3. Open a UNIX shell, type xwinnmr П. The xwinnmr window should come up. Use the mouse to set the XWINNMR window to proper size and location. The spectrum from the previous experiment using this account will be displayed.

4. Type edcП (edit current dataset) if you want to create a new file:

NAME Type in your preferred file name (up to 14 characters) in the upper small window followed by П. A good example is: YKdemo001 (Y. Kishi demo sample 001). Hint: Use your initials from your name in front of the file name to ensure that we can identify the owner of the file. The name must not contain any of the characters: !"#$>&/( )=?`^*|@_-Ј.,:;Ї' '}{фіх][ or other curious characters you might find on the keyboard.

EXPNO 1

PROCNO 1

DU disk03 (or x or n).

USER GROUPNAME (your UNIX user group name)

TYPE nmr

Click on save with the left mouse button.

5. Type rparП, Select H1std or C13std. Press copy all with the mouse left button. Type ii П.

(You can also type rpar H1std all or rpar C13std all.)

6. Click on Windows on the pull-down menu bar and then click on lock. (typing lockdisp П has the same effect). The Lock display window comes up. Resize it if needed.

7. To change sample, press LIFT on/off button to on the BSMS panel to eject the sample (typing ej П or e Пhas the same effect). Change sample and adjust the height of the new tube with the depth gauge and put the sample back on top of the magnet. Press LIFT on/off again (or typing ij П or i П). Press the SPIN button to spin the sample tube.

8. Type rshП, select a default shim file (use default shim files cdcl3, acet, d2o, etc. depending on the solvent you use) . If you want to save a personal shim file the wsh command can be used; do not overwrite existing parameter and shim files please.

9. Move the lock signal to the center of the lock display window using the FIELD button and the universal knob on the BSMS panel. Lock the field by pressing the LOCK ON/OFF button.

10. Press Onaxis on shim console if no light on that button.

11. Maximizing the lock signal by adjusting the Z1, Z2, and Z3 shims.

B. Probe Tuning

1. Go to the acquisition window by typing acqu (ret).

2. Type wobb(ret) to show the tuning curve, which should have a dip along a vertical line marker when the probe is tuned.

3. Use the tuning rod to adjust the tuning capacitor and the matching capacitor accessible underneath the probe until the number of the corresponding LED displays on top of the preamp housing is minimized, or the tuning curve on the screen is dipped all the way down to the baseline and centered on the line marker.

4. When done, type stop [ret] ( or click on the stop button).

C. Starting the data acquisition

1. Type rgaП. Receiver gain adjustment is performed, wait until message "rga finished" is seen.

2. Adjust ns and other parameters needed. Type exptП to show how long the experiment will take.

If needed, type edaП to check all acquisition -related parameters, and edpП to check all processing-related parameters. ( Type asП to set up all parameters used in the pulse sequence, if desired).

3. Type zgП .

(You can also type gaП to do data acquisition, FT, and autophasing all together.)

4. You can watch the accumulated FID by typing acquП.

D. Data Processing

1. Type efpП, after the acquisition is done, to perform exponential multiplication, Fourier transformation, and phasing of the data. If you want to see a spectrum before the acquisition is finished type trП (transfer from memory to disk) and do efpП. Type haltП if you need to stop the data acquisition before ns scans are collected.

2. Type apkП, the computer performs automatic phasing. Type absП (automatic baseline correction) if baseline is distorted. apksП is an alternative if apk behaves badly. Manual phasing can be done by clicking on phase, then biggest buttons, and by clicking and holding ph0, and ph1 buttons to adjust the phase. Use ph0 to phase the biggest peak, and ph1 the other peaks.

3. To assign the reference peak, expand the region around the reference peak with the left mouse button and the middle button (move the cursor to the spectral window, click the left mouse button and glide the new cursor to the left edge of the desired spectral region, click the middle mouse button and glide the second cursor to the right edge of the desired spectral region, and click the middle mouse button again), then click on calibrate, move the cursor to the top of the reference peak, click the middle mouse button, enter the right chemical shift and П.

4. Click on |<>| to regain the full spectrum. Adjust the spectral amplitude with the р button.

5. To integrate the peaks, click on the integrate button to enter the integration subroutine. Move the cursor to the spectral window and click the left mouse button, a new type of arrow cursor appears on the spectrum baseline. use the middle mouse button to define the integral regions one by one. Click with left button to get out of integral selection mode.

Use the button: р under all to adjust integral heights with the left mouse button.

Select an integral, which represents a known number of protons, by placing the red arrow on the signal and click the left mouse button twice, you should see a star (*) on the top of the integral. Click on calibrate, and type in the number of protons and П.

Click on return. Click on save as intreg & return. You don't see the integrals any more but they can be plotted as shown in the display later.

E. Plotting the spectrum:

Method A:

a. Select the plot region by using the mouse buttons.

b. On the pull-down menu click on Output, then Define/show plot region, then Retain CX. Auto- adjust Hz/cm (or click on the DP1 button), and answer all the questions with П.

(Steps a and b can be replaced by defining F1P and F2P parameters).

c. Check all plotting related parameters by typing edg П.

d. type plot to plot the spectrum. (You can type view to preview the plotted spectrum.)

Method B:

Type xedplot П and use the interactive graphic window to edit the plot page.

F. Finishing Up

1. To store data on the disk using different filename and disk unit, type wrdП.

NAME: YKdemo002 (your own expname)

EXPNO: 1 (use the expno seen in the top part of the xwinnmr window).

PROCNO: 1 (use the procno seen in the top part of the xwinnmr window).

DU: disk03, which has the largest disk space, there are two other disks, x and n, available).

USER: user name (your UNIX user name)

(Other commands like wrpa, wra, etc. can also be used. Check the pull-down menu under File, Copy).

2. When you finish the experiment, type halt П again to stop data acquisition. Replace your sample with a reference sample. Type exit П to exit from xwinnmr program. Move the mouse cursor to the desktop with no icon or window and click and hold the right mouse button and select logout, click on the OK button to finish the logout procedure.

3. Log out on the RESLOG terminal.

II. Gradient Assisted COSY Experiment

A. Data Acquisition:

1. Check and tune the probe using wobbП (in acqu window) and determine the 90 degree pulse width. Run a standard 1H spectrum in expno 1 with properly adjusted spectral width.

2. Create expno 2 from expno 1 by edcП.

3. Type edaП. change PARMODE to 2D (by clicking on the 1D button with the left mouse button, select 2D with the left mouse button), click SAVE. Click OK when asked "Delete 'meta.ext' files?" and "update:/u/exp/stan/nmr/lut/meta.ext." Type edaП again, the eda window should be in 2D format.

4. Change ND0 to 1 (it is important to change this first before changing other parameters), TD to 1024, NS to 1 (or larger), DS to 8, TD(F1) to 256, SWH(F1) to SWH(F2), GRDPROG to 2sine. Click SAVE.

5. Change both cnst21 and cnst22 to 10.

(Steps 3-5 can be replaced by rpar cosygsП , followed by entering the O1 SW, SW(F1) and RG determined in expno 1.)

6. Type asП, change PULPROG to cosygs, and assign the following parameters:

p1 p0 p16	90А pulse, in Еs 45А or 90А pulse Gradient pulse	(9 u) (9 u) 1m
d1	Relaxation delay = (1-5)*T1	(1s)
d0 d16	Initial evolution delay, in s Recovery time after gradient pulse	3u 200u
DE	Receiver dead time	4.5u
SFO1{F2}	Proton frequency	П
PL[1]	Pulse level	0.00dB
SWH{F2}	Spectral width	SW(1D)
TD{F2}	Total data points for F2 acquisition	1k
NS	Number of scans	1
DS	Dummy scans	8
TD{F1}	Total data points for F1 acquisition	256
IN	Incremental time, in s =	П

7. Check the experiment time by exptП. Start the experiment by zgП.

B. Data Processing

1. Type edpП and check the processing parameters:

SI(F2) = SI(F1) = 1024

OFFSET(F1) = OFFSET(F2)

MC2 = QF

WDW(F2) = WDW(F1) = SINE

SSB(F2) = SSB(F1) = 0

PH_mod(F2) = no

PH_mod(F1) = mc (magnitude calculation)

Click SAVE

2. After about 64 FIDs have been collected, you can examine the spectrum by xfbП.

3. Use buttons *2, /2, *8, /8, -, - to adjust the amplitude of the spectrum ( - requires pressing and holding left mouse button and move the mouse up or down).

4. Use mouse buttons to select and expand a region of the spectrum.

5. Click on DefPlot or Limits to define the plot limits, the lowest contour level, and the number of contour levels to be plotted.

C. Plotting

1. Type edgП, and check all plotting parameters:

CONTPLO yes

X1AXIS yes

X2AXIS yes

PROJ1 yes

PROJ2 yes

TITLE yes

PARAM yes

2. Click on EDCONTP and make sure CX1 = CX2 = 15 cm (numbers up to 22 cm are allowed if no projections and parameters are needed).

3. Click on EDAXIS and make appropriate changes when needed.

4. Click on EDPROJ1 and check the parameters for the F1 projection:

PF1CY (adjust it so that the peaks of interest have adequate height)

PF1EXT external(1r)

PF1DU m (disk unit containing the 1D spectrum)

PF1USER your account name

PF1NAME filename of the 1D spectrum

PF1EXP 1 (usually the 1D spectrum is in EXPNO 1)

PF1PROC 1 ( PROCNO 1).

5. Click on EDPROJ2 and check the parameters for the F2 projection.

(should be the same as the above for F1 projection).

6. Click SAVE.

7. Type settiП, and type in any message you want to be as the title in the edit window and save it.

8. Type plotП to plot the spectrum.

III. Phase-sensitive NOESY Experiment

A. Data Acquisition:

1. Check and tune the probe using wobbП (in acqu window) and determine the 90 degree pulse width.. Run a standard 1H spectrum in expno 1 with properly adjusted spectral width.

2. Create expno 3 from expno 1 by edcП.

3. Type edaП. change PARMODE to 2D (by clicking on the 1D button with the left mouse button, select 2D with the left mouse button), click SAVE. Click OK when asked "Delete 'meta.ext' files?" and "update: /u/exp/stan/nmr/lut/meta.ext." Type edaП again, the eda window should now be in 2D format.

4. Change ND0 to 2 (it is important to change this first before changing other parameters), TD to 1024, NS to 16 (or larger), DS to 16, TD(F1) to 256, SWH(F1) to SWH(F2). Click SAVE.

(Steps 3-4 can be replaced by rpar noesytpП , followed by entering the O1, SW, SW(F1), and RG determined in expno 1.)

5. Type asП , change PULPROG to noesytp, and assign the following parameters:

p1	90А pulse, in Еs	(9u)
d1	Relaxation delay = (1-5)*T1	(1s)
d0 d8	Initial evolution delay, in s Mixing time	3u (400m)
DE	Receiver dead time	4.5u
SFO1{F2}	Proton frequency	П
PL[1]	Pulse level	0.00dB
SWH{F2}	Spectral width	SW(1D)
TD{F2}	Total data points for F2 acquisition	1k
NS	Number of scans	16
DS	Dummy scans	16
TD{F1}	Total data points for F1 acquisition	(256)
IN	Incremental time, in s =	П

6. Check the experiment time by exptП.

7. Start the experiment by zgП.

B. Data Processing

1. Type edpП and check the processing parameters:

SI(F2) = SI(F1) = 1024

OFFSET(F1) = OFFSET(F2)

MC2 = TPPI

WDW(F2) = WDW(F1) = SINE

SSB(F2) = SSB(F1) = 2

PH_mod(F2) = no

PH_mod(F1) = no

Click SAVE

2. After about 64 FIDs have been collected, you can examine the spectrum by xfbП.

C. Phasing the Spectrum

1. Click phase button, which creates a new window with the whole 2D spectrum displayed on the left and three small windows on the right.

2. Click row button, use the cross cursor to select 3 rows (well separated and containing large diagonal peaks) of the 2D spectrum for phasing, and load them into the 3 small windows on the right.

3. Use ph0 to adjust the spectrum that has its number highlighted, and ph1 button for the other two spectra.

4. Click return, and save & return buttons, and OK when asked starting xf2p? The phased spectrum will be displayed.

5. Repeat steps 11-13 with col button to phase columns. Clip on return to go back to normal 2D display mode.

6. Use buttons *2, /2, *8, /8, -, - to adjust the amplitude of the spectrum ( - requires pressing and holding left mouse button and move the mouse up or down).

7. Use +/- button to toggle display of positive and negative peaks.

8. Use mouse buttons to select and expand a region of the spectrum.

9. Click on DefPlot or Limits to define the plot limits, the lowest contour level, and the number of contour levels to be plotted.

D. Plotting

1. Type edgП, and check all plotting parameters:

CONTPLO yes

X1AXIS yes

X2AXIS yes

PROJ1 yes

PROJ2 yes

TITLE yes (no)

PARAM yes (no)

2. Click on EDCONTP and make sure CX1 = CX2 = 15 cm (numbers up to 22 cm are allowed if no projections and parameters are needed).

3. If both positive and negative peaks are to be plotted, change

CPLIN linetype

CPLTYPP enter 0 for all levels

CPLTYPN enter 1.003 for all levels

4. Click on EDAXIS and make appropriate changes when needed.

5. Click on EDPROJ1 and check the parameters for the F1 projection:

PF1CY (adjust it so that the peaks of interest have adequate height)

PF1EXT external(1r)

PF1DU disk03 (disk unit containing the 1D spectrum)

PF1USER your account name

PF1NAME filename of the 1D spectrum

PF1EXP 1 (usually the 1D spectrum is in EXPNO 1)

PF1PROC 1 ( PROCNO 1).

6. Click on EDPROJ2 and check the parameters for the F2 projection.

(should be the same as the above for F1 projection).

7. Click SAVE.

8. Type settiП, and type in any message you want to be as the title in the editing window and save it.

27. Type plotП to plot the spectrum.

IV. Gradient-HMQC (magnitude mode)

1. Check and tune the probe. Get 1D 1H spectrum in expno 1and make a note of the O1, SW, and RG values. Determine the 90А pulse width for both nuclei.

2. Create a new expno by edcП.

3. Type rpar_hmqcgs_allП. Type edaП. Change PULPROG to hmqcgs.

4. Make sure that ND0 = 2 and MC2 = QF.

5. Set SW2 = SW (1H) from 1D spectrum in step 1 and SW1 = SW(X).

6. Set O1 = O1 (1H) from 1D spectrum in step 1. Set O2 to 13000 for a window centered around 100 ppm for 13C.

7. Set const21 = 25, cnst22 = 15, cnst23 =20 (for 1H-13C HMQC)

8. Type asП and assign the following parameters:

p1	90А 1H pulse, in Еs	(9u)
p3 p16 d11	90А X pulse, in Еs gradient pulse delay for disk I/O	(12.0u) 1m (30m)
d1	Relaxation delay = (1-5)*T1	(1.0s)
d2	Refocusing delay = (2JHX)–1	(3.45m)
SFO2	X channel resonant frequency
PL[2]	Pulse level for channel 2	0.00dB
d12 d13 d16 d20	Power-switching delay short delay delay for gradient recovery d2-p16-d12-d13	20u 3u 200u (2.45m)
d0	Initial evolution delay, in s	3u
p2	180А 1H pulse, in Еs	(18u)
DE	Receiver dead time	4.5u
SFO1{F2}	Proton frequency	П
PL[1] PL12 PCPD2	H1 Xm power C13 Xm power for decoupling 90А X pulse, in Еs with PL12	0.00dB 14 (60u)
SWH{F2}	Spectral width	SW(1D)
TD{F2}	Total data points for F2 acquisition	1k
NS	Number of scans = 1*N	1
DS	Dummy scans	8
TD{F1}	Total data points for F1 acquisition	256
IN	Incremental time, in s =	П

The pulse sequence is as follows:

1H d1 p1 d2 d0 p2 d0 d20 acq

13C p3 p3 garp

gradient p16-d16 p16-d16 p16-d16

V. Gradient-HMBC (Magnitude mode)

1. Check and tune the probe. Get 1D 1H spectrum in expno 1and make a note of the O1, SW, and RG values. Determine the 90А pulse width for both nuclei.

2. Create a new expno by edcП.

3. Type rpar_hmbcgs_allП. Type edaП. Change PULPROG to hmbcgs

4. Make sure that ND0 = 2 and MC2 = QF.

5. Set SW2 = SW (1H) from 1D spectrum in step 1 and SW1 = SW(X).

6. Set O1 = O1 (1H) from 1D spectrum in step 1. Set O2 to 13000 for a window centered around 100 ppm for 13C.

8. Type asП and assign the following parameters:

p1	90А 1H pulse, in Еs	(9u)
p3 p16 d11 d16	90А X pulse, in Еs Gradient pulse delay for disk I/O Gradient recovery time	(12.0u) 1m (30m) 200u
d1	Relaxation delay = (1-5)*T1	(1.0s)
d2 d6	Refocusing delay = (2JHX)–1 delay for long-range J, (2^2,3JHX)–1	(3.45m) (40m)
SFO2	X channel resonant frequency
PL[2]	Pulse level for channel 2	0.00dB
d12 d13	Power-switching delay short delay	20u 3u
d0	Initial evolution delay, in s	3u
p2	180А 1H pulse, in Еs	(18u)
DE	Receiver dead time	4.5u
SFO1{F2}	Proton frequency	ј
PL[1] PL12 PCPD2	H1 Xm power C13 Xm power for decoupling 90А X pulse, in Еs with PL12	0.00dB 14 (60u)
SWH{F2}	Spectral width	SW(1D)
TD{F2}	Total data points for F2 acquisition	1k
NS	Number of scans = 4*N	4, 8, or 16
DS	Dummy scans	8
TD{F1}	Total data points for F1 acquisition	256
IN	Incremental time, in s =	-

The pulse sequence is as follows:

f1:	d1	p1	d2		d6		d0	p2	d0		go
f2:				p3		p3				p3

VI. Gradient-1D Difference-NOE (GOESY)

1. Check and tune the probe. Run a standard 1H spectrum in expno 1 with properly adjusted spectral width, and determine the 90 degree pulse width.

2. Determine the offset of the peak to be irradiated with respect to O1 using the spectral cursor.

3. Create expno 2 from expno 1 by edcП.

4. Type rpar goesyП, then change SW and O1 to proper values.

5. Type asП. to set up all the parameters used in the pulse sequence goesy.

p1 p2 p11 p16	90А pulse, in Еs 180А pulse 90А shape pulse Duration of gradient	(9u) (18.0u) (100m) 1m
d1	Relaxation delay = (1-5)*T1	(1s)
d8 d11 d12 d16 d20	Mixing time I/O delay RF switching time Gradient recovery time =p16+d16	(400m) 30m 20u 200u 1.2m
DE	Receiver dead time	4.5u
SFO1{F2}	Proton frequency	П
PL[1] PL18 SP1 SPOFFS1 SPNAM1	Pulse level Default power for shape pulse Power for the shape pulse Offset of the shape pulse Shape of the selective pulse	0.00dB 120 dB (68 dB) (from step 2) gauss
SWH	Spectral width	SW(1D)
TD	Total data points for acquisition	32k
NS	Number of scans	16
DS	Dummy scans	4

6. Type zg to start the acquisition.

7. Phase the spectrum with the peak that was irradiated negatively phased. Any peaks that are

positive in phase indicate positive NOE.