- NMR Operation Manual for Department of Chemistry -
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ADDSUB:SPECTRA ADDITION / SUBTRACTION ON THE GEMINI
1. In experiment k, display spectrum A. Make sure the reference peak is at the
correct chemical shift. Type NM to normalize the vertical scale to standard, and
adjust VS if necessary, then type ADDI(CLEAR), to delete spectrum in addsub buffer.
Enter ADDI(START), this command puts the current spectrum A into the addsub buffer.
2. Join experiment j by typing JEXPJ. In this experiement, prepare the spectrum B
which is going to be added to or subtracted from spectrum A. Display the spectrum,
make sure the reference is correct as well, and the FN for spectrum B should be
the same as that for spectrum A. Type NM and adjust vertical scale.
3. ADDI. This macro leads you into an interactive mode in which you can do the
following to the three spectra (A,B,result ) that are displayed on the screen
simultaneously:
(1)F2: Use this function key to select the spectrum you want to control.
(2)F8: Change addition or subtraction mode.
(3)At most time, you need to adjust the WP, SP and VS for the spertra A and B to make sure they are added/subtracted in the right chemical shift. To do this, normally one common peak in both spectra is used as an indicator. Especially in subtraction, this peak shoud become a flat line (or close to) in the result spectrum.
(4)F7:Use this key to save the result spectrum when you are satisfied with it.
4.DS(RESULT) shows you the result spectrum. You can process with this spectrum
as usual except saving it on disk, because it is ONLY a spectrum without any FID.
NOTE:Addition or subtraction of spectra works best when two spectra are acquire
under identical condition (i.e. frequency, spectral width, etc.) and on the same
machine.
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APT:ATTACHED PROTON TRANSFER
The PW90 value has to be up-to-date for this experiment.
1.Acquire a good carbon spectrum in Expm, move parameters from Expm to Expn by typing
MP(m,n). Ensure that the carbon spectrum is properly phased. This step allows you to
return to Expm to reobtain the parameters whenever you need to.
2.Type APT.
3.The instrument will setup APT experiment parameters and give a prompt with a series
of particulars to check. After you check those, type TIME to check the duration of
the acqusition. Adjust NT so that the experiment will fit in your time slot.
4.Type GA to start acqusition.
5.At the end of acquisition, process th spectrum just like you would for any 1D carbon
spectrum.*
*:Do not type APH or perform any other phasing because they will randomize the phase
information that you are looking for!
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BYF19:MODIFIED SEQUENCE FOR 19F NMR SPECTRUM
Usually 19F NMR spectra have wave-like baselines, especially when the signals are very
strong. The following procedure can provide a flat baseline for 19F spectra without
losing much signal-to-noise ratio.
1.Run a standard proton NMR as usual to ensure appropriate locking and good shimming.
2.Type macro BYF19 to set up a 19F experiment that contains a modified S2PUL sequence
with an extra delay D3 between the PW pulse and acquisition time AT. This will allow
the system to avoid the first very short period (1x10-4 sec, or so) to get rid of
breakthrough or too strong signals in FID.
3.Use the shortest delay D3 as you possibly can to get the least phasing distortion.
Suggested starting value for D3 is 1x10-4 sec, type D3=1e-4.
4.Use sufficient number of transients NT. Start acquisition by typing GA.
5.After a spectrum is displayed. Examine the baseline first, if it's still a little
wavey,rerun the spectrum with longer D3 delay, like 5x10-4 sec. Otherwise,type QP to
get into the quick phasing mode. Use the right phasing RP and left phasing LP knobs
to obtain a normal spectrum with flat baseline #.
6.If a good phasing can not be archieved, shorten D3 to rerun the experiment until
satisfied. Usually, there are not too many peaks in 19F NMR spectrum, therefore,
quick phasing works very well even when D3 is relatively long, like 1 msec.
#: Spectrum has already been referenced to CFCl3 ( 0 ppm).
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CCC2D: 13C-13C CONNECTIVITY (INADEQUATE)
The 13C PW90 value has to be up-to-date for this experiment.
1.Acquire a good carbon spectrum in Expm, move parameters from Expm to Expn by typing
MP(m,n). Ensure that the carbon spectrum is properly phased. This step allows you to return
to Expm to re-obtain the parameters whenever you need to.
2.Type CCC2D.
3.The instrument will setup parameters for an INADEQUATE experiment. The PW should equal
to PW90 and P1 should equal to 1350 or 900. NI should be a multiple of 32. Type LK and F1
to stop the spinning. Type TEMP=30 to setup temperature regulation. Use TIME to check the
time will be consumed, adjust NT or NI if necessary.
4.Type SU and GA2D to start acquisition.
5.At the end of acquisition, a rough 2D NMR spectrum is displayed and process the it
just like you would for any 2D carbon spectrum.*
*: Refer to COSY part of this booklet.
NOTE: INADEQUATE experiment tests the 13C-13C connectivity and requires large amounts of
sample. A signal/noise ratio of over 25 by one transient is required in 1D spectrum if
a 24 hour 2D experiment is expected.
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HOMCOR: 1H-1H CORRELATED 2D SPECTROSCOPY
(ACQUISITION AND PROCESSING)
You need 3 experiments (Expk (128k), Expl(128k) and Expm(>512k) ) for maximum efficiency.
1.Acquire a good proton spectrum in Expk. Ensure that PW90 value is up-to-date.
2.Set cursors at both ends of the region of which you need to observe the COSY. Type
MOVESW.Parameters will be setup so that this is the only region that is going to be
observed. You may leave out solvent and TMS peaks, etc.. Do not reference after the
MOVESW.
3.Move parameters from Expk to Expl by typing MP (K,1). Re-acquire a proton spectrum
and ensure there are no folded (aliased) peaks. If there are, then repeat step 2
and/or change TO value until no folded peaks are observed.
4.Type MP(k,m) to move parameters from Expk to Expm.Then type HOMCOR. The instrument
will setup the parameters and give a text file with a series of particulars to check.
After you check these type TIME to check the duration of the acquisition. Adjust NT
and NI to make the experiment fit in your time slot.
5.Type GA2D or AU to start acquisition. If an error message GO FID FILE TOO SMALL
appears, reduce NP by factors of 64 and try to start acquisition again.
6.At the end of acquisition and computing, a very rough contour plot will appear on
the screen. Type FOLDT to symmetries data.
7.Change values of VS and/or TH (threshold) to reduce noise and enhance signals.
Use color bar on the side of the plot as reference.
Some useful commands and their function:
DCON: Display contour plot
DCONN: Display contour plot without clearing previous screen
AXIS=PP: Set both of the axes in ppm
FULL DCON:Contour plot fills the screen
DCONI: Display contour plot in the interactive mode
SP and WP: Start of plot and width of plot
SP2 andWP2: Same as SP and WP but in the second dimension
DS2D: Display a stacked plot of 2d spectra (whitewashed)
HOMCOR: 1H-1H CORRELATED 2D SPECTROSCOPY
(PLOTTING)
1.When you have a satisfactory contour plot on the screen, set the label that you
want to be plotted on the spectrum by typing TEXT('text') followed by DCON.
2.Set the number of pens you want to use by typing MAXPEN=n. Commence plotting
by RCONPP. Do not attempt to do other operations while the plotting is in progress
as it will crash the computer.
3.At the end of the 2D plot, join the experiment that contains the 1D spectrum
(Expk) and type PCON1D. This will place the spectrum on the top left of the
screen. Adjust VS so that the tallest peak is on scale and type COSYPL. This will
plot the 1D spectrum on both sides of the 2D plot.
4.If you require expanded regions of the 2D plot, input the desired SP, WP, SP2
and WP2 from the appropriate 1D spectrum and type DCON.
5.Follow step 2. Then join 1D experiment, type in appropriate SP and WP and
follow step 3.
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DEPT:DISTORTIONLESS ENHANCEMENT BY POLARIZATION TRANSFER
1.Acquire a good carbon spectrum in Expm. Ensure the PW90 and PP values are up-to-date.
Remember that TPWR and DHP are related to PW90 and PP values, respectively. If you are
not sure about these two parameter, do a PW90 pulse* and PP calibration before going to
the next step.**
2.MP(m,n) to move parameters to Expn, which has more than 512K words,JEXPN. This step
allows that you can always come back to get the original parameters whenever DEPT
experiment fails.
3.Type DEPT. Standard parameters will be setup. Use a suitable NT to meet your time
solt, NT should be a multiple of 8.
4.Type GA. At the end of processing, type DEPTP. This macro will do all the spectral
editing for yor, make sure the plotter is ready before you type this command.
*:Refer to PW90 part of this nmrbooklet.
**:PP calibration:
1. Select a compound which has a CH carbon, and is concentrated enough to get a carbon
NMR easily. (e.g. Ethyl Benzene)
2. Set up a DEPT experiment following the steps above down to step 3. Before go to step
4 above, type MULT=1 and a PP array (Current: PP is arround 30 when DHP=43 and PPLVL=60).
3. GA. At the end of acquisition, look for the CH peak(s) of maximum intensity while
other protonated carbons are at minimum. The PP value corresponding to that spectrum
is the required PP value (Proton 90o pulse width with power from decoupler)
4. PP value can also be obtained using D2PUL sequence experiment, refer to system manual
for description.
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HETCOR:1H-13C CORRELATED 2D SPECTROSCOPY
(ACQUISITION AND PROCESSING)
You need 4 experiments (Expj (128k), Expk(128k),Expl(128k) and Expm(>1024k)) for maximum
efficiency.
1.Acquire a good proton spectrum in Expj. Follow step 2 and 3 in COSY section of this
nmrbooklet to check folded peaks (in Expl). Print out the parameters by typing DG PRINT DG1
PRINT for later use.
2.Acquire a good carbon spectrum in Expk. Check for foldovers in reduced spectral window.
(as in step 1 above, in Expl). Ensure that all PW90 and PP values are accurate.
3.Move parameters from Expk to Expm by typing MP(k,m). Type HETCOR.
4.The instrument will setup the parameters and you can use the printout generated in step
1 to check the parameters pertaining to the proton spectrum. Type TIME to display the
duration of the acquisition. Adjust NT or NI to make the experiment fit in your time slot.
5.Type AU or GA2D to start acquisition. If an error message GO FID FILE TOO SMALL appears,
reduce NP or NI and try to start acquisition again.
6.At the end of acquisition and computing, a very rough contour plot will appear on the
screen. Change values of VS and/or TH from the keyboard to reduce noise and enhance
signals. Use the color bar on the side of the plot as your referrence.
Some useful commands and their function:
DCON:Display contour plot
DCONN:Display contour plot without clearing previous screen
AXIS=PP:Set both of the axes in ppm
FULL DCON:Contour plot fills the screen
DCONI:Display contour plot in the interactive mode
SP and WP:Start of plot and width of plot
SP2 andWP2:Same as SP and WP but in the second dimension
DS2D:Display a stacked plot of 2D spectra (whitewashed)
HETCOR:1H-13C CORRELATED 2D SPECTROSCOPY
(PLOTTING)
1.When you have a satisfactory contour plot on the screen, set the label that you want
plotted on the spectrum by typing TEXT('TEXT')followed by DCON.
2.Set the number of pens you want to use by typing MAXPEN=n. Commence plotting by typing
RCONPD. Do not attempt to do other operations while the plotting is in progress as it
will crash the computer.
3.At the end of the 2D plot, join the experiment that contains the 1D carbon spectrum
(Expk) and type PCON1D.This will place the spectrum on the top left of the screen. Adjust
VS so that the tallest peak is on scale and type PL(PAGE1).This will plot the 1D spectrum
on one side of the 2D plot.
4.Join the experiment with the proton spectrum (Expj) and type RCON1D. Adjust VS and type
PL to plot the proton spectrum on the other side of the contour plot.
5.If you require expanded regions of the 2D plot, input the desired SP, WP, SP2,and WP2
from the appropriate 1D spectrum and type DCON.
6.Follow step 2. Then join 1D experiment, type in appropriate SP and WP and follow step 4.
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HMQC:HETERONULCLEAR MULTIPLE QUANTUM COHERENCE
You need 3 experiments(Expk(128K),Expl(128K) and Expm(>512K)) for maximum efficiency.
1.You have to replace the regular "blue box" LP filters with band-pass filters in the X
channel and lock channel. Install a carbon (or 15N or 31P) filter between the
probe and the J5302(13C) connector on the magnet leg. Install a 2H band-pass filter between
the probe and the J5202(2H) connector on magnet leg. A significant change in lock phase will
be observed when the lock filter is placed in-line, adjust lock phase LPHASE to regain
a good lock signal.
2.Type JEXPk and SETUP(C,solvent) to set up a 13C experiment with suitable solvent. Obtain
a 13C spectrum. Do a reference calibration. Move the cursor the signal on which you want to
put the transmitter offset and type MOVETO. Record the new TO value. Do not recalibrate the
reference at this time. Do a 900 pulse determination for carbon.
3.JEXPl to move into experiment l. Set up a 1H experiment and get a proton experiment. Narrow
the 1H spectral width to include just the peaks of interest. Set DO=@TO and type D2PUL to set
up the D2PUL pulse sequence. The D2PUL sequence uses the decoupler to pulse protons. The power
level will now be controlled by the parameter PPLVL. Initially make sure that PPLVL is equal
to TPWR for protons. Acquire a 1H spectrum to ensure that the DO and SW values are correct.
Measure the 1H 900 pulse width using PW array in the normal manner, and put PW90 at the value
you obtained.
4.Turn the spinner off. Type MP(l,m) and JEXPm to move the parameters from the proton
experiment into experiment m and join the experiment. Type HMQC(EXPk), where EXPk is the number
of the experiment that contains the 13C data, to set up the HMQC experiment.
5.Set PHASE=0,suitable NT and PWX(1)=0,15. Acquire the spectra. The data should show the proton
spectra with and without carbon pulses. In other words, the first spectrum shows you inverted
peaks for all protons, the second shows inverted peaks for protons that not attached to 13C
but normal peaks for 13C attached protons. Now determine the 900 X pulse (13C,31P,15N etc)
value by constructing a PWX array. The PW90 for X nucleus should be close to the value of
90 pulse measured in step 2.
6.Set the PWX to the value obtained in step 5. Set NULL=0 and get a spectrum. The doublet for
protons coupled to 13C will appear at approximately 150Hz apart, and the center peak from
protons not coupled to 13C will appear in the center of the doublet. Array NULL to establish
the value the provides maximum cancellation of the peak from protons not bonded to 13C. Usually
NULL varies from 0.2 to 0.4. Enter the value of NULL that corresponds to minimum center
peak intensity.
7.If X-coupled indirect spectra are desired, there is no need to calibrate the decoupler
power,and you can go on to step 8. If you need X-decoupled spectra. You need to set the
WLVL(decoupler power level) at an appropriate value. Refer "Indirect Detection
Experiments" by Varian on page 30-31 for WLVL calibration. (Approximately WLVL=48 DMF=16200).
Set DM=NNY, WOFF=@TO.
8.Type GO to initiate acquisition. When the acquisition is complete, type WFT to Fourier
transform the first few increments. Display the first increment of the 2D data by typing
DS(1).Phase this spectrum in the normal manner using RP and LP.
9.Perform the 2D transform by typing WFT2DA. At this point, data can be manipulated like
any other 2D data set.
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HOMDEC:1H-1H HOMONUCLEAR DECOUPLING
Two experiments are needed for good efficiency, expk and expm (> 512k).
1.In experiment k, obtain a good proton spectrum as usual. Use commands S1,S2, etc. to
save each region you want to observe later. Copy the current parameter setting into experiment
m by typing MP(k,m). Join experiment m by JEXPm.
2.In experiment m, move the cursor to the center of the peak(s) which you wish to decouple,
type SD. This command changes the DO(Decoupler Offset) so that the decoupler will irradiate
at the desired frequency.* The decoupler power is controlled by DHP in broadband system which
range from 0 to 50 (highest power). Input an array of DHP value by DHP(1)=5,10,15,20,25....
Type DM=YYY, DM=C. Start the acquisition by GA. At the end of it, DSSH to display all spectra.
Examine the spectra and decide whichpower level is the appropriate one to use. A good power
level setting should diminish the target proton peak without disturbing the others. Currently,
a good DHP value is around 20-30.
3.Still in experiment m, use the DHP value determined in step 2. Input a sufficient NT number.
Type GA. Examine each region of the resultant spectrum by R1, R2, ... commands.
4.If different frequencies needed to be decoupled, change the DO by SD command as in step 2.
Determine the DHP similiarly. Go back to experiment k to retrieve the original parameter
setting whenever necessary.
*. DO is a fine adjustment of decoupler frequency while DBO controls the base setting.
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DETERMINATION OF HOMONUCLEAR NOE ENHANCEMENT
Samples have to be adequately degassed with N2 or Ar before the NOE experiment.
1.Obtain a good proton spectrum in an experiment with more than 512k words. Display the peaks
you wish to determine NOEs and save each region.
2.Set the decoupler on one of the peaks you are going to saturate by putting single cursor
on that peak and typing SD.
3.Determine the lowest power that is needed to saturate that peak, using a DHP array experiment.
(The higher DHP number, the lower decoupler power).
4.Set DHP to the value determind in step 3. Set a DO array of all the peaks you wish to
saturate. Then set an additional DO value by typing DO(n)= -10000, where n is the next
index number on the array.
5.Set DM=YYN, DMM=C, IL=Y, SS=2 BS=1/4 NT ,D1=10xT1, PW=PW90.
6.Type GA to start acquisition.
7.Recall the regions of interest. Type DSSH to display all the DO array spectra.
8.Measure the intensities of the peaks in all spectra by typing DLL or LL. The differences
between the peaks before the last peak and the last peak give NOE results for each DO value.
9.Moreover, you can set DO value at one you used in step 4. Use a DM array ,DM(1)=NNN, YNN
in step5. After you get the two spectra, generate a line list using DLL or LL. Type FPS(#),
followed by NOE(#). All these give you NOE results for different peaks on the printer (only
one peak saturated).
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NOEDIF: NOE ENHANCEMENT EXPERIMENT BY NOEDIF SEQUENCE
1.Run the proton spectrum in the usual way, store the spectrum in Expk, then enter MF(k,l) to
move the FID to Expl.
2.Enter WFT to display the spectrum, followed by macro NOEDIF. This macro converts the pulse
sequence from S2PUL to NOEDIF and created the parameters to setup the experiment.
3.Find the peak or multiplet to be irradiated and expand it.Display a cursor and select the
frequencies for cycling during irradiation as follows:
a.If the line is a single line, set the cursor at the point halfway up the peak on the left
side, then enter SD followed by F1=@DO. Reset the cursor at the center of the peak, then enter
SD followed by F2=@DO. Next, set the cursor at the point halfway up the peak on right side,
enter SD followed by F3=@DO. Enter TAU=0.1 unless it is already set to this value.
b.If the proton to be irradiated is a doublet, F1 through F4 are normally used, leaving F5 at
0. Set up the frequencies F1 to F4 as described above, setting the cursor in sequence on the
side of the first line, then on the second, then on the other side of the first line, and
finally on the other side of the second.
c. Triplets and higher multiplets are usually cycled over the frequencies of each of the
lines in the multiplet. Set the frequencies F1 to Fn (where n is the number of lines in the
multiplet) at the top of each peak.
4.Enter SS=4, D1=4. For samples with very long T1, longer D1 can be used.DM=YNN. DOFF=-10000Hz.
5.Set an array for CTRL at N and Y. Enter CTRL(1)=N,Y. N gives a NOEDIF experiment while Y gives
a normal proton spectrum.
6.GA. If the observed proton peaks have positive NOE, the CTRL=N spectrum will show positive
peaks and comparison with the CTRL=Y spectrum can give NOE values. Note the irradiated peaks
will appear to be large negative signals.
NOTE: For broadband Gemini system the homonuclear decoupler field strength is controlled by
the parameter DHP,setable in 0.5 dB from 0 to 50. NOE difference experiments will require a
setting near 25. Calibration can be performed if necessary, look up in the Gemini Operation
Manual for more information (p123-5).
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NOESY: NOESY LABORATORY FRAME OVERHAUSER EXPERIMENT
Samples have to be adequately degassed with N2 or Ar before the NOESY experiment.
PW90 should be also up-to-date to ensure best results.
1.Obtain a good proton spectrum in experiment n , narrow the spectral width SW to retain
only the desired peaks by MOVESW. Move parameters to experiment m with more than 1024k words
by MP(n,m), followed by JEXPm which activates experiment m.
2.In experiment m, setup the 2D exchange experiment by typing NOESY.
3.Examine data parameters after DG command. Among them, D1 should be at least 1.0 second,
but no more than 3 times T1. If phase sensitive NOESY is desired, set an array for PHASE
at 1,2 (namely, PHASE(1)=1,2 ),leave PHASE=0 otherwise.# The most important parameter is
the mixing time MIX, which is usually between 0.1 to 0.2 sec. Sometimes, different MIX times
have to be used to determine the best MIX.
4.Set DM=NYYN which makes the decoupler on during evolution and mixing periods. NT sould be
multiples of 8 when PHASE=1,2 and 16 when PHASE=0. NI should equal to FN2/2 or FN2/4,
for slightly better data and speed, respectively.
5.Type AU to start acquisition. At the end of the experiment, a rough 2D contour plot,
just like a COSY spectrum, will be displayed. Do a FOLDT, and process it same as for a
normal 2D spectrum*, use WFT2D command for PHASE=0, and WFT2DA for PHASE=1,2 experiment.
#When PHASE=0, NOESY is actually an NOE2D experiment.
*Refer COSY section in this nmrbooklet.
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PW90: 90°Pulse Width Calibration
1.In an experiment which has more than 512 words, obtain a routine proton spectrum. Display
the region which has a singlet or 'well-defined multiplet'. Type S1 to save that region.
2.Set up a PW array from 5 to 50 ms in increments of 5 ms using PW(1)=5,10, ..., 50,5.*
3.Set NT at enough transients to get a good spectrum, SS=1, D1=10.
4.Type GA to start acquisition.**
5.At the end of acquisition, retrieve the region you have saved in step 1 by command R1.
6. Type DSSH followed by DA1 DSSN to display all PW values and spectra. The trace through
the peaks should form a sine wave. The PW value corresponding to the tallest peak is the 90
degree pulse width and that corresponding to the null should be the 180 degree pulse.
7.Reset the PW array in 1 ms increment, put 180o pulse near the middle of the array.Do the
acquisition again and get more accurate result from the 180o null time.
8.Adjust VP (Vertical Position) and VS (Vertical Scale) values to make spectra fit into
whole window. Type PL(ALL) to plot all spectra on the plotter. When adjust VS, the tallest
peak can be displayed by DS(#), where # is the number corresponds to that peak.
9.To determine the PW90 value for carbon, follow the same procedure but work with carbon
parameters and use enough NT for carbon spectra. Ensure that D1 is long enough (usually
has to be more than 30 sec).
*:Repeat the first value at the end of the array. The spectra corresponding to the value
1 and n should be the same, otherwise, prolong D1 to get full relaxation.
**:Gemini 300 has a parameter TPWR (Transmitter Power Output), different TPWR values are
related to different PW90, do not change TPWR during the calibration. Whenever use
calibrated PW90 value, make sure TPWR is correct.
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SELDEC:SELECTIVE PROTONDECOUPLING IN CARBON SPECTRA
1.Follow the steps in HOMDEC part of this nmrbooklet to determine the DHP level and decoupler
frequency setting (DBO and DO value) in a proton spectrum. Record the DBO and DO value.
2.In another experiment, setup the standard parameter for carbon by SETUP(C, solvent). Input
DHP, DBO and DO the same values as determined in step 1.Type DM=YYY. DMM=C.
3.GA. The spectrum will show the coupled carbon spectrum with some proton(s) selectively
decoupled. For comparison, DM can be set to NNN and the resultant spectrum shows the fully
coupled carbon spectrum. *
4.If desired, go back to proton spectrum to make other settings of DO and DHP to decouple
different protons.
*: Coupled carbon spectrum takes much longer time, make sure it fits your time slot.
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STANDARD OPERATION ON GEMINI 300 NMR SYSTEM
Part I: Standard operation on Gemini 300 system includes the following steps:
1. Sample preparation and placement into the probe.
2. Logging into the system.
3. Setting up experiment with standard parameters and appropriate modification if required.
4. Locking and shimming.
5. Acqusition.
6. Data processing, spectrum plotting and storing on disks if required.
7. Replacement of standard sample, relocking and logging out of the system.
Part II:System operation in detail:
Sample:Usually, to obtain a good spectrum on the Gemini 300 within 30 minutes or less, a
minimum of 2 mg of sample is needed for a 1H NMR spectrum and 50 mg for a 13C NMR spectrum.
The sample must be dissolved into a deuterated solvent, like CDCl3 or Acetone-D6,etc. We
use the deuterium in the solvent to LOCK the field, and keep the spectrum frequency from
moving. Additionally, acquisition can be done without locking, ask a coordinator for
instruction.
Placement:We always keep a standard sample, which contains TMS, CHCl3 and CDCl3, in the
probe. Before the experiment, that standard sample must be ejected from the probe, and
the sample to be tested is placed into the probe. The height of the NMR tube in the
spinner is very important. Done incorrectly, an NMR sample could break in the probe.
Probe repairs starts at $4000, not to mention other troubles and inconvenience. Standard
instructions on how to measure the appropriate height of the nmr tube in the spinner
are given by an NMR coordinator before you get an account. When the tube is in the probe,
make sure the green spinning indicator lights up, and the meter reads speed around 20-25 rps.
Login:If the system is not prompting " LOGIN: ", type LOGIN to get that prompt. Input your
own username (should be less than six letters, including numbers, etc.), then the password
when asked for. The password when you are initially given an account is identical to your
username. After you log in, type PASSWD, follow the prompts and then you can have your
own password. Do this as soon as possible to protect your account.
Setting up:Normally, an experiment starts with 1H NMR. Type SETUP(H,solvent) to obtain
the parameter setup, where solvent = D2O, CDCl3, C6D6, CD3OD or other appropriate deuterated
solvents. The system will set up the standard parameters for 1H NMR with different
Transmitter Offset (TO) values and/or reference calibration. Alternatively, the command
RT(STDPAR.H) does the same but much faster for samples using CDCl3 as solvent. Since
CDCl3 is the most common solvent, this command is very useful. For 13C ,19F or 31P NMR,
use similiar command but change H to C, F or P,respectively.
Locking: One reason to use a deuterated solvent is to trace the 2H signal through the
whole experiment, this maintains a stable frequency. A lock signal monitoring window is
obtained by typing LK. In that window, you can manually turn the spinning off and on by
the F1 and F2 keys, and the lock off and on by F3 and F4 keys. At the bottom of the lock
window, there are four lock signal adjustment parameters, corresponding to the five black
knobs below the screen (starting from the left, Z0, LPOWER,LGAIN and LPHASE,
the fifth knob is not functional at this time).
Z0: Main magnetic field B0 base adjustment. ( around -370)
LPOWER: Lock power. The power level to be used to irradiate 2H signal. (0-40)
LGAIN: Lock gain. The gain level to detect 2H signal. (0-30, in steps of 10)
LPHASE: Lock phase. Phase of the lock signal.(usually around 110)
To avoid saturation, LPOWER is usually less than LGAIN. When a good combination of parameters
is archieved, the locking signal which is in the middle of the screen should show a stable
flat line with little oscillation. Do not let the line go beyond the window, because you can
not judge how stable the locking is. After you obtain a good lock signal, you are ready to
start shimming.
Shimming:Shimming can be very tricky for new users. The strategy of shimming is to use the
deuterium resonance as an indication of the magnetic field homogenity, and when the homogenity
is high, persumably the intensity of 2H signal is high too. Therefore, obtaining the highest
lock signal under given LPOWER and LGAIN is the target of shimming. This is also the other
reason of using a deuterated solvent. Other shimming methods are not discussed here.
Type S or SHIMI to get into the shimming window. There are many shimming parameters. Use F1
through F5 key to choose those you want to adjust. We usually keep most of the parameters
same for routine works. Non-spinnings shims are only to be adjusted by an NMR coordinator.
Users should only adjust Z1 and Z2. Press F1 key, to adjust Z1 and Z2. The five black knobs
are labeled (from left to right): Z1, Z2, Z1C,Z2C and LPOWER. Z1 and Z2 are for fine
adjustment, Z1C and Z2C are for coarse adjustment. On the right side of the screen are two
colored bars indicating the lock level, the left one shows large scale ( x1) while the right
one shows small scale(x5). It's not easy to describe the tricks of shimming in a short
paragraph. A good start is to try Z1C first, turn it to make lock signal higher, ( tell
from the two colored bars) and when you find the signal drops down, try Z2C ( Hint: first
try the direction opposite to the one you just used for Z1C), to have the lock signal go
up again. After several rounds like this, you are able to obtain a relatively high lock
level. Now start shimming with Z1 and Z2 for fine adjustment. The closer to the summit,
the slower and more careful you have to be when turning those knobs. In addition, always
adjust Z1 last.
Acquisition:When you are satisfied with shimming, you can start acqusition by typing GA or
GO (assuming other parameters are correct, including NT). GA performs both acquisition and
Fourier-Transform, whereas GO does acquisition only. If GO has been used, type WFT after
the acqusition to obtain the spectrum.Since the spectrum has been displayed, the shimming
result can be examined by inspecting line shape of any peak(s). Usually, it's the easiest
to inspect the solvent peak. If the shape of that peak is not Lorentian, and
sometimes even distorted or split into two or more peaks, try shimming again. If you want
to measure the linewidth of a peak, get one cursor by pressing the F3 key and move it close
to the peak by the knob coresponding to CR, type NL, which puts cursor in the middle of that
peak, if DRES is then typed, the linewidth of that peak will be shown on the screen. Usually,
a linewidth below 1 Hz is considered adequate for 1H spectra in most solvents. You will have
to balance between the value of shimming improvement and the time you can spend to decide
whether to go on or back to shimming.
If a higher S/N ratio is desired (this means a nicer looking baseline), input in a greater
number of transients, type NT= ##, followed by RA. At the end of the acqusition, a better
spectrum with higher S/N will be presented. Signal to noise ratio S/N is proportional to
the square root of the number of transients NT, in other words, if you want double your
S/N ratio, you have to have four times of NT than you already have (Assuming fully relaxed).
Spectrum editing:
Referencing: Use NL to put one cursor in the middle of the peak to be referrenced. Type
RL(###P), in which ### is the correct chemical shift in ppm for that peak. For instance,
the 1H chemical shift of TMS methyl group is set at 0.00 ppm, if you find that peak in the
spectrum, put the cursor on that peak using NL and type RL(0.00P), that peak is then set at
0.00ppm. Similiarly, use RL(7.24P) for CDCl3 solvent peak (in fact, CHCl3 in CDCl3).
Spectrum window: SP and WP are the two parameters which control the the displayed starting
chemical shift and width of spectrum (in ppm), respectively. If input SP=-0.2P and WP=12.2P,
a spectrum starts at -0.2 ppm (including TMS peak) and ends at 12 ppm will be displayed.
Vertical scale: First type NM and then use the knob corresponding to VS to adjust vertical
scale.
Intergration: Press the F1 key to display the yellow intergration line. Type CZ to
reinitialize the intergrations to zero. Get one cursor (F3 key) and use the F7 key to cut
at both sides of each region to be intergrated. Press the F10 key to get partial display
of the intergration if necessary. Then type DLI to get a line list of each region and its
intergration. Very often, you need to modify the intergrations to make them integrals (like
1, 2 or 6 etc.). To do so, input INS="unit value" followed by DLI , where "unit value" is
the figure the original intergration will be divided by. For example, if the original
intergration is 24.6, when INS=12.3 is input, DLI will show the new intergration as 2.0.
Threshold: The F6 key displays threshold line (a straight yellow line). Use the middle
knob to adjust the height of it. Only peaks above the threshold are considered true peaks.
Type DLL to get a line list of peaks on the screen and LL to send the list to the printer.
Moreover, DPF displays peak frequencies above each counted peak. These commands are
frequently used in 13C NMR spectra.
Printing and plotting: When a nicely edited spectrum is displayed on the screen (with scale,
peak frequencies,etc), simply type BDUMP to get a copy on the printer. On the plottor, usually
two kinds of plotting, NPLOT and RPLOT, can be executed. NPLOT performs plotting based on
11"x17" paper while RPLOT on 8.5" x 11" paper. Also CPLOT can be used which is a macro
contains RPLOT and CUTOFF to eliminate any part which is higher than a certain level.
(It is highly recommended to use CUTOFF in RPLOT, because otherwise a peak that is way
off-scale on the display will be plotted entirely on the paper!)
After the plotting mode has been chosen, type PL PSCALE to plot the spectrum and the scale.
You may wish to have an expanded region on the same paper (called inset). To do this, save
the current display by S1 (so you can always come back from the inset), use the F3 key to
get two cursors and move them at the two edges of the region to expand, type INSET to get
a display of that region above the original one. Use the five black knobs to move it
arround, expand it or adjust height of it. When you are satisfied with it, use PL PSCALE
or other plotting commands to get an insert plot. Use R1 command to return to the full
spectrum.
Data Storing and Retrieving:Currently, The Gemini 300 takes only 5.25 inch floppy disk
(double sides). Like any other system, formatting has to be done prior to other processing.
Use FORMAT(DSK5) to format new floppy disk, DCT(DSK5) to display content of the floppy disk,
SVF(DSK5.filename) to save FIDs onto the floppy disk with the name filename, and RT(DSK5.filename)
to retrieve a file from floppy disk. The retrieved is the FID only which can be converted to
spectrum using WFT command.
Logging out: Replace the standard sample into the probe. Setup a proton NMR parameter, use
lock signal monitoring window to gain a decent lock. Type LOGOUT or BYE to log out. This ends
standard operation on the Gemini 300 system.
Part III: Other frequently used commands(C) and parameters(P).
Setting Up:
SFRQ(P):Spectral Frequency, ~300 MHz for 1H, ~75 MHz for 13C, etc.
SW(P):Spectral Width (in Hz), coverage of frequency.
TO(P):Transmitter Offset (in Hz), center frequency.
AT(P):Acquisition Time (in sec.).
NP(P):Number of Points in FID decay.
TN(P):Testing Nuclei,shown by mass of nuclei.
BS(P):Block Size,new FID collected and placed in memory after each Block Size.
PW(P):Pulse Width (in msec), duration of pulse.
D1(P):Delay time 1 (in sec), delay time before every pulse.
NT(P):Number of transients, repetition of FIDs.
TPWR(P):Transmitter Power, controls power output of transmitter.
DO(P):Decoupler Offset (in Hz), decoupler center.
DN(P):Decoupler Nuclei, usually 1 for 1H.
DM(P):Decoupler Mode, usually NNN for 1H and YYY for 13C NMR.
DHP, DLP(P):Decoupler High and Low Power, control decoupler power output.
PW90(P): 90o Pulse Width (in msec), value associated with TPWR.
Acqusition and Processing:
SA(C): Temporarily stop acquisition, can be resumed by RA.
/A(C): Permanently stop acquistion, can also be resumed by RA.
TIME(C):Shows remaining acqusition time.
WFT(C): Weight and Fourier-Transform, effective after new FID collected.
LB(P):Line broading (in Hz), line broadening used in Fourier-Transform.
FN(P):Number of points used for Zero filling.
Spectrum Editting and Plotting:
DS(C): Display current spectrum.
FULL(C):Display spectrum over whole screen.
DSCALE(C):Display scale under spectrum.
F FD(C):Display whole spectrum, clear CUTOFF if present.
VP(P):Vertical position, the baseline position of spectrum.
PPF(C):Plot Peak Frequency for each peak above threshold.
PIR(C):Plot Intergration Ratio at the bottom of each region.
LABEL(C):Get into Labelling mode.
Ctrl-U: Move cursor up.
Ctrl-D: Move cursor down.
Ctrl-L: Move cursor let.
Ctrl-R or Space bar: Move cursor right.
Carriage Return:Change to the next line.
Ctrl-E: Exit Labelling mode without doing anything.
Ctrl-P:Plot what has been edited as label.
Data Restoring and Retrieving:
SVP(C):Similiar to SVF but save parameters only.
Miscellaneous:
MP(1,2)(C):Copy parameter from exp.1 to exp.2 (usable for other experiments).
JEXP2(C):Join experiment 2 (usable for other experiments).
EXPLIB(C):Show current available experiments.
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T1:DETERMINATION OF T1 BY INVERSION RECOVERY
Samples have to be adequately degassed with N2 or Ar and sealed before T1 experiments, and
PW90 value should be correct. The accuracy of T1 obtained by this experiment is limited by
the accuracy of PW90.
1.Obtain a routine proton spectrum in an experiment that has more than 512 k words.
2.Display those peaks you wish to determine T1s, and save each region.
3.Type DOT1. You will have a series of prompts, input the minimum expected T1 (usually 0.5 s),
then the maximum expected T1 (usually 8 s). For the expected experiment time, put in 1 hour.
4.Type in a suitable NT to make the acquisition time fit in your time slot and resulted
spectra will be good enough as well.*
5.Type GA. After the acquisition, Display the regions containing the peaks of interest and use
suitable threshold to print a line list by command LL.
6.Type DSSH to display all spectra. Type FBS(#) to measure the height of peak # in all spectra.
Followed by T1(#) to print out the T1 value of peak #. (# is the number in the list generated
in step 5).
7.For carbon T1 values, follow the same procedure except working with carbon and using
different expected T1s (usually more than 10s for maxium T1).
*:In T1 experiment, peaks should form a reverse exponential decay curve.The first inverted peak
and the last several peaks should have almost equal height. You can tell from the curve whether
the expected T1 settings in step 3 are good or not. Therefore, T1 experiments have often to be
run more than once before satisfactory results are obtained.
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CPMGT2:CARR-PURCELL MEIBOOM-GILL T2 TEST
SAMPLE SHOULD BE PROPERLY DEGASSED PRIOR TO THE EXPERIMENT, AND PW90 FOR THE OBSERVED
NUCLEI SHOULD BE UPDATED AS WELL.
1.Obtain a routine proton spectrum in expk. Save the regions for peaks you want to
test T2 values using the S# commands.
2.Type MP(k,l) and JEXPl. Check PW90 for proton and then type in appropriate PW90.
Use macro CPMGT2 to setup parameters for T2 test, type SPIN=N and LK to get into the
lock signal monitoring window, use F1 key to stop the sample spinning. Locking should
be still on.
3.Type D1=20 or the value of 3 times of T1. D2 is usually from 1 to 10 msec. Parameter
BT ( "BIG TAU" ) is the total spin-echo time between PW pulse and the acqusition.
It should be set at 4xNxD2, in which N is the repetition number of spin-echo. Input an
array of BT, the following can help you choose suitable BTs.
(a)Assuming the longest T2 in your sample is T2max and shortest, T2min. The
maximum BT should be about 4 times of T2max, and the minimum BT should
be about 1/4 of T2min. For example, if the estimated T2max is 5 sec and T2min
is 2 sec, then you can arrange a BT array from 0.5 to 20 sec.
(b)Because the signal decays in a single-exponential manner, it's better to array
the BT in such a way that the signal decays steadily, in other words, increase
BT slowly at the beginning and faster in the later part. Using the same example
as in part a), the BT array can be 0.5,1.0,1.5,2.0,3.0,4.0,6.0,10.0,15.0 and 20.0.
(c)The computer will modify BT so as to fit BT=4xNxD2 equation, and use N number
of flip-flops in the sequence.
4.Start experiment by GA. When it finishes, display the whole spectrum of the first
one in array by DS(1) and F FD. Use suitable threshold to cover all the peaks of interest
followed by FPS(ALL), and then DLL or LL to obtain a line list of each peak.
5.Retrieve each region with R# command. At this point, you can get T2 value for each region.
Type T2("peak number") to print out the T2 value of all peaks. Alternatively, you can
use DT2("peak number") to display the result and PRINT to have a hard copy.
6.Carbon nuclei T2 determination follows the same procedure with some changes for D1, D2
and BT array.
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VNMR:Processing of NMR spectra on the SUN SPARC Station
INTRODUCTION
Up to date, there are two ways to transfer NMR spectra to the SUN station, one is to use
ESAVE or XSAVE macro on GEMINI, the other is to retrieve spectra from streaming tape
which obtains spectra from XL300. It is expected that we will have many other ways to
do the transfering in the near future. Ultimately, we will be sitting before the SUN
(or even in your office at your micro! ) doing all the acqusition and data processing.
Log into the SUN as vnmr2 (lower case) or other username you have been given, type in
the correct password (case sensitive). The system has been set to go into the SUN
OpenWindowsTM directly from login shell, and Varian's VNMR software will be executed
right after that.
PROCESSING AND DISPLAY
From top to bottom in the VNMR are the message bar and command section (commands will
be in bold later on ), and menu buttons (will be in italic later on) which can be
activited by mousebutton, and then spectrum display window and text window.
Use mouse's left button to click main menu,(do this whenever you get lost in the process),
and file, then the current directory's file contents (including subdirectories)are shown
in display window. You can find out what directory you are in by typing pwd and it will
be shown in the message bar at the top. Highlight any directory and click change to get
into your own directory, and then highlight the file you want to work with, click load,
then process, weight and transform. The spectrum will be displayed. Sometimes you have
to type this command:convert('filename') in the command bar before doing process, weight
and transform or typing wft.
If a larger full screen display of your data is desired, click large, return,size and
full screen. The spectral display can be controlled through a combination of cursor
positioning and mouse buttons. Using the left mousebutton will display and control
a single (or left) cursor which will track with the parameter CR observed at the
bottom of the display window.Depression of the right mousebutton will display and
control a second (right cursor). The parameter DELTA (observed at the bottom of screen)
is the difference in position between the two cursors. When both cursors are displayed,
the left mousebutton will move together such that DELTA does not change. DELTA is
changed by moving the right mousebutton. The region defined between the two cursors
as described above can be expanded on the screen by using the expand menu button.
Full returns the whole spectrum.
Optionally the inset command will allow the expanded region to be manipulated more
readily for positioning on the screen without clearing the original display from the
screen. (Suggest you use s1 to save original display before doing inset so it can be
retrieved with r1.)
The middle mousebutton in almost all cases functions to control scaling. When a
spectrum is displayed, pressing the middle mousebutton will adjust the spectrum
intensity (VS) such that the spectral feature (either a peak or baseline) at that
position will reach the position of the mouse arrow. Placing the mouse arrow at the
left edge of the display will vertically position the display to that point (VP).
When an integral is displayed, the middle mousebutton functions the same way with the
integral scaling (IS) and baseline (IO).
To see a scale under the spectrum, type dscale, or from the display menu, use
interactive, next then scale. The units are controlled by the parameter AXIS=
p, h or k (ppm, Hz, or KHz respectively).
PHASING
Type aph or choose display then massage, then autophase to do automatic phasing.
If improved or customized phasing is desired, use the display, then interactive
menu buttons to reach the phase key. Position the mouse arrow over a spectral region
on the right side of the spectrum, about half way up the screen and click the left
mousebutton. The special display observe allows you to adjust the zero order
(frequency independent) phase (RP) of theis region. Postitioning of the mouse
button within the two cursors determines the direction of the correction (above or
below line: clock wise or counter clockwise peak rotation). the left mousebutton
will make large (coarse) pahse changes relative to the right mousebutton (fine).
Generally holding the right mousebutton down and slowly moving the mouse up or
down will give a smooth continuous phase adjustment. When this region is adjusted,
move the mouse arrow outside of this region to a spectral region towards the left.
Repeat the phase process as before, which will adjust the frequency dependent phase,
LP, holding the phase constant at the previously phased region.
Any clicking of the middle mousebutton will apply the phasing to the entire
spectrum as well as adjusting VS. Choose any other button (i.e. box) to leave this
mode.
If you have any problems, recover the last phase correction before you
went into the phasing mode by clicking the left mousebutton on the horizontal line,
or typing aph.
REFERENCING
If a non-zero ppm reference is to be used, use the mousebutton to place the cursor
on the reference peak. Type nl (nearest line) then rl (##p) with the reference shift
## in the parenthesis. Or, from the main menu, use display, then more and reference
to set TMS to 0 ppm.
INTEGRATION
If blanking of baseline integral regions is desired, make intmod='partial'.From the
main menu, click display, interactive, integral, return, next and then resets. Now
desired regions can be defined by positioning the mouse arrow and pressing the left
mousebutton.
Another alternative is to use a single cursor to define reset points and type z at
each position. Keep in mind that two reset points are needed to define each integral
region ( for intmod='partial' ).
The drift correction (dc) can be optimized manually using the lvl/tlt menu button (from
interactive menu). The left and right mousebutton function similiar to the phasing routine.
To display (dpir) or plot (pir) the values of the integral regions under the spectral
display, first type vp=12 to allow enough room.
To find peak position, first use the threshold key (within the interactive menu) to set
the lowest absolute value for listed peaks with left mousebutton. Typing dpf will show
pointers to listed peaks. Display a line list in the text window with dll.
PLOTTING
You must remember all plotting jobs are not sent to the plotter until the page command is
typed. From the display menu, click plot which then gives a range of plotting options.
plot ( or pl ) will plot th spectrum (as shown and integral as well if displayed). params
( or pa ) gives a brief listing of main parameters. all params (or ap ) gives a complete
listing parameters (upper left of plot). scale ( or pscale ) places a scale under the
spectrum.
pir ( or pir ) plots integral values under the specified regions.The command pll will
plot a column of line lists on your page, this should probably be on a seperate sheet
from your spectrum.
TEXT MESSAGE
Text messages help document both your plotted and stored data. A new text
message is inserted into the active experiment using the format text('put your message
here and use \ to start a new line') .
Additional text can be added to an existing message using atext('this will be added
at the end of message')
STORING DATA
To save an FID in a hard disc file, change the current directory as desired by menu buttons
from main menu, file, click on the directory chosen, then setdir, chdir. Then after
clicking on data, save fid, type in the name you choose for the file. (The .fid extension
will be added automatically.)
If the FID file is already under current directory, storing it onto a 3 1/2" floppy disc can
be done by typing bar cvf /dev/rfd0 filename.fid under UNIX system shell. Remember the disc
should already be a formatted one by command fdformat under UNIX system shell.
Retrieving the FID needs another bar command bar xvf /dev/rfd0 filename.fid, also under the
UNIX shell.
Storing data to streaming tape needs command tar cvf /dev/rst0 filename.fid and retrieving
tar xvf /dev/rst0 filename.fid.
USING MULTIPLE EXPERIMENTS
You may use as many of the 9 possible experiments as you wish during your session. Use the
workspace menu to create any new experiments not defined in your directory. The amount of
space created in each experiment is flexible and will adjust to whatever parameters you set.
Keep in mind EXP5 is used for add-subtract operations and data may be overwritten if you use
that option.
Parameters are moved from one experiment to another by mp(i,j), move parameters from
experiment i to j. Your working experiment is changed by jexp(j), join experiment j.
Display parameters can be saved and retrieved within an experiment by s#
and r#, respectively (# =1...9). Display parameters can be moved between experiments
by md(i,j) (move display from exp i to exp j).
FINISHING UP
When you are ready to leave the SUN data station, use the menu item main menu, more and
then exit or type exit to leave VNMR.
Then placing the mouse over the logo (right) portion of the screen, press the right
mousebutton, and slide the mouse down the pop-up menu until "exit". Release the
mousebutton, then confirm your choice to leave sun view.
Type logout to end your session elapsed time.
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XSAVE:Transfer Spectrum from Gemini to SUN data Station
1.When you have obtained a 1D or 2D spectrum in any experiment on Gemini-300, you can
transfer the results to the SUN data station via Limnet by typing in XSAVE *, which is a
macro stored in the MACLIB on Gemini dsk1.
2.During the data transfering, you will see a number somewhat like " A000### " appear on the
screen which is going to be the filename under directory " /home/vnmr/tmp/ " on the SUN data
station ( the extanded name will be your username on Gemini) .
3.You now can go to the SUN station, log in as VNMR2 (the password is available) and work in
the VNMR window. Go to the directory " /home/vnmr/tmp/ ", your file should be there and can
be retrieved and processed **.
4.Your spectrum in that directory will be deleted after some period of time to save disc
quota, therefore it is highly recommended that it be saved into your own directory which
you may create with permission of the management.
*:ESAVE does similiar job except it excludes username at the end of the filename.
**:Instruction on how to use the VNMR software on the SUN data station is
available upon request, also refer to VNMR section of this nmrbook.
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ZETA:USE OF ZETA PLOTTER
1.There are two main plot modes: NPLOT and RPLOT (Normal plot and rotated plot). NPLOT plots
the spectrum along the paper lengthwise, and RPLOT across. NPLOT is the standard plotting
mode which fits 11x17" paper. RPLOT is recommended because of the consumption of paper.
2.Refer to the attached paper. In RPLOT mode, there are four important scales:
WC: Width of the spectrum (in mm, normal value 220)
WC2: Height of the spectrum(in mm, normal value 175)
SC: Start of chart in direction 1 (in mm)
SC2: Start of chart in direction 2 (in mm)
Note:Vertical scale VS is also measured in mm, after typing command NM.
3. Other useful command (C) and parameters(P):
CUTOFF(C): Cuts off the spectrum at appropriate WC2 value. A line will be dispalyed on the top of screen and only the part of the spectrum that is below that line will be plotted.
MAXPEN(P):Number of pens to be used for plotting.
LABEL (P):Any desired information can be printed on the paper beside the spectrum after typing
LABEL.Use ^U,^D,^L,^R,^P,^E to change the cursor up, Down, Left,Right,start plotting and abort
abelling, respectively.
CPLOT(C):A macro, sets up RPLOT,SC=0,WC2=190,CUTOFF.Type NPLOT and FD to go back to NPLOT mode
from either CPLOT or RPLOT.
PL (C):Plots the spectrum, intergration lines if present.
PSCALE(C):Plots the scale.Use PSCALE(##) to adjust the vertical position of scale on the paper,
where ## is the VP value, and the default number is same as the VP for the spectrum.
PIR (C):Plots the intergrated ratio under each wanted region.
PPF (C):Print the peak frequency on top of each peak which is above the threeshold.
INSET(C):A nice macro which can let you plot a part of spectrum beside the
main spectrum. To do this: (1) After you plot the main spectrum, type S1 to save the main window, this allows you to return later. (2) Put two cursors at the region you want to do
"INSET", and then type INSET. (3)The desired region will be displayed above its original,
use the five black knobs to adjust this region on screen, then PL PSCALE...to plot it.
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