to:

 

Parameter	F2	F1
SI(F2)	512
WDW(F2)	EM
LB(F2)	2
PH-mod(F2)	pk
PHC1(F2)
MC2
SI(F1)
WDW(F1)
SSB(F1)
PH-mod(F1)
PHC1(F1)

• Phase the rows and columns as presented in the gs-COSY experiment.

 

---


Selective Pulse Lengths and Powers:
 
• ¹H 90^o 50 ms @70db
• ¹³C 90^o ____  @ ____