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1D Proton NMR on Bruker NMR

There is now an NMR web site accessible from the Rockefeller University web page:http://www.rockefeller.edu/nmr. On it you can find copies of many handouts as well as

descriptions of some experiments as well as some useful links.

What is included in this Handout?

Spectrometer locking and shimming Datasets Parameters and acquisition Phasing, display, integration, plotting Configuration of TopSpin Plotting using TopSpin Plot Editor Fixing problems

Archiving data Variable temperature NMR Automated processing and integration Importing spectra into other

applications

List of NMR Techniques for structuredetermination

Magnet Safety

The magnetic fields within about one meter of the magnetic are extremely strong. Do NOTbring any iron containing materials (tools, gas cylinders, etc.) near the magnet. The forceexerted by the magnet rises dramatically within a certain critical distance. You may not evennotice an attractive force until it is too late and the object is stuck to the magnet. Magnetic mediasuch as floppy disks, tapes, and credit cards can be erased if brought close to the magnet.Mechanical watches (those with hands) can be damaged.

Sample PreparationThe tube should be filled with a deuterated solvent to a minimum depth of 5.0cm (about 0.60ml).Lesser depths will make shimming the magnet homogeneity difficult. Greater depths are OK,except for variable temperature experiments. The amount of sample required for a protonspectrum ranges from less than 1mg/ml to about 20mg/ml (M.W. = 400). Too much sample canresult in a loss of resolution or a distorted spectrum. This includes not just the sample of interest,but any proton source such as protonated buffers, residual protonated solvents, and water. About5mg/ml is sufficient. The solution should be free from any solid, such as undissolved solute, ordust. Filter the solution, if necessary.

Starting up and exitingWhen the instrument is free, the screen will show the login screen. Never use the instrumentunder a different persons login. If someone has just finished using the instrument, exit to thelogin screen.

The NMR program, which is called TopSpin, will begin by typing topspin in a terminalwindow, or by double clicking the TopSpin 1.3 icon on the desktop. If the program does not

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start, or showing any error messages, you should exit the program, and in a terminal window,type shmrm command. Run shmrm 3-5 times, and then restart TopSpin. A terminal windowcan be called from RedHat icon (bottom left) System Tools Terminal.

It is very important to exit the program in the correct manner. To exit TopSpin, select Exitfrom File menu, and then click on OKbutton. To logout from your account, select Log outfrom RedHat icon. Simply typing exit at the terminal does not log you off the computer. Do notlogout before exiting from TopSpin (this causes problems).

Loading the sample and locking

All the lock and shim controls are located on buttons on the BSMS (Bruker ShimMicroprocessor System).

Push lift on/offto eject sample (typing ejwill also do it, ijfor insert).

Always use the depth gauge to set the position of the tube within the spinner. Failureto use the depth gauge will result in your tube being broken inside the NMR.

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problems. However spinning is not recommended for most experiments.

Type lock, choose the solvent and wait several seconds. Picking the wrong solvent may resultin your peaks appearing in the wrong place or not at all. Type lockdisp to bring up the lockwindow. Alternatively, double click the lock icon in the acquisition toolbar. Adjust lock gain

such that the horizontal line is approximately two-thirds of the way up the screen. You mayadjust lock phase for a maximum.

Shimming

If necessary, reset the shims to the standard best values, by typing rsh today. Optimize Z andZ2. Be sure thefinebutton is illuminated. The goal is to maximize the lock level by adjusting theshim values (lock gain is not a shim value; use lock gain to keep the lock display in the middle ofthe display range). Adjust the shim values slowly since there is a delay in the lock response. Theprocedure for shimming is as follows:

1. Adjust Z1 for maximum lock level.2. Maximize Z2,3. Repeat until no further improvement is seen. Reduce lock gain if the lock signal goes to

the top of the screen. Always end with Z1.4. Press standby when finished.

These steps are generally sufficient. Do not adjust Z3 or Z4. The proper value of Z4 requireshours of shimming. If you have followed the above procedure, any change in Z4 should reducethe lock level.

If and only if, the sample is not spinning, you should adjust x and y.

Data Sets

All data is automatically written to disk. If you do not change the data set name before acquiringdata, the new data will over-write existing data. Thus defining a data set is one of the first stepsand should be done prior to reading in parameters. New data sets are defined by the edceditor(typing edcto invoke it), as shown in the figure below.

The five parameters must follow these guidelines:

NAME name of data set usually describing your sample. The use of severalspecial symbols in the name, such as /, !, $, * and . are not permitted and

will cause errors. EXPNO experiment number (can only be numbers) PROCNO process number (can only be numbers) DU disk unit, should always be /opt/topspin USER must be set to your exact login ID Solvent Choose the solvent from the list Experiment Choose from the list or type a parameter set name. For standard proton, the

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Bruker instrument, all TopSpin program and data are stored in the /opt/topspin partition. All dataare located in /opt/topspin/data/username/nmr (replace username with your actual login ID).

Your dataset will show up in the data browser/portfolio from which you can browse, select, andopen data (see example above).

The parameters represent directory names on the computers disk and are arranged in a treestructure with the disk unit at the top. For example, under a user name (yours) there may bemany names of data sets. Under one name, there may be many experiment numbers, and under agiven experiment number, there may be many process numbers.

Raw data, that is, FIDs, are identified at the experiment number level. Completely independentdata can be identified by different names or experiment numbers. For example, assume there aretwo data sets defined by the parameters and . In there could be proton data while in there could be carbon data.

Spectra are identified at the process number level. Different process numbers are only used fordifferent processing (window functions, FT, difference spectra) of the same raw FID. Forexample, assume there are two data sets defined by the parameters and . These are transformed spectra of the same FID.They may have been transformed with different values of LB, or with some other processingchange. A transformed spectrum is defined by a complete set of the above parameters.

File Commands

To see the commands that act on data sets, click on File at the top. The most useful choices areSearchand Open data sets by name.Search brings up the portfolio editor which shows all NMRdata sets on the disk. Select a data set and click on append. This puts the selected name into the

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portfolio. Multiple sets can be put into the portfolio. To read in the data set, make sure the dataset is highlighted and click on apply.

Data sets have UNIX permissions. You only have permission to operate on (phase, delete, etc...)data sets that you have created. Do not attempt to read in and operate on data sets that you have

not created. You will get errors.

Set-up and Acquisition

Type rpar protonstd (and click on copy all) to read in the standard proton parameters. Thisoverwrites existing parameters. If you wish to change parameter values, such as for ns, youmust do so after you have given this command.

At this point it may be necessary to tune the probe. Tunning the probe refers to adjusting capacitorrods on the probe in order to make the resonance frequency of the probe circuit equal to the NMRfrequency. Usually this is not necessary for routine proton. It is necessary for more advancedproton experiments (COSY, NOE) and all heternuclear (13C, 15N, 31P, etc.) experiments. To tune

the probe, type wobb, and underAcquireat the top, select Observefid window (or type acqu).In a few seconds a tuning curve with a dip will be displayed. The tuning and matching rods arenow adjusted to make the dip both centered and as deep as possible. Type halt to end thistuning procedure. This will be demonstrated.

Type rga and wait several seconds to automatically set the receiver gain, rg. This step isnecessary for proton NMR. Values of rg range from 1 to 32,000 with 1 being the minimum . (Donot use the rga command for heteronuclear spectra.) Type zgto start an acquisition and it willcontinue until NS scans are acquired. Typing haltstops an acquisition while retaining the data.Typing stopaborts an acquisition and discards the data. Typing exptcalculates the length oftime the experiment will take. After the acquisition is finished or halted, type ef to performexponential multiplication and transform your data. During long acquisitions, such as for dilutesamples, it is desirable to look at the transformed spectrum without stopping the acquisition.Type tr, followed by ef(or other suitable command) to transform the FID.

Summary of procedure to lock, acquire, and transform data

1. edc - define the data set (be sure the user (owner) name is set to your login id!)2. lock - choose solvent and lock spectrometer3. lockdisp - generates lock display4. rsh today - If necessary, this reset the shims to the standard best values Shim z1 and z25. rpar protonstd - reads in default proton parameters6. rga and zg set receiver gain and start acquisition7. halt - stop FID collection before ns scans are completed8. tr - transfer data to processing computer without interrupting acquisition9. ef - exponential multiplication (em) and fouier transformation (ft)

Parameters

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The parameters can be accessed from the editors eda (acquisition), edp (processing), andedg(plotting). Alternatively, each parameter may be changed by simply typing its name, andthen changing the value. Some important parameters are as follows:

sw spectral width o1p transmitter center (center of spectrum) ns number of scans rg receiver gain d1 relaxation delay p1 pulse width (for simple 1D acquisition) aq FID acquisition time td number of points used to define FID si number of points used during FT (can be different from td)

Processing

The following commands are useful for 1D NMR:

em - exponential multiplication on the FID, uses the parameter LB. This improves signalto noise at the expense of resolution.

lb - this controls the degree of broadening added and affects your signal-to-noise. To seeits effect, simply change its value and re-Fourier Transform with ef.

gm - Gaussian multiplication on the fid, uses the parameters LB and GB. ft - Fourier transform ef- combines em and ft gf- combines gm and ft pk - phase correct, applies the last phase correction to the spectrum. Useful when youhave phased a preliminary spectrum, (with only a few scans) and wish to apply the same

phase correction to the final spectrum.

efp - combines em, ft , and pk. abs- automatic baseline correction apk automatic phase correction

To investigate different window functions, click on Process and selectManual window adjust.

Display

There are buttons that control the display of spectra, as shown here:

Buttons for vertical scaling:

Increase the intensity by a factor of 2

Decrease the intensity by a factor of 2

Increase the intensity by a factor of 8

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Decrease the intensity by a factor of 8Increase/decrease the intensity smoothly

Reset the intensity

Buttons for horizontal scaling (zooming):

Reset zooming (horizontal scaling) to full spectrum

Reset zooming (horizontal scaling) and intensity (vert. scaling)

Zoom in (increase horizontal scaling)

Zoom in/out smoothly

Zoom out (decrease horizontal scaling)

Exact zoom via dialog boxRetrieve previous zoom

Retain horizontal and vertical scaling when modifying dataset or changing to different

dataset. Global button for all data windows

Buttons for interactive manipulation

Phasing

Click on . By default, the phase pivot point is set to the biggest magnitude intensity of thedisplayed region of the spectrum. The pivot point has a vertical red line going through it. Thechange the pivot point, right-click on the desired pivot point position, and choose Set pivot

point from the popup menu. Click-hold the button and move the mouse until the reference

peak (the pivot point) is exactly in absorption mode. Click-hold the button ay in absorption mode. Click the button

nd move themouse until the entire spectrum is exactl to save andxecute the phase correction and return.e

Expansions

The easiest way to do horizontal expansions on the screen is to click-hold on the left mouse

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button at the left edge of the region you want expanded and move the cursor to the right edge,nd release the left mouse button.a

Calibration

Click . Move the cursor to the reference peak and click the left mouse button. Type in thechemical shift value, and then click OK. The spectrum will be calibrated and re-displayed.TopSpin will automatically leave calibration mode.

Integration

Click . To define integral regions interactively, Click the button (button turns green). Putthe red cursor line at one edge of a peak or multiplet, and Left-click-hold and drag the cursor lineto the other edge of the peak or multiplet. Repeat to define all regions. Click the green button toleave the define region mode (button turns grey). To phase or reference an integral, they must

first be selected. To select an integral, right-click in the integral region, and chooseSelect/Deselect from the popup menu. Selected integral regions are ind ated by a color filled

integral label. To phase an integral, select it, click-hold the

ic

(slope)or (bias) buttons, movethe mouse to phase it. A properly defined integral should extend beyond the apparent ends of thepeak (if there is no other adjacent peak). A properly phased integral should be horizontal beforeand after the peak. Good integration requires careful attention. To reference an integral, right-click in the reference integral region, and choose Calibrate from the popup menu. Enter the

desired value for the reference integral and click OK. To leave integration, click on (Saveintegrals and Return).

Peak picking

Click on . Put the cursor at the upper-left corner of a peak picking range.Left-click-hold anddrag the mouse to the lower-right corner of the range. The peak picking range will be marked

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green. The minimum nd maximum intensity are set and the peaks in the range are picked ana d

isplayed. Click thed button to save the peak region and peak list and return to normal mode.

Plotting

There are two ways to plot in TopSpin. The first method is very simple but rather limited. In thismethod, plotting is done directly from the bottom of TopSpin. The second method uses a submodule called TopSpin Plot Editor. First, the simple method from TopSpin will be described.

The user must explicitly set the region and intensity to be plotted. Click the bottom in thepper toolbar to open the Print dialog box. Select Print active window, then click OK.

on aifferent page. Two spectra cannot be plotted on the same page using this plotting method.

xamined by typing edg. Common plotting parameters that you

re inverted)

plunit ppm or Hz units for peak-picking tick marks.u may unselect peak-peaking, integrals, a title, all of which are, by default,

lected for printing.

Plotting using TopSpin Plot Editor

uTo plot horizontal expansions, one must repeat the above procedure and plot the expansiondPlotting parameters may be emay wish to change include:

cx- x axis length of plot in cm (plot can extend over several pages) szero - vertical offset of spectrum (useful when peaks a

From within edg, yose

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TopSpin Plot Editor is a very versatile graphic object editor. It allows several different spectra tobe plotted on the same page, expansions to be plotted on the same page, and many other features.It requires a bit of learning, however. To start TopSpin Plot Editor, type plot in the commandline within TopSpin. An important idea to remember about this editor is that it is only a displayeditor. All objects, i.e., integrals, peak lists, etc, must be defined from within the main TopSpin

rogram before you enter Plot Editor.

s performed by the mouse. The mode is set byclicking on its icon. The most useful modes are:

mode. This is used to draw spectra and is always the first mode used.

jects can be movednd resized and the spectra within them can be moved and expanded.

n with thet button you draw out a rectangle which defines the region to fill the object.

ra mode. This is one way to bring in several spectra and allows control theiralignment.

eful modes include Title and Annotate (ABC), which is found by clicking on the Basicutton.

of theurrent data set. Hold the mouse down until the spectrum covers most of the main space.

ndle, depressinge mouse and dragging. Selected objects can be deleted by clicking on delete.

xpansions can beade in this way. This is also where integrals and pick peaking are turned on.

pA picture of the Plot Editor interface is shown above. The current mode is shown in the lowerleft corner. The mode controls the type of action

Spectrum

Object Selector mode. This is used to edit already drawn spectra. Oba

Expand mode. This is just one way of making an expansion. By mousing dowlefMulti-spect

Other usbTo bring in a spectrum, set the mode to spectrum. Move the cursor into the main drawing area,hold down the left mouse button and, as you move the mouse, it draws out the spectrumc

Object selection and editing

Each spectrum brought into the editor is considered an object. The object as well as the spectrumwithin it can be moved, resized, deleted, and edited. Set the mode to object selector. Then clickwithin the drawn spectrum to edit it. Green dots should now surround this object. Using the leftmouse button, the object can be resized. Using the middle mouse button, the object can bemoved. These tasks are accomplished by placing the pointer on a green dot hath

To edit the spectrum within the object, while the green dots surround a spectrum, click on 1D-Edit. From here you can expand, or move the spectrum within the object. Em

Insets and expansions

An inset is an expansion of part of the spectrum. To make an inset, you must bring in second

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complete spectrum and then edit it so that only the region of interest is displayed. To do so, setthe mode to spectrum, bring in a spectrum, change the mode to object selector, select the newspectrum, and then resize it by grabbing its green dot handles. Then click on 1D-edit andxpand and shift the spectrum to display what you want.

ouse button and while keeping it depressed, draw out a box around thexpansion you want.

s you doot need to exit from Plot Editor. You simply need to go back to the TopSpin window.

remaining spectrum. The onlyroblem with this method is that they are not necessarily aligned.

ector, highlight the second spectrum and clickn SET. The second spectrum should now appear.

le spectra should now be displayed. Spectra Offsetadjusts both the verticalnd horizontal offset.

mode to Title, and

e

Alternatively, once the second spectrum is brought in, it can be expanded by using the EXPANDmode. Click the left me

Integrals and peak picking

To display integrals or peak peaks for a given spectrum, they are turned on within the 1D-EDITeditor. Integrals and the threshold for peak peaking must be defined from within the mainTopSpin program prior to displaying them here. If you have forgotten to do these stepn

More than one spectrum and aligning them

One can also bring in several different spectra. For this to work, you must follow this procedure:click on data, and then edit. This brings up the portfolio editor. Select a data set and then click onappendand then apply.The name of the data set is now in the data set selector window. Repeatthis for each data set to be brought in. Now within the data set selector, highlight a data set andclick on apply. Draw out the spectrum. Now, highlight a different data set in the data set selector,click on apply and draw out the next spectrum. Repeat for eachpAn easy way to get two spectra to have the same displayed dimensions (and thus aligning them)

is to do the following: Copy the first spectrum and then replace the copy with the secondspectrum. To do this, follow the above procedure to load the data sets names into the data setselector. Draw out the first spectrum. Switch to green-dot-mode and select the spectrum and thenclick on copy. Move this spectrum by placing the cursor on a green dot and using the middlebutton, drag the spectrum. Then, in the data set seloA third method utilizes the multi-spectra mode. This is useful for displaying spectra withconsecutive experiment numbers. Follow this procedure: First, add all desired spectra into theData Set Selector using the method described above. Then, select mulit-spectrum mode, anddraw out your spectrum. Only one spectrum will be drawn out initially. Then, select object

selector mode, select the spectrum, and then click edit. Towards the bottom of this edit list, arethe parametersNumber of Stacked Spectra and Spectra Offset. Enter the number of spectra andclick onApply. Multipa

Title and annotation

To display the title assigned in TopSpin with the xau settitle command, set the

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then click on the left mouse. Place the title and click again on the left mouse.

left mouse, type your comment,nd click the left mouse again. This is an easy way to add a title.

e TIFF format which can beansferred to PCs and Macs. See the end of this handout for details.

o exit TopSpin, click on File and thenExit.

roblems

he following are common problems on Bruker spectrometers and their solutions:

dow with the upper left menu keys. To close the lock window, click on the quit

lem persists exit TopSpin, type shmrm in a terminal

s

o the right of your name. If that doesnt solve the problem repeat until thescreen is free.

(which includes this one) without appropriatenowledge. Notify NMR staff of all problems.

To annotate, set the mode to ABC. Place the pointer, click on thea

Plotting

There is a dotted black box around the screen. Only things within this box will actually beplotted. To plot, click onfile, thenprint... Selectprint. If you are interested in importing spectrainto Word or other programs, Plot Editor can write the spectrum in thtr

xitingET

PT

1. Typing lockdisp gives a time out error. The solution: open up the console and push thered button labeled reset on the CCU board located in the upper left of the console. Waitfor 1 minute while the spectrometers computer reboots. Exit the TopSpin program andrestart it by typing topspin. Type ii. Proceed. This problem is caused by closing thelock winbutton.

2. The proton FID or spectrum shows no signal and you know your sample should. TheSolution: Type ii. If the probwindow, and restart TopSpin.

3. The computer display looks fuzzy, unreadable, and not normal (not the normal blurrinescaused by the magnets). Reboot the computer by selecting Restart from RedHat icon.

4. The screen is frozen (caused usually by clicking outside one of the pop-up boxes). Firsthit the escape button. If this does not work try the following: Go to another computer, ordata station. Bring up a terminal shell. Type ssh dpx400a. Login. Now you can do oneof two things: a) type reboot to reboot the computer, or b) type top to get a list ofprocesses. Pick one of the processes and kill it type by typing kill -9 xxxx where xxxxis the number t

Never turn off any Linux based computerk

rchiving DataAData backup will be the responsibility of each individual user, however the University-wide

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computer back-up system is employed on this instrument. It is suggested that the data on the hardrive be backed up frequently.

y DVD-/DVD+R/DVD-RW/DVD+RW disk (4.4 GB uncompressed) can be used in the drive.

you make an archive, youill save space on disk and the archiving process will be much faster.

u want to delete only theaginary parts of processed data, type deli and select delete all.

break if dropped (cost $200). Ask forssistance the first time you set up a VT experiment.

e instruments onockefeller Campus have software buttons, found within edte, the VT editor.

thencreases or decreases the sample heater current so as to maintain the desired temperature.

on change next to target temperature and enter the desiredtemperature in Kelvin.

d

DVDRW Drive

A DVDRW drive is available for creation of individual laboratory back-ups. AnR

Processed and Raw Data

Data consists of both raw FIDs and processed (FT) spectra. The imaginary parts of processeddata (1i for 1D data, 2ii, 2ir, 2ri for 2D data) do not need to be archived. Only the raw data andreal part of the processed data need to be archived. Once the raw data is read back in, it can beretransformed (FT, XFB, etc). By deleting the imaginary data beforew

To delete processed data, go into the TopSpin, the NMR program. Type delp and select deleteall. You will only be deleting processed data, not raw data. If yoim

ariable TemperatureVEach of the instruments has a different variable temperature range. The variable temperature

range is limited to -80 C (193K) to +80 C (353K). Do not exceed these ranges. Fortemperatures above about 40 C, the ceramic spinner should be used. The plastic spinner willexpand, deform and become stuck when heated above room temperature. The ceramic spinnershould only be for VT work since it is fragile and willa

Operating the Variable Temperature Controller

There are two types of VT controllers: manual buttons and software buttons. ThRThe VT controller can be used for both low temperature and high temperature experiments. Acooling system generates cold air going through an insulated hose that is attached to the probe bya ball-and-socket joint. This cold air is then heated, by the sample heater, to the desired

temperature. A sensor, located near the sample, sends a signal to the controller, whichin

Type edte (or double click the Temperature icon in the acquisition toolbar)to invokethe VT editor. Click

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least 10 minutes before taking an NMR spectrum. Although the controller may indicatethe sample temperature is at the target value, the sample will lag behind the sensorbecause of its greater thermal mass.

d the sample shimmed.

Both shimming and probe tuning depend on sample temperature.

s, evoke edte again, and set target temperature back to roomtemperature (298 K).

e boiling point of your sample solvent and keep the temperature 10 K

elow this value.

in shift between the

ethyl and hydroxyl resonances of 100% methanol, in ppm, is given by

T(K) = 403.0 - 29.53 - 23.87 2

between the methylene and hydroxyl resonances

f 100% ethylene glycol, in ppm, is given by

T(K) = 466.0 - 101.6

irbrother, Palmer, and Skelton, ProteinMR Spectroscopy, Academic Press: New York, 1996.

r spectrum, save it, and type multiefp and enter the number of spectra

to be processed.

Once you are at the target temperature, the probe must be tuned an

After the experiment

Caution: Use the ceramic spinner for high temperature experiments (>40 C).Caution: Note th

b

Temperature Accuracy

The controller does not necessarily provide an accurate temperature. To know the actualtemperature it is necessary to use an NMR thermometer the temperature dependent shifts ofmethanol or ethylene glycol. Over the range 250-320 K the difference

m

Over the range 300-370K, the difference in shift

o

both of these equations are taken from Cavanagh, FaN

ulti-Spectra ProcessingMThere are several programs that allow multiple spectra to be processed automatically.

multiefp this does efp on successive experiment numbers using the phase correctiondetermined in the first experiment number. To use this, go to the first experiment number,phase correct you

multi_integ this integrates of series of spectra in successive experiment numbers usingthe same integration regions. The data must be in successive experiment or processnumbers. To use this, go to any experiment number, phase correct, and integrate yourspectrum (exit integration by saving it as intrng). Then type wmisc, select intrng andgive or select a file name. This saves your integral regions as a file. Then type edo andgive a file name for CURPRIN (any name will do). Type multi_integ, enter 0 if your

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data is in sequential experiment numbers (the usual case), enter the first experimentnumber, the number of experiments to be integrated, and then the name of the file(above) containing integral regions. The results are written to a file int.all in the directorylisted in the completion message.

ou with your desired file format, thenne can convert it with Photoshop or freeware converters.

directory are not automatically deleted, please delete those files yourself when you are finished.

porting Spectra into other ApplicationsImIf you are scanning printed spectra in order to use them in Word documents or elsewhere, there islikely a better route. Plot Editor can write spectra to disk in a variety of formats. Then, using aFTP client that supports secure shell, such as Fetch (Mac) or WinSCP (PC), one can transfer thefiles to Macs or PCs. And if Plot Editor does not provide yoTo print to a file within Plot Editor, under Options, Printer setup, scroll down through the printertypes. At the bottom there are TIFF, EPSI, and PCX. Select your preferred format (I know TIFFworks when converting to JPEG images), and click OK at the bottom. To print, under File, select

Print, To File and use your home directory (/home/username/). Since files in your home

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NMR Techniques for Structure Determination

The following techniques are implemented on the NMR instruments at Rockefeller. Please askthe director for assistance. Other techniques can be implemented upon request.

1. H-H connectivity through J coupling COSY COSY-DQF TOCSY-2D selective TOCSY-1D

2. J measurement (H-H)

H-H Homonuclear decloupling Spectral simulation (Xsim, gNMR

program)

COSY-DQF ECOSY

3. H-H spatial proximity

NOESY-2D ROESY-2D 1D-gradient selectived NOESY

4. Carbon-proton connectivity throughJ coupling

HMQC - direct correlation: one bond HSQC - direct correlation: one bond HMBC - long range correlation: 2 to 3

bond

INADEQUATE - proton detected longrange correlation

5. J measurement (C-H long range)

Exside6. Water suppression

watergate presaturation

7. Exchange - Relaxation

2D EXESY (NOESY) selective magnetization transfer 1D T1 measurement

8. Heteronuclear NMR (DPX 400 only)

13C, 15N, 31P, 2H, 29Si, etc. DEPT spectra

9. Heteronuclear multidimensional NMRfor protein structural determination

HNCA/HNCO HN(CO)CA

HNCACB CBCA(CO)NH HBHA(CO)NH (H)CC(CO)NH H(CC)(CO)NH Heternoculear NOE experiments

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