NMR FACILITY DEPARTMENT OF CHEMISTRY

University of Washington

XWINNMR 3.5 TRAINING NOTES

08/8/2005 rajan paranji

CONTENTS

Views of XWINNMR INTERFACE 4 Operating the Bruker Spectrometer – FLOW CHART 5 LOGON 6 SAMPLE HANDLING 7 DATA SET DEFINITION 8 PARAMETER EDITING 10 LOCKING AND SHIMMING 12 ACQUISITION 14 PROCESSING FT 15 PHASE CORECTION 16 INTEGRATION 17 PEAK PICKING 19 PLOTTING 21 LOGOUT 26 DATA ARCHIVAL 27 APPENDIX A LIST OF COMMONLY USED XWINNMR PARAMETERS 29 B COMMONLY USED COMMANDS IN XWINNMR 33

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TYPOGRAPHIC CONVENTIONS USED IN THIS NOTES:

• boxed text corresponds to a button to be clicked with mouse on screen OR a key to be pressed on the BSMS keyboard.

LIFT ON/OFF

• Commands to be typed appear with the following font: edc

• Mouse buttons are depicted like this : LEFT

3

,

ACQUISITION WINDOW

SPECTRUM WINDOW

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OPERATING THE BRUKER SPECTROMETER

LOGON Username Password

SAMPLE INSERTION Positioning sample in spinner Inserting (or) ejecting sample

DATA SET DEFINITION Creation of new dataset

Loading standard parameters

PARAMETER OPTIMIZATION & PREPARATION

Tuning and Matching probehead Locking and Shimming Spectral width, FID size

ACQUISITIION Receiver Gain optimization

Beginning Acquisition Halting/Stopping Acquisition

PROCESSING Spectrum size

Window function FT and phase correction

Integration Peak Picking

PLOTTING / OUTPUT xwinplot

DATA ARCHIVAL / BACKUP FTP data to your lab

LOGOUT Stop spinning Eject sample

Logout of XWINNMR

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LOGON

Username: genuser Password: nmr301

Password for any instrument is “nmr” followed by the 1H frequency of that instrument.

Instrument

Name Host Name Used for

FTP Base

Frequency AV300 av300.chem.washington.edu 300.13 MHz AV301 av301.chem.washington.edu 300.13 MHz AV500 av500.chem.washington.edu 499.044 MHz DRX499 drx499.chem.washington.edu 499.85 MHz DMX750* dmx750.chem.washington.ed

u 750.13 MHz

* For DMX750 alone, different research groups are provided with individual login accounts. Contact the Facility Manager for details.

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SAMPLE INSERTION To Lift the sample out of the magnet: • Press the LIFT ON/OFF key on the BSMS keyboard

OR • type ej on the command line and enter To insert the sample into the magnet again: • Press the LIFT ON/OFF key on the BSMS keyboard again

OR • Type ij on the command line and enter. Points to Note:

• When the sample is seated properly inside the magnet, a GREEN colored LED next to the label DOWN will be lit on the BSMS keyboard.

• When the sample is floating on top of the magnet, a GREEN colored LED next to eh label UP will be lit on the BSMS keyboard.

• While the sample is being transported in between DOWN and UP positions, a RED colored LED next to the label MISSING will be lit. This light should not be lit at any other time. Even after injecting the sample into the magnet, if you see this RED LED on, there is some problem with the sample transport system. PLEASE CONTACT THE FACILITY MANAGER OR ASSISTANT IMMEIDATELY.

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DATA SET DEFINITION • Enter the command edc. • Next to user, type your name • Next to name, enter a meaningful name for the dataset. • Next to expno, enter a numerical value (1, 2,...10,...) • Next to procno enter a numerical value (1, 2,...10,...) • Click on SAVE

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DATA STRUCTURE OGRANIZATION IN XWINNMR

/opt/xwinnmr

exp conf data

prog bin

User 1 User 2 User 3

nmr

Dataset 1 Dataset 2 Dataset n …

…10 20 27 101

pdata

1 3 42

Experiment Number

Processed data number

$NMR

User directories

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PARAMETER OPTIMIZATION & PREPARATION

Assuming that you are going to record a 1H ( i.e. proton) spectrum first, proceed as below:

• Type proton on the command line • Type ased to access the window that shows you the parameters that

are relevant to a 1D proton experiment. • Modify the parameters that are of importance to you. Click the SAVE

button to exit the table.

• Enter edte on the command line. Choose the appropriate temperature. Wait for the temperature to

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stabilize. You must wait at least 5 minutes. • Enter wobb on the command line.

Look at the wobble curve. The ‘cusp’ of the wobble curve should touch the bottom and it should also be aligned with the blue vertical line at the center of the screen.

edte WINDOW

wobb CURVE

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Locking and Shimming • Enter lock • Choose your solvent from the drop down list.

Wait for the Automatic Locking process to complete. You will see the message “Lock finished” just below the command line.

• Enter lockdisp as shown here. LOCK DISPLAY SOLVENT LIST

• Start shimming. You can use the BSMS keyboard to vary the different shim currents. If you prefer not to use the BSMS keyboard for this, there is another option. Enter the command bsmsdisp on the command line and you will be given a graphical interface that will have buttons that mimic all the functions of the BSMS keyboard.

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BSMS DISPLAY WINDOWS:

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ACQUISITION

• Enter rga For proton observation the receiver gain i.e. ‘rg’ can be anywhere from 10 to 250. If the receiver gain is a larger number like 1800, for example, this indicates that the signal is weak. This implies a problem somewhere.

• Enter zg o You can switch to the acquisition window by typing ‘acqu’ on

the command line. Here you will be shown a status window while the data is being acquired. Status window shows how many scans are complete and how much more time is left to complete the data collection.

o Wait for the message “Checklockshift finished” to appear below the command line.

o You can enter at any time during the acquisition, the command “tr” to transfer the FID that has been recorded so far and process the same to see how your experiment is progressing. This is useful, for instance, in 13C experiments.

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PROCESSING 1. FT

• Enter efp to process the FID and get a spectrum.

efp actually stands for three operations : em + ft + pk em – multiply the FID with an exponentially decaying function. ft – perform fourier transformatioin of FID pk – apply phase correction as defined by the parameters phc0 and phc1

What is shown above is a default command which serves well to get solution NMR of small molecules. But you can change these behaviors if you need to. You can choose a different window function by invoking the command winfunc on the command line.

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2. PHASE CORRECTION

• Click on the phase button to enter the phase correction routine. • Click on the cursor button. The pointer will turn into an inverted

white colored arrow that is tied to the spectrum. Move it to the rightmost part of the spectrum.

• Use the PH0 button to interactively adjust the phase till all the peaks look symmetric and positive (Lorentzian lineshape).

• If necessary, use the PH1 button also. • Click on return button. You will get three buttons of choice.

Choose Save & Return.

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3. INTEGRATION A: Basic Steps

• Click on the integrate button to enter the integrate routine • Click on LEFT mouse button anywhere inside the spectrum. The

pointer will turn into an inverted semi-transparent arrow that is tied

to the spectrum. • To integrate an isolated peak, move this cursor onto the left side of

the peak. Click on the MIDDLE button. The inverted arrow now leaves a copy of itself, in the form of an opaque white pointer that is fixed at the position where you clicked.

• Move the transparent arrow to the right side of the peak. Click the

MIDDLE mouse button once again. Now, you will see a blue colored integral appear, between the limits you have selected.

• You simply have to repeat this operation for integrating all the peaks.

• Once you are done, click the LEFT mouse button to come out of this integrating mode.

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B: Calibrating integrals:

• Double click under the integral you want to choose as your standard peak for calibration (example: a methyl group peak corresponding to 3 protons). A white opaque arrow sticks onto the integral you have chosen. This is called the “current” integral.

• Click on the calibrate button. You will be given a dialog box. You can enter the number of protons that gives this integral. In the example above, you will enter 3. All the areas shown for other integrals will be recalculated based on the above calibration.

C: Saving and Recalling integrals:

• Once you are finished integration and calibration, click on return button. Among the choices given, you must choose save & return now. Then only the integrals are stored with your dataset.

• Let us say you have already integrated the spectrum a few days ago. How do you recall those integrals now and see on the screen ?

o After you click the integrate button in your main spectrum window, you can click on the pull down menu “File”.

o You will see the first choice in this pull down menu as read intrng. If you click on this, you will get your saved integrals displayed once again on the spectrum.

D: Hard Copy output of Integrals :

• You can print the integrals as a list. Enter the command li on the command line. By default the list of integrals goes to the currently chosen printer or plotter.

E: Comparing Integrals Across spectra :

• Instead of choosing a given peak under the current spectrum for calibration, if you click on lastcal, all the integrals will be calibrated in reference to the previous integration operation. This can be very useful in kinetics studies or comparing two different compounds as they evolve over time.

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4. PEAK PICKING A: Basic Steps

• Enter pscal global • See if the vertical scale of the spectrum is in units of ‘cm’. If not,

click on the YU button. This should switch the vertical scale units to cm.

• Click on the utilities button. You will be taken into a display shown below.

• Click on MI button. A horizontal line appears. If you click and

hold the mouse cursor on the position of MI button and move up and down, the horizontal line also moves. Position this line where you want the lower threshold to be. Peaks that appear below this line will not be considered for picking.

• Now click on MAXI button. Another line appears and this you can use to define the higher threshold. Peaks that are taller than this level will not be considered for picking.

• Now, enter pps to see a listing of the picked peaks in a separate window. You can send this list to the printer if you choose.

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B: Optimizing Peak Picking

• Enter pc. By default pc is 1. If you want more number of peaks to be considered for picking between MI and MAXI, decrease the value of pc below 1.

• If you want lesser number of peaks to be picked, increase the value of pc to something larger than 1.

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PLOTTING – XWINPLOT EDITOR

XWINPLOT is a stand alone Plot editor that is quite versatile and user friendly. It is called a WYSIWYG plotter. What You See Is What You Get. Any existing XWINNMR dataset can be imported into XWINPLOT and edited. When you start XWINPLOT from within XWINNMR, the current dataset is automatically loaded.

A: Basic Steps:

• Enter xwp from XWINNMR. The current spectrum is loaded and XWINPLOT window opens. An example is shown below.

• In the simplest case, once you have opened the XWINPLOT with the current data set, you just have to click on the “File” pull down menu, choose print and your spectrum will be plotted on the plotter. That’s it !

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B: Optimizing XWINPLOT Features:

XWINPLOT BUTTONS AND THEIR FUNCTIONS:

Select a Graphics Object (Spectrum, Title Box, etc.)

Expand a region of the spectrum using mouse

Zoom onto a region of the display – On Screen only

Insert Title from the current data set

Insert the list of parameters for the current data

Single spectrum display mode

Spectral Array display mode

Text insertion box • Any of the above buttons can be clicked once to choose that particular mode of

editing. The mode you have chosen currently, will be displayed in the lower left corner of the xwinplot area, as shown below:

• To switch between modes, you have to click the corresponding button once.

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XWINPLOT shows a “white” area in a cyan background. This is a “virtual sheet of paper” on which you will place your graphics objects. By default, XWINPLOT includes the following objects of display, when you open it with a valid dataset.

• Spectrum in landscape orientation • List of parameters used in a two column format on the right side of the spectrum • Integrals • Title of the spectrum from the dataset.

The default behavior can be changed completely by editing the objects of display individually. Do the following for the editing.

1. Click on the button. Move the cursor onto the spectrum display and click once. The spectrum area is surrounded by “green” handles or hot spots, like below.

2. Now click on the button on the top right corner, next to the

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button. You will get an edit window like below:

Size of the spectrum window displayed.

Edit font type, sizes, curve thickness, etc.

Size and position of displayed integrals

X-axis Units

1. If you want peaks to be displayed, you can turn on the Show Peaks button. 2. If you want to change the limits of X-axis, you can modify the Xmin, Xmax

range. 3. For Y-axis limits, edit the Ymin, Ymax entry.

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4. Once you are finished, click OK or Apply You can edit pretty much any other graphics object that was placed inside the “white” area of XWINPLOT by using the edit mode.

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LOGOUT

To end your run do the following :

• If you are using a temperature that is different from ambient temperature, then set the temperature to 298 K.

• Stop spinning the sample.

• Click on the LOCK button the BSMS keyboard to end locking.

• Click on the LIFT ON/OFF button to eject your sample.

• Transfer the dummy sample into the spinner and insert that into the magnet.

• Enter exit on the command line. Click OK to the questions asked. XWINNMR automatically logs you out of the system once the session is over. You must do this to enable the next user in line to use the instrument. If you do not log out there is a very real possibility of your data being overwritten by the next user, unknowingly.

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DATA ARCHIVAL & RETRIEVAL XWINNMR stores all the FID that are collected but automatically erases the spectra once they are a week old. To regenerate the spectrum you need to just enter efp.

• You can use a third party FTP (File Transfer Protocol) software that runs on any platform like, Microsoft Windows, Mac-OS or Unix like systems, to transfer all the data you have collected so far to your lab computer.

Here are the points to note for getting data by FTP:

• Do NOT use a secure FTP software. The FTP software that runs on NMR machines are anonymous FTP servers that have restricted access to only your data directories.

• Use a FTP software that has a graphical user interface. XWINNMR stores data in a complex tree structure and it will be very difficult if not impossible to use a text based FTP program to transfer all the directories successfully. An example of a FTP program with graphical interface is given below:

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Left side of the display shows the local computer’s disk. Right side shows the user data directories on the NMR computer: av301.chem.washington.edu

• While configuring the FTP software, use the connection Port number: 21. • For username, enter: ftp • For password enter: ftp • Also, make sure that your FTP program is configured for passive mode of

transfer.

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APPENDIX A

LIST OF COMMONLY USED PARAMETERS

p1

5 us to 12 us - 1H 12 us to 18 us - 13C F1 channel 90o pulse width

p2

10 us to 25 us - 1H25 us to 40 us - 13C F1 channel 180o pulse width

p3

5 us to 12 us - 1H 12 us to 18 us - 13C F2 channel 90 pulse width

p4

10 us to 25 us - 1H25 us to 40 us - 13C

p6 25 us to 40 us TOCSY 90 1H pulse length; calculated based on the p1

p7 50 us to 80 us

TOCSY 180 1H pulse length; normally derived internally from p6

p10 1000 us gradient pulse length

p11 variable - shaped pulse

usually employed in water suppression schemes: WATERGATE, WET, water Flipback

p15 250 us ROESY spin lock soft pulse

p16 1000 us homospoil/gradient pulse length

PU

LSE

S

d0 incremented delay in 2D

d1 1 to 1.2 s relaxation delay : 1 to 5 times T1

d2 J for CH: 125 to 222 Hz 1/2J

d3 1/3J

d4 1/4J : used in HSQC

d6 delay for evolution of long range couplings

d7 delay for inversion recovery

d8 200 ms to 1 s NOESY mixing time

d9 60 ms to 80 ms TOCSY mixing time

d11 30 ms : default delay for disk I/O

d12 20 us : default delay for power switching

d14 delay for evolution after shaped pulse.

d16 200 to 500 us delay for gradient recovery

DE

LAY

S

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d19 100 to 200 us

delay for binomial water suppression in WATERGATE : 1/[2*d19] gives the position of next null

cnst0 used in different contexts.

cnst1 J (HH) proton-proton J coupling stored as default

cnst2 J (XH) ex: proton-carbon 1 bond J coupling

cnst3 J (XX) ex: carbon-carbon 1 bond J coupling in labeled solute

cnst11 1 or 2 for multiplicity selection in DEPT or APT

cnst12 1 or 2 for multiplicity selection

CO

NS

TAN

TS

vc variable loop counter

vd variable delay list VAR

IAB

LES

l1 MLEV loop counter

l2 GARP loop counter

l3

loop for phase sensitive 2D or 3D using States or States-TPPI method l3 = td1/2

LOO

P

CO

UN

TER

S

lb <line broad. Factor> for exponential window function

td 4k to 64k number of FID points

si 4k to 64k number of points in the spectrum M

ISC

ELLA

NEO

US

30

ns 1 to 10000 number of scans of a given experiment

td0 integer value data is automatically written to disk after [ns/td0] scans.

sw 10 ppm to 300 ppm

spectral width ppm units. Depends on the nucleus studied.

swh

spectral width Hz units. Depends on the nucleus studied and the spectrometer's base frequency

ds 4 to 32

number of dummy scans. Experiment is performed but the FID is not recorded

aq 0.1 s to 5 s length of FID in seconds. Solvents have long FID length.

o1 ( proton) 1500 to 3000 Hz

Offset of the spectrum center with respect to the base frequency (BF) of the spectrometer. Depends on the spectrometer's absolute base frequency. - CHANNEL F1

o1p (proton) 2.5 ppm to 7.5 ppmoffset of the spectrum center in ppm units. - CHANNEL F1

o1p (carbon) 75 ppm to 125 ppm

o1p (fluorine) 0 ppm to -95 ppm

o1p (phosphorous) 0 ppm to 150 ppm

o2 (proton) 1500 to 3000 Hz Same as o1, but on CHANNEL F2

o2p (proton) 2.5 ppm to 7.5 ppmSame as o1p, but on CHANNEL F2

o2p (carbon) Same as o1p, but on CHANNEL F2

o2p (fluorine) Same as o1p, but on CHANNEL F2

o2p (phosphorous)

Same as o1p, but on CHANNEL F2

solvent

ensure that this is the same as the lock solvent you used, before starting the acquisition.

pc 0.1 to 100

peak picking sensitivity factor: pc < 1 will pick more peaks; pc > 1 will pick less number of peaks; pc = 1 is default

MIS

CE

LLA

NE

OU

S

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cy 10 to 10000 Height of the largest peak in cm.

PH_mod

pk : phase sensitiveps : power spectrummc : magnitude spectrum

rg

1H : 1 to 200 13C (natural abundance) : 750 to 3000

depends on : solute concentration nucleus studied temperature

phc0 0 to 360 zero order phase correction factor

phc1 0 to 180 first order phase correction factor

MIS

CE

LLA

NE

OU

S

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APPENDIX B

COMMONLY USED COMMANDS COMMAND

NAME COMMAND ARGUMENT DESCRIPTION

acqu to switch to acquisition window

ased show and allow editing of the limited set of parameters, relevant to the current experiment

eda edit acquisition parameters in table format.

edte start the temperature controller interface

getprosol

load the calibrated pulse and power levels for the current hardware

go start acquisition, but do not zero the contents of the memory buffer

halt halt the acquisition after the current transient is completed.

lock perform auto-locking of the spectrometer field, using the lock solvent signal (usually 2H)

popt run the parameter optimization program

rga automatically find out the optimal reciever gain

rpar <parset> all to load an existing parameter set

rsh <shim file> to load an existing shim file

show proc show the list of commands that are being executed by XWINNMR

stop abort the acquisition immediately 'go' will not work after this

tr transfer the transients collected so far to the disk

wobb tune and match the probehead

wpar <parset> all to save the current dataset's parameters with a unique name

wsh <shim file> write current shims to a unique shim file name

xau <file> execute the automation program

zg begin acquisition of the current experiment

AC

QU

ISIT

ION

dir show the list of datasets recorded so far, for the current user

diro show a list of valid users FILE

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re m n

switch to a different [Exp. No, Proc. No.] combination of the current data set. m : experiment number n: processed data number

wra m

copy the raw data (FID) from the current experiment number to a different one. m: experiment number

wrp n

copy the current processed spectrum to a different proc.number within the same dataset n: processed data number

wrpa m n

copy the current dataset in its entirety (including the FID and the spectrum) to a new [Exp. No, Proc. No.] combination. m : experiment number n: processed data number

FILE

setti open an editor for creating a title for the current dataset.

xwp launch XWINPLOT plot editor opens the editor, with the current dataset for display, as a default. PL

OTT

ING

abs perform the default baseline correction

apk automatically correct the phase of the spectrum

cfbsms software reset the bsms system, when locking is not successful.

edp edit processing parameters in table format.

efp Perform FT after doing 'em' and apply the existing phase correction values i.e. phc0, phc1

ej eject the sample from the magnet

em apply an exponential window function to the FID, using the value of 'lb'

fid to display free induction decay in the spectrum window

ft Fourier transform the FID.

ij inject the sample into the magnet

maxi maximum threshold in cm for peak picking

mi minimum threshold in cm for peak picking

PR

OC

ES

SIN

G

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pps display the picked peak list on screen.

pscal global for peak picking

sino calculate the signal to noise ratio you must define the signal and noise regions prior to this.

sref automatically perform spectrum calibration based on lock solvent and TMS info.

winfunc enter manual window function manipulation program P

RO

CE

SS

ING

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