Bruker AC/AM NMR User’s Guidecic/nmr/Guides/BUG/bug.pdf · 2002-08-20 · UWChemMR Facility Page...

Bruker AC/AM NMR User's Guide

by

Charles G. Fry

University of Wisconsin�Madison

Chemistry Department

(updated August 20, 2002)

BUG User�s Guide Page ii

Copyright © 1994-2002 Charles G. Fry All Rights Reserved.

BUG User�s Guide Page i

Table of Contents

1. UW Chemistry Magnetic Resonance Facility ............................................1 I.Facility Layout (2nd floor Matthews) ........................................................................ 1 II.Facility Personnel ................................................................................................... 2

2. Bruker Acquisition Basics ..........................................................................3 I.Description .............................................................................................................. 3 II.Deuterium Lock ...................................................................................................... 3 III.Shimming .............................................................................................................. 4 IV.Acquisition Parameters ......................................................................................... 5

3. Introduction to AC+ Spectrometers..............................................................6 I.Summary of 1D Acquisitions on AC Spectrometers................................................. 6 II.Locking and Shimming with the SCM ..................................................................... 7 III.Job Files and Lock Power ..................................................................................... 8 IV.Data Collection ..................................................................................................... 8 V.File Transfer: NMRLINK........................................................................................ 8 VI.Departure.............................................................................................................. 9 VII.Adjusting for SW and O1...................................................................................... 9 VIII.Spectra showing folding and unfolding of deshielded proton. ............................ 10 IX.Caveats .............................................................................................................. 11

4. Primers for Areas of Common Interest....................................................12 I.Primer on phasing ................................................................................................. 12 II.Locking and Shimming Primer #1......................................................................... 14 III.Primer on Bruker EP Mode � Most Common Uses............................................ 15 IV.Primer for Obtaining Accurate/Consistent Proton Integrations in DISNMR.......... 16 V.Primer for Applying Pearson�s Gaussian Apodization in DISNMR........................ 17 VI.Primer on Lorentzian and Gaussian (Bruker) Multipliers [MathCAD import] ........ 18 VII.Primer for Decoupler Modes on Bruker AC and AM Spectrometers ................... 25 VIII.Primer for Estimating T1 Values Theoretically................................................... 26

5. AM/AC Spectrometer Software.................................................................28 I.ADAKOS (Aspect Disk And Keyboard Operating System)..................................... 28 II.DISNMR............................................................................................................... 28

A. Control Keys and System Commands 28 B. Files in DISNMR 29 C. Common Acquisition Commands (WJ, RJ) 29 D. Common Pulse Sequence Commands and Parameters (WJ, RJ) 30 E. Common Data Processing Commands (WJ, PJ) 30 F. Common EP Commands 30

III.Plotting with DISNMR.......................................................................................... 31 A. Overview 31 B. Standard Plots 32 C. Stack Plots 32

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IV.Data Transfers .................................................................................................... 32 A. UW Network 32 B. NMR-Link 32 C. FTP to/from NMRSnap 33

V.Formatting Floppy Disks ...................................................................................... 33

6. 2nd Order Shimming and High Resolution Check-Out ............................34

I.2nd Order Lock Shimming (Match Tuning) on Z2.................................................... 34 II.High Resolution Check-Out .................................................................................. 34

A. FID Shimming (Line Tuning) 34 B. Window Set-Up and Acquisition 34 C. Lorentzian to Gaussian Line Shape Transformation (Resolution Enhancement) 35

7. 1H NMR on AM-500/ACs............................................................................36 I.Sample In.............................................................................................................. 36 II.Shim Control Module (SCM): Locking on AM Spectrometers .............................. 36 III.Coaxial Shims ..................................................................................................... 37 IV.Nonspinning Shims............................................................................................. 37 V.Taking 1H NMR Spectrum.................................................................................... 37

� EP (Expansion and Phase) mode: 37 VI.Plotting 38 VII.FID Shimming (Line Tuning) .............................................................................. 38 VIII.Baseline Correction........................................................................................... 39 IX.Linefitting ............................................................................................................ 39

8. 13C NMR on AM-500/ACs ..........................................................................40 I.Sample In.............................................................................................................. 40 II.Probe Tuning........................................................................................................ 40 III.Decoupler Tuning................................................................................................ 40 IV.Shimming............................................................................................................ 41 V.Taking 13C NMR Spectrum.................................................................................. 41 VI.DEPT 41 VII.Data Processing ................................................................................................ 42

9. AM/AC QuickGuide for X-Nucleus Experiments .....................................43 I.Probe Changes ..................................................................................................... 43 II.Probe Tuning........................................................................................................ 43 III.Acquire Data ....................................................................................................... 44

10. Saving Group-Specific Jobfiles, Shim Files, etc.....................................45 I.Formatting 8" Floppies with ADAKOS ................................................................... 45 II.Create or Modify .EXE File ................................................................................... 45 III.Run Backup to Floppy ......................................................................................... 45

11. Variable Temperature Measurments ........................................................46 I.Variable Temperature on Phoenix (Bruker AC-250) .............................................. 46

A. Initial Equipment Checks 46

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B. Changing set-point 47 C. Taking data 47 D. Finishing up 47 E. Self-tune (if control is erratic) 47

II.1H NMR Chemical Shift Thermometers................................................................ 48 A. Low Temperature: methanol (with 0.03% concentrated HCl) 48 B. High Temperature: ethylene glycol (neat) 48

III.Automated VT Runs ............................................................................................ 48 IV.Example Automation Routine Listing................................................................... 49

12. HOMODEC on AM/AC Spectrometers......................................................50 I.Setting Up ............................................................................................................. 50 II.Taking 1H NMR Decoupling Spectrum.................................................................. 50 III.Processing FIDs.................................................................................................. 51 IV.Listing of Automation Routine ............................................................................. 51

13. 1H Spin-Lattice Relaxation, T1 ..................................................................52

I.Discussion............................................................................................................. 52 II.Rapid Determination of T1 by Inversion Recovery Null Method ............................ 52 III.Rapid, approximate measurement of T1 by Repetition-Rate method ................... 53 IV.Quantitative measurement of T1 by Inversion Recovery Method......................... 53

A. Comments 53 B. Acquisition Set-up 53 C. T1 Analysis 55

14. 1D NOE Measurements .............................................................................56 I.Discussion............................................................................................................. 56 II.Critical Parameters for NOEDIFF.AU ................................................................... 57 III.Setting Up For Well-Resolved Spectra: NOEDIFF.AU........................................ 58 IV.Critical Parameters for NOEMULT.AU ................................................................ 59 V.Setting Up Best-Case Spectra: NOEMULT.AU ................................................... 59 VI.Acquiring NOE Difference Spectra...................................................................... 60 VII.Processing FIDs................................................................................................. 60 VIII.Obtaining NOE Difference Spectrum................................................................. 60

A. via AT (additive Transfer): 60 B. via dual display: 60

IX.Listing of Automation Routines............................................................................ 61

15. HOMONUCLEAR COSY.............................................................................63 I.Quick Summary for Acquiring and Plotting COSY Spectra .................................... 63 II.Detailed Explanation of COSY Set Up.................................................................. 65

A. Take high-resolution1D profile spectrum 65 B. Setup 2D parameters 65

III.Acquiring COSY Spectra..................................................................................... 67 III.Processing 2D Data in DISNMR.......................................................................... 67

A. Setup 1D projection file 67 B. Setup and plot .SER 2D files 67 C. Plot .SMX 2D files 68

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IV.Listing of Automation Routine ............................................................................. 70

16. X-Nucleus Decoupling on Athena (AC-300a)...........................................71 I.Discussion............................................................................................................. 71 II.Critical Parameters�Broadband Decoupling ....................................................... 71 III.Acquisition of Inverse Spectra ............................................................................. 71

A. Initial Measurements 72 B. Hardware Changes for Inverse Experiments 72 C. Software Setup for Broadband Decoupling 72 D. Switching Hardware Back to Normal Detection 72

17. Heteronuclear Correlation on AM Consoles............................................73 I.Discussion............................................................................................................. 73 II.Critical Parameters............................................................................................... 73 III.Acquiring 2D HETCOR Spectra........................................................................... 73 IV.HETCOR 2D Processing..................................................................................... 75

18. NOESY ........................................................................................................77 I.Quick Summary for Acquiring and Plotting NOESY Spectra.................................. 77

19. Polarization Transfer Experiments (INEPT and DEPT)...........................79 I.General Discussion ............................................................................................... 79 II.Setting Up INEPT Experiments ............................................................................ 80

A. Critical Parameters 80 B. Coupled Experiments 80

III.Setting Up DEPT Experiments ............................................................................ 81 A. Critical Parameters 81 B. Coupled Experiments 81

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1. UW Chemistry Magnetic Resonance Facility by: cg fry: � updated 20.Aug.2002

I. Facility Layout (2nd floor Matthews)

Vorlon500

Narn500

ESR

Athena300a

Homer300

2221

2221c 2221d

2224

2202

2237

22302207

2201

2209

2210

charliefry

NM

R P

Cs

Phoenix250

VirSS-300

360NaToth

Xirth600

Adva

nced

Pr

oces

sing

Sun,

PC

sSG

I Wks

tn.

Matthews 2nd floorAsst.Director

marvkontney

Studentprep lab

Samplestorage/prep

As of August 2002:

ATHENA � AC+ 300 routine 1H/19F/31P/13C � auto-sample changer, quad-nucleus probe

HOMER � AC+ 300 routine 1H/13C � 1H/13C dedicated

PHOENIX � AC+ 250 routine BB VT � routine BB (29Si/11B/2H/199Hg/etc.), variable temperature

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NATOTH � Avance-360 non-routine BB VT � long-term VT, kinetics, concentration limited samples; 5 and 10 mm BB probes, 5 mm inverse probe

VIR � UNITY-300 solid-state NMR � conformational, motions, solid-state (not currently available for use) packing, catalysts, amorphous and glassy

compounds

NARN � UNITY-500 non-routine 1H/BB VT � high-sensitivity, sample-limited (1H < 5 mg, 13C < 15 mg), short-run, sophisticated experiments (e.g., HMQC, DQCOSY, gCOSY, gNOESY); limited access

VORLON � INOVA-500 inverse exps, 2D studies � long-term, sophisticated, gradient-enhanced experiments; combi-chem MAS probe; limited access

XIRTH � INOVA-600 long-term 2D studies � long-term, most sophisticated, gradient-enhanced experiments (e.g., NOESY/ ROESY, HMQC, DQCOSY); limited access

ESPY � ESP-300 electron spin resonance � paramagnetism, free-radical chemistry

PC�s � six PCs surround the main printer GKAR in rm 2224 � NMRSNAP.CHEM.WISC.EDU is a Snap server available via password from anywhere via

the web � BABYLON5 is the Win-2000 server

Sun�s � three Suns are available for data workup � NARN, VORLON and XIRTH are hosts for the respective instruments

SGIs .......................................................................................................................................................� NATOTH (Avance host computer) and GQUAN (for off-line data workup)

II. Facility Personnel Director, Chemistry Instrument Center Prof. Paul M. Treichel Rm. 6359A 262-8828 [email protected]

Director, Magnetic Resonance Facility Dr. Charles G. Fry Rm. 2203 262-3182 o [email protected] (Charlie) 276-0100 h [email protected]

Associate Director, MRF currently vacant

NMR Engineer Marv Kontney Rm. 2210 262-0563 [email protected]

ESR Engineer Roger Clausen Rm. 2225 262-8196 [email protected]

Teaching Assistants Ryan Nelson Rm 7361 262-0216 [email protected] Mike Birkeland 9th floor new add. 265-7048 [email protected]

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2. Bruker Acquisition Basics by cg fry: created 2/17/94 � updated 12-Sep-00

I. Description [See the Primers chapter for more specific information on certain areas]

Acquisition on Bruker AM and AC spectrometers is based on the command GO. GS (go-setup), ZG (zero-go), and CO (continue) all use the GO command; ZG zeroes memory and coadds acquisitions until NS and NE are fullfilled, whereas CO coadds new acquisitions to the current memory. GS displays only the most recent acquisition.

Four events are initiated when GO is issued, as shown in the figure above:

i) The spectrometer waits a time RD seconds; relaxation delay.

ii) A strong transverse rf pulse is applied with a duration PW µs.

iii) A delay of duration DE µs occurs to allow for probe ringdown.

iv) Acquisition begins, and lasts a time AQ seconds.

II. Deuterium Lock Locking on a deuterated solvent involves adjusting four spectrometer variables:

i) Change Z0 shim (FIELD) to find the lock signal. Deuterons track closely with protons, so CDCl3 will be 5.2 ppm downfield of (CD3)2CO. It should be clear (if you don't know, please ask!) that changing the magnetic field strength by 5.2 ppm will require a change in the spectrometer frequency by the same amount to keep a 10 ppm sweep width running from TMS at δ=0 ppm to δ=10 ppm.

ii) The second adjustment involves the lock phase. The lock uses a phase-sensitive loop to keep the frequency of the spectrometer stable:

The lock in sweep mode on Bruker spectrometers should always have a positive initial slope.


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Changes in solvent, temperature, and even in adjustments from poor to good shims can change the

phase. Good lock phase is critical to obtaining a stable, good lock, and thus a good shim and lineshape! So you should adjust the lock phase after any such changes. Initially use the sweep display, but later carefully adjust the lock phase to maximize the lock amplitude.

iii) The other two adjustments are the lock gain (lock amp on WP�s) and lock power. Although the apparent effect of both controls is similar, to increase/decrease the lock intensity, the actual changes are fundamentally different. The lock gain is the lock receiver gain, changing nothing involved with the sample magnetization. The lock gain can therefore be changed at will with little danger.

iv) The lock power, on the other hand, must be carefully adjusted to achieve the best lock performance. This control changes the radiofrequency (rf) power to the sample. Too little power will give a weak response, and a noisy lock. Too much power will cause saturation, which must be avoided. Proper settings can be found for any solvent using the following technique:

Increase/decrease the lock power, and watch for a �bounce� in the signal: the signal will go up/down in response to the change in power, but if it then rebounds (perhaps just slightly) down/up as in a bounce, the power is too high. Decrease the power until the bounce cannot be observed, and decrease the power another 20% to be safe.

Suggested settings for the lock power for the most common solvents are posted next to each AC spectrometer in the facility.

III. Shimming Once lock is achieved, the magnetic field inhomogeniety must be shimmed out. For experimental

planning, one must keep in mind differences in solvents. Acetone-d6 has a very narrow natural 2H linewidth, and therefore gives good performance for shimming. DMSO-d6 and pyridine-d7 both have much broader natural 2H linewidths, and are therefore relatively poor solvents for lock shimming. D2O can give very broad 2H lines due to exchange. Of course, the last three solvents might be the only choices for solvation and cost reasons, but shimming on the FID might then be necessary if high-resolution 1H spectra are needed.

A number of good discussions about shimming strategies are available. Since every change of sample requires shimming, every spectroscopist should take advantage of strategies to lessen the time spent shimming. Especially useful is G. A. Pearson, �Shimming an NMR Magnet,� Chem. Dept., Univ. IA, Iowa City, IA 52252. Many practical examples are given that show how to visually determine which shim needs adjusting, and in which direction. These quick determinations can save much time at the spectrometer and lead to improved spectra. More detailed discussions of shimming can be found in Derome, p. 42-50, and in G. N. Chmurny and D. I. Hoult, �The Ancient and Honorable Art of Shimming,� Concepts Magn. Reson. 2, 131-149 (1990).


THE UNIVERSITY OF WISCONSIN�MADISON Magnetic Resonance Facility�Chemistry

IV. Acquisition Parameters The most important acquisition parameters are briefly

described in this section. The mnemonics are specific to Bruker spectrometers:

PW � pulse width of GO command (see Section I)

RD � relaxation delay

DE � probe/filter ringdown delay

SW � sweep width in Hz

O1 � sets center of spectrum by changing absolute frequency

DW � dwell time in µs; time per digitized point; set by SW → SWDW 21=

TD � data acquisition size; number of points digitized; TD/2 = # complex data pairs

SI � Fourier transform size; zero fills if TD<SI; only half SI used for real part, so

# points in spectrum = SI 2 digital resolution

sweep width

# points in spectrum= = =

⋅SW

SI

TD

AQ SI2 AQ � acquisition time; related to obtainable resolution (i.e., best resolution possible) unless

sophisticated analytical procedures, such as linear prediction, are used. The digital resolution can be improved by zero-filling, but not the actual obtainable resolution (appropriate Gaussian multiplication perhaps can take advantage of one zero-fill, but further zero-fills will not help).

obtainable resolution = 1

AQ AQ DW TD

TD

SW= ⋅ =

2


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3. Introduction to AC+ Spectrometers by cg fry: created 1/10/94 � updated 21.Aug.2002

I. Summary of 1D Acquisitions on AC Spectrometers The following list progresses similar to taking data on any sample. These are not inclusive for all occasions that occur in the laboratory, and cannot therefore replace an understanding of the basics of NMR. Many limitations will be self-imposed by the student that utilizes this list too strongly. That said, this section should provide a useful tool while learning to use Bruker AC spectrometers.

a) Unless you are immediately following another user, the spectrometer should be locked on a CDCl3 standard. Remove the CDCl3 standard:

! Turn up display intensity using knob on lower right corner of display.

! Use CNTL-L and CNTL-D until the display has the proper information on screen.

! Unlock and stop the spinner: press LOCK and SPIN ON/OFF; the light goes off for both buttons (this step is not really necessary).

! Eject the cdcl3 standard sample: press 2ND LIFT .

b) Insert your sample:

! Put the sample/tube into a sample spinner; lower it to the bottom of the depth gage, or if tje solvent height is less than ~0.5ml (always use ≥ 0.35ml!!), center the solvent column on the rf center mark drawn in red on the depth gauge.

! Clean the bottom of the tube using the ethanol and chemwipes: do not get ethanol on the sample spinner (it will ruin it, at $75 each!).

! Make sure the lift air is on: if it is not, press 2ND LIFT .

! Place the spinner+sample in the lift stream at the top of the magnet; make sure the tube is floating freely before letting go of it!

! Press LIFT OFF and allow the sample to seat (you should hear a click as it seats).

! Press SPIN ON/OFF; the sample should spin up, but after a ~5−20s delay.

c) Lock and shim the sample to a good line shape:

Follow section II below, and see the Primer on Shimming in Chap. 4.

d) Read in the proper jobfile, set RG, and acquire:

Follow section IV below: e.g., RJ CDCL3.1DJ↵ II↵ ZG↵ ^H RG↵change↵ ZG↵

e) Remove your sample, and lock on the CDCl3 standard:

Follow section VI below; turn on the decoupler as needed using the DO↵ command.

f) Transfer data to PC server, work up and plot:

Follow section V below (e.g., ^X TOPC filename.xxx ^X), and use the NUTS Cheat Sheet for help with data work-up and plotting.

g) Backup data:

Use Windows Explorer to copy data to a Zip disk; data will reside on the spectrometer for a few weeks (possibly longer, but we cannot assure that this will be true).

Intro to AC+ Spectrometers Page 7


The primary functions for obtaining routine 1H spectra are contained in the Shim Control Module (SCM) and with preset job files that setup acquisition.

II. Locking and Shimming with the SCM • Users should not use any controls except those shown

darkened on the right!

• <CNTL>-L toggles lock/data display

• Samples are ejected by pressing the orange 2ND button on the lower right, and the LIFT button. Insert the sample by pressing the LIFT OFF button.

• For samples with a change in solvent:

a) turn LOCK off

b) change LOCK POWER to appropriate setting (see table next page)

c) set SWEEP AMPL to 24, and center sweep on screen with FIELD control; reset SWEEP AMPL back to 16

d) adjust LOCK PHASE if necessary; DUAL SWEEP helps here

e) press AUTO LOCK and wait for LOCK GAIN to stop blinking

f) press LOCK; check LOCK POWER

g) adjust LOCK GAIN to get signal in top fifth of screen

h) adjust LOCK PHASE just as you would any shim (no interactions occur; but LOCK PHASE should be readjusted whenever Z2 is moved substantially)

i) shim with Z and Z2 shims; turn off spinning and adjust X and Y shims if spinning sidebands are large (all other shims should not need to be adjusted)

• Shim settings can be reset to PREVIOUS SET value by repressing the current shim key. E.g., start in Z at -140, and realize after going to -80 that you're going in the wrong direction. Pressing Z key again will reset the Z shim to -140.


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III. Job Files and Lock Power

Deuterated Solvent Job File Lock Power unlocked locked

1H δ (ppm)* 13C δ (ppm)*

acetone ACETONE.1DJ 25 15 2.2 30.2 205.1

acetonitrile CD3CN.1DJ 20.0 10.0 2.0 0.3 117.2

methanol CD3OD.1DJ 20.0 10.0 3.5 49.3 deuterated water D2O.1DJ 25.0 15.0 4.8 dichloromethane CD2CL2.1DJ 5.3 54.2 dimethylsulfoxide DMSO.1DJ 35.0 35.0 2.6 39.5 chloroform CDCL3.1DJ 35.0 25.0 7.3 77.7 benzene C6D6.1DJ 22.0 12.0 7.4 128.7 *from Bruker Almanac 1994, p. 119-120.

IV. Data Collection A. Read in a job file for your solvent; use a job name in the table above: e.g., for CDCl3, enter

RJ CDCL3.1DJ . Type II to initialize the interface (i.e., set the hardware up correctly).

B. Set the receiver gain, RG, by watching carefully the size of the 1st FID following a ZG.

i. Type ZG. If the 1st FID is larger than ±1 division on the AC/AM display, stop the acquisition using <CNTL>H . Increase RG, and redo the ZG, again watching carefully the size of the 1st FID.

ii. If the 1st FID is much smaller than ±1 division, increase RG.

iii. Can also use automatic gain adjustment: RGA. This method is not recommended for various reasons too detailed to go into at this point (ask if you really want to know).

C. Use EP mode to adjust the sweep width and offset using <CNTL>O (see next page)

D. Set the number of scans, NS, to a multiple of 8. Collect data by using zero-go, ZG.

E. Write the data to the spectrometer hard disk using the write command: WR filename.xxx.

F. Transfer the data to the network server via NMRLink (see next section).

G. Log usage on the Chronos PC Excel spreadsheet.

V. File Transfer: NMRLINK (usually store FID�s; RE FID1 after FT; spectrum is SPC1)

� NMR-Link NMR-Link is bi-directional, Bruker image format

Fastran is unidirectional, LYBRICS format

� To send file to network (following for Phoenix and Athena; use USR FASTU for Homer)

i) take data as normal (ZG, then WR filename.xxx)

ii) type <CNTL>X to go to 2nd session

iii) enter topc filename.xxx

iv) type <CNTL>X to go back to 1st session and DISNMR



� To retrieve file from network back to spectrometer

- same as sending, but use frompc filename.xxx instead of topc command

VI. Departure A. Following Section II, insert lock standard and relock.

B. Check spin rate and shims, turn screen intensity down.

C. Clean up the console area.

D. Log time on the Excel spreadsheet on the PC Chronos (currently in rm 2230); note any problems on Chronos such as difficult spinning, ejecting sample, spinning sidebands etc. Any problems such as computer crash, disk full, or sample breaking, find TA or the NMR Director immediately.

VII. Adjusting for SW and O1 The following notes summarize commands to allow quick manual setup of SW and O1 to get appropriate spectra for special cases:

� Suppose you want to look at a portion of the normal spectrum with high digital resolution (e.g., aromatic region of ODCB). Take the normal spectrum, and then follow the steps shown below to narrow the sweep width to obtain just that region.

� Suppose instead that you have an acid group or heteroatom that causes chemical shift outside the normal 0-10 ppm region. In these cases, you would see �folded� peaks in spectra obtained with SW=10ppm (2500 Hz on Phoenix); see the spectra on the next page. Folded peaks will almost always have unusual phasing that cannot be corrected with the 0- and 1-order phase corrections in PCNMR.

A. For folded peaks, double or triple the sweep width, SW. On the AC-250, 2500 Hz = 10 ppm, so use SW=5000 (standard setup) or 7500. Retake the spectrum using ZG.

B. Perform an FT at the spectrometer, by entering FT .

C. Go into EP mode by entering EP<RET> .

D. Note in EP mode that the region of text has changed from the top of the screen to the bottom. EP mode is now ready to accept a variety of commands; use the following two (see also Primers Chapter):

P - phase adjust: i. expand using B-knob on a large peak on one side of spectrum ii. enter phase mode by entering P (do not press <RET>) iii. correct 0-order with C-knob iv. move to large peak furthest away and correct 1st-order with D-knob v. use M to save

A-knob moves spectrum horizontally B-knob expands/contracts region on screen C-knob performs 0-order phase correction (<CNTL>C reverses) D-knob performs 1st-order phase correction (<CNTL>D reverses) M will memorize phase adjustment and return to EP mode <RET> will ignore phase adjustments and return to EP mode


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<CNTL>O - automatic SW and O1 redefinition i. expand using B-knob to region you want as full spectrum ii. move cursor to center of screen using C,D-knobs iii. press <CNTL>O. The computer will set new SW and O1 values.

E. Press <RET> to exit EP mode, and enter ZG to take new spectra.

VIII.Spectra showing folding and unfolding of deshielded proton.



IX. Caveats A. Watch the lock power: if it�s too high, saturation will occur, and poor line shapes will likely

result. Can check by watching that lock signal increases consistently as lock power increases.

B. [Note: we are now using NMRLink for all data transfers, so all transfers since yr 2000 are bi-directional. The following would apply to data transfered and backed-up prior to yr 2000.] The USR FASTU command invokes a FASTRAN one-way transfer. The data following the transfer will appear on the network, but is in PCNMR-specific (Lybrics) format. You cannot get this data back onto the spectrometer, or into any other software package besides PCNMR or NUTS. Store the data to 3.5" floppy at the spectrometer if you want more versatile storage.

C. The hard disks on the AC spectrometers fill multiple times per year. If you get a DISK FULL error message, see NMR Director or a TA to clear space. Always make backups immediately!


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4. Primers for Areas of Common Interest

by cg fry: created 2 May 1996 � revised 13-Sep-00

I. Primer on phasing Phasing involves turning all lines in a spectrum into pure adsorption lines:

Zero-order phasing rotates all lines in the spectrum identically:

First-order phasing rotates lines progressively faster as one moves away from the �pivot� line. The pivot line is assigned in NUTS by placing the cursor on a line in the spectrum, and holding the left mouse button down while pressing the P key (left-hold P). Since the pivot line does not change angle under 1st order phasing, this is the line that should be correctly phased when performing 0-order phasing:

Primers for Areas of Common Interest Page 13

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In the other primary method for phasing spectra in NUTS, the PS-mode, the tallest peak the zoom region 1 is automatically assigned as the pivot. Chosing between PH or PS modes depends for the most part on the number of points and resolution of the peaks. For most 1H spectra the PH method works fine. But for very high digital resolution spectra, PE can work better�the data reduction done on entering is less dramatic because of the expansion. See the NUTS help and tutorials at the Acorn website at http://www.acornnmr.com for assistance with both methods. But the primary philosophy stays the same:

� increase the vertical scale of the slope until the baseline is obvious,

� perform 0-order phasing (using the left mouse button) on the peak at the pivot point; the best indicator of reliable phasing is that an extrapolation (by eye) of the baseline would have it connect smoothly under the peak (note in the figure above how the baseline of the 270° dispersive peak runs in essentially opposite directions�curving down the page coming from the left-hand side and up the page coming from the right-hand side�but connects under the absoptive 0° peak,

� perform the 1st-order phasing on a region far from the pivot point,

� ignore the phase of TMS and other solvents peaks (often may be slightly out-of-phase with solute peaks due to saturation effects).

In PCNMR4Windows the pivot line is chosen automatically, and colored red. There are a number of ways to phase spectra in PCNMR4Windows, but the method I (cgf) prefer is:

i) always perform 0th order phasing on the red peak: start by expanding about the red peak, enough to give clear definition to the peak, but not so much that you cannot see a good amount of baseline on both sides of the peak

ii) scale up by clicking the the button three or four times (enough to give the baseline better definition)

iii) I like to press and hold the left mouse button while phasing; letting go of the button then fixes the phasing. The other method of click the left button, phasing, and then clicking the left button again to fix the phasing I have problems with; I find that often on the second click, the phase jumps slightly ruining the phasing I had worked to get. Note that only up-and-down (away-and-towards you) motions of the mouse effect the phasing; sideways motions do nothing.

iv) use Display Reset the expand about the peak furthest from the red peak

v) click the button to get 1st order button (blue)

vi) click and hold the left button: up and down motions of the mouse adjust the phasing; release the button to fix the phasing

vii) use Display Reset ; if the phasing looks good�the baseline should appear continuous if

followed through all peaks�then press the button


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II. Locking and Shimming Primer #1 by cg fry: revised 20-Sep-00

Make sure the LOCK POWER is changed to reflect the solvent. Once set correctly (note the 10 unit difference between unlock and locked power�s!), do not change the LOCK POWER; use the LOCK GAIN to adjust the vertical position of the lock signal on the monitor.

Lock solvents have unique characteristics depending on their spin-lattice relaxation; at this point, it is sufficient that you realize the difference between groups of solvents:

� D2O and DMSO can be shimmed relatively quickly (i.e., changes in shims values are reflected by quick changes in the lock signal), but will often be �insensitive� to shim changes, which can lead to poorer lineshapes.

� C6D6, CDCl3, and CD3OD are more intermediate: changes in shim values are accurately reflected within 2-4 s wait in the lock signal. This wait after adjusting the shim value is longer than with DMSO-d6, but better lineshapes will result under careful shimming.

� CD3CN and acetone-d6 usually will provide very good lineshapes by careful shimming; care must used with these solvents, however, as changes in shim values may not be accurately reflected in the lock signal before 6-8 s have past, and 10 s may be needed to see the final lock position.

Typically, I will move a shim by 10 units (the exact amount is spectrometer dependent, with the values stated here OK for the AC-250), then stop all movement with the knob. For D2O/DMSO, I wait just 1 s or so, CDCl3/C6D6 2-4 s, and CD3CN/acetone 5-6 s, before moving another 10 units. Shim Z1 first, then Z2, then back with a check on Z1 (important if Z2 changed a lot). Once I find a maximum, I reduce the shim value change to try to get ~2 units accuracy (typical) at the end. Observing a well-resolved 29Si doublet from the 28Si singlet of TMS is a useful measure of whether you�ve attained a good shim or not.


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III. Primer on Bruker EP Mode � Most Common Uses by cg fry: revised 22-Sep-00

Enter by typing EP <RET> at DISR 1. 2. or 3. prompt. Note in EP mode that the region of text has changed from the top of the screen to the bottom. EP mode is now ready to accept a variety of single letter commands, used without pressing <RET>. The <RET> key will back you out of command levels in EP: e.g., in phasing <RET> will back out to top EP level, and another <RET> backs out to DISR. The following five commands are the most common:

! E - toggles between three different cursor display modes

! : - toggles between Hz and ppm display

! P - phase adjust:

A-knob moves spectrum horizontally B-knob expands/contracts region on screen C-knob performs 0-order phase correction (<CNTL>C reverses) D-knob performs 1st-order phase correction (<CNTL>D reverses)

i. expand using B-knob onto a large peak on one side of the spectrum

ii. enter phase mode by pressing P (do not press <RET>)

iii. phase 0-order with C-knob (if hit end of knob, press <CNTL>-C to continue phasing in same direction)

iv. move to large peak furthest away using A-knob; correct 1st-order with D-knob

v. use M to save

M will memorize phase adjustment and return to EP mode <RET> will ignore phase adjustments and return to EP mode

! G - set reference

i. use A- and B-knobs to center and expand about reference peak

ii. use C-knob (course) and D-knob (fine) to set cursor on reference peak

iii. press key G and enter reference (e.g., 0P for TMS, 7.24P for CDCl3)

! <CNTL>O - automatic SW and O1 redefinition

i. expand using B-knob to region you want as full spectrum

ii. move cursor to center of screen using C,D-knobs

iii. press <CNTL>O. The computer will set new SW and O1 values (check AQ is SW is substantially changed!).

8 - estimate signal-to-noise (S/N); make sure cursor is on peak of interest, and if possible

have that peak be the only one on the display (use A � A command) 9 - toggle between points connected or points only display


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IV. Primer for Obtaining Accurate/Consistent Proton Integrations in DISNMR by cg fry: revised 28-Oct-98

Accurate integrations in NMR is a non-trival task. In all cases where quantitation is required, care should be used. I highly recommend �playing around� with the software, i.e., experimenting with various options in the software, and document how the integrals are changed. Be especially careful of baseline correction. I have seen instances where > 100% differences in integrations seemed quite easy to achieve. These have always been cases with a narrow large peak compared to a broader smaller peak. A lot of care must be exercised to get reasonably accurate integrations (and I wouldn't be over optimistic about how good you can get; i.e. assign reasonable error bars, reflecting your experience with the software). The best method of obtaining accurate intensities (actually areas), not discussed here (see the NUTS or VNMR help), is to use deconvolution methods in NUTS or VNMR.

Through trial and error, I have yet to see a way to use any automation sequence or macros to get consistently good integrations for quantitative work. These comments apply when small and/or broad lines exist in the spectra; automatic procedures seem to work well for "routine" spectra where only semi-quantitative integrations are sufficient. I recommend the following procedure for performing integrations within DISNMR (and I currently prefer NUTS much more than PCNMR4Windows; advanced users are fine with VNMR, but be careful(!) with baseline corrections):

1. Obtain the data with a large sweep width (e.g. 20ppm) to help reduce baseline rolling in the data region. Make absolutely certain that no signal clipping is occurring (reduce RG by a factor of 2 if uncertain!). Take one data set with a relaxation delay 5 times longer to make certain T1 effects are not affecting the results.

2. Take all data if possible at one time using identical (RG, RD, etc.) settings. If this can be done, then PK will work well and phasing should not be adjusted differently for different spectra. Use an automation routine to EM;FT;PK and write out spectra (see FT.AU on the spectrometers). If data is taken at different times, phasing should be performed manually, with care taken to try to get the baseline to have an identical slope over the region of interest (use some defined criteria to keep from becoming overly subjective).

3. You can use a matched Lorenztian line broadening for simplicity, but for best results use Gerald Pearson�s Gaussian apodization (not the same as DISNMR�s GM!!); see the next primer for more information.

4. Baseline corrections always have to be performed, and I have never gotten consistent autobaseline routines to work sufficiently well. For a lot of spectra, performing the manual baseline corrections is the most tedious and subjective part of the process. Do _not_ rely on slope adjustments to correct a poor baseline!!

5. I have found that with good baseline correction, the slope adjustment is straightforward and only has to be done once. The integrals are not nearly as sensitive to slope as they are to phasing and baseline correction (at least in my experience).

For small numbers of spectra, DISNMR works quite well, albeit a bit tediously.


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V. Primer for Applying Pearson�s Gaussian Apodization in DISNMR [The following is taken directly from an e-mail message from G. Pearson, U. Iowa; see in addition the

reference given below � revised 29-Oct-98]

A Gaussian apodization envelope works much better than an EM [in particular, for integrations]. For the same full width at half height, a Gaussian will have much narrower feet than will a Lorenzian. The narrower feet mean that the "steps" in the integral will be significantly sharper, so the _height_ of the steps will be much better defined. Felix and Nuts (and presumably other off-line stuff) allow you to multiply by a Gaussian without necessarily multiplying by an increasing exponential at the same time, but the Bruker software incorrectly assumes that no one ever has any good reason to do just a simple Gaussian apodization.

You can lie to DISNMR and DISMSL so as to use a combination of GM and EM to produce a desired Gaussian broadening, without any Lorenzian garbage. Basic equations are in:

Gerald A. Pearson, "Optimization of Gaussian Resolution Enhancement", J. Magn. Resonance 74, 541-545 (1987).

In particular, eq. 11 gives the height of the shifted gaussian produced by GM, in terms of the intrinsic line width and the fractional sharpening which is wanted.

Let's take an example. Say you wanted to do a GAUSSIAN broadening of 5 Hz, and your acquisition time is 4.096 sec.

1. First, multiply by an envelope which will transform a 1-Hz Lorenzian into a 5-Hz Gaussian. [The 1-Hz Lorenzian is arbitrary, and will eventually cancel; you could use any other convenient LB.] By eq. 11:

tmax = 2 ln2 / ( π * 1 Hz * (5/1)2 ) = 2 * .693 / (3.14 * 1 * 25) = 0.0176 sec.

So you want to set

LB = -1.0 and GB = (0.0176 / 4.096) = 0.00431

and then do a GM.

2. Now, get rid of the increasing exponential:

set LB = +1.0 and then do an EM.

This is a real round-about way of doing such a simple thing, isn't it? That's one of the reasons why we [the Univ. of Iowa] routinely process stuff off line on a PC, using either Felix for Windows or NUTS (so use NUTS here at UW; if you are stuck with DISR, however, the above should help).


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VI. Primer on Lorentzian and Gaussian (Bruker) Multipliers [MathCAD import]

by cg fry: revised 28-Oct-98


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VII. Primer for Decoupler Modes on Bruker AC and AM Spectrometers by cg fry: revised 28-Oct-98

PO � power off; typically leave spectrometer in this mode, although DO is OK

DO � �decoupler on�; same as a power switch�decoupling delivered to probe only through AU sequences (no decoupling delivered when type in command DO)

� recommend going through DO from and to PO to get to other modes

HG � homonuclear gated decoupling; used in NOE and PRESATuration experiments; decoupler off during acquisition; � DP (typical) = 20-40L (AC�s), 30-50L (AM�s)

HD � homonuclear decoupling; used in HOMODEC experiments; decoupler is gated during acquisition (see Figure below);

� DP = 0-30L (AC�s), 10-40L (AM�s)

CW � continuous wave decoupling; used in selective heteronuclear experiments; decoupler can be on during acquisition (depends on .AU sequence)

� DP = 0-30L (AC�s), 10-40L (AM�s)

BB � broadband decoupling; old form, replaced by CPD � do not use BB mode

CPD � composite pulse decoupling (hardwired WALTZ-16, A. J. Shaka, J. Keeler, R. Freeman, J. Magn. Reson. 53, 313-340 (1983)); used for broadband decoupling of 1H in heteronuclear experiments; reasonable 90° pulse calibration required, input as P9 for AC�s and AM�s; gives clean decoupling over ~5 kHz (10 ppm on 500 MHz spectrometers), so O2 must be centered in 1H spectrum on AM-500 (dof on Unity-500).

� DP = 18-20H for 5mm probes, 12-14H for 10mm probes, � P9=100-130 µs.


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VIII.Primer for Estimating T1 Values Theoretically

[by cg fry: revised 28-Oct-98: the following is a problem given in Chem. 626, followed by the solution.]

A. Estimate from theory T1 for the singlet aromatic 1H of pamoic acid.

Assume relaxation is dominated by dipole-dipole interactions and the correlation time for pamoic acid, τc # 10-11 s. From pg. 193 of T. C. Farrar, Introduction to Pulse NMR Spectroscopy we can derive:

where γ Ht = 2 67. , r has units of Å and τc is in sec.

B. Quickly measure T1 of the 1H�s of interest for Pamoic acid.

1. Follow directions in section II (Rapid Determination of T1, by Inversion Recovery Null Method, p. 44) of the T1 chapter in the Bruker User�s Guide.

2. Include with turned-in solutions null times and T1 values for longest and fastest relaxing 1H on pamoic acid. Give two reasons why the T1 estimate made in part A might differ from the value you just measured [hint: think about assumptions made in part A, and how the sample has been prepared].

Solution:

Using the last equation on pg. 193 of T. C. Farrar, Introduction to Pulse NMR Spectroscopy (Farragut, 1989):

( )R

TS S

rs

It

St

IS

c

c

c

c

c

c1

1

2

6 2 2 2 2 2 29 11

11

3

1

6

111126 10≡ = +

++

++

+

× ×− +

−( ) .γ γ τ

ω ττ

ω ττ

ω τ

where . Since ωo = 2.5×108×2π and τc = 10-11, we can see that ω τo c ≈ −10 12 « , so that

On pg. 192, Farrar gives the correct units for this equation, where γ Ht = 2 67. , so all we need is r in Å.

Use C-H = 1.1Å, and C-C(aromatic) = 1.4Å then elementary geometry gives:

H4 to H6 distance = 2×1.4×cos30° = 2.4Å


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=T s1 45

(Actually, I believe the 1.1126 constant is a mistake in Farrar�s text, and should be 1.48... giving T1 = 33 s, which I�ll use now.)

Similarly, we could have calculated the protons, as

H6 to H7 distance = 1.4 + 2×1.1×sin30° = 2.5Å

=T s1 18 (add rates for both H7 and H4 = + = −R s111

33

1

420 054. !!)

�CH2� to H7 distance = 2.4 - 1.1 + out-of-plane ~ 1.7 to 2.1Å

so for H7 we have R1(H7) = 1/42 + 1/8[for 1.9Å]

Finally, we have H-C-H = 2×sin(109.5°/2)×1.1Å = 1.80Å, so

for �C(H toward H7)H� R1(toward) = 1/8 + 1/5.8[for 1.8Å]

and �C(H away H7)H� R1(away) = 1/5.8[for 1.8Å]

The primary reasons for differences in the estimated values and measured values are:

i) lack of careful degassing to remove oxygen, or other contamination in solution;

ii) incorrect estimate for the correlation time (in fact, measuring T1 is one of the best methods for obtaining correlation times).


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5. AM/AC Spectrometer Software by cg fry: created 12/10/94 � updated 20.Aug.2002

I. ADAKOS (Aspect Disk And Keyboard Operating System) � ADAKOS is the base operating system of the ASPECT computer, the acquisition and analysis

computer on the AM spectrometers. Some functions of ADAKOS include listing of directories, batch copying or deletion of files, renaming of files, initialization of new disks and restoration of deleted files. This program is started automatically whenever the computer is rebooted (<STOP><CLEAR><DISK>).

All ADAKOS commands have two or three letters, e.g., DIR, COP, and DEL.

MO (monitor) to exit to the ADAKOS subprogram from DISNMR.

* DSP ON ("*" is the AM-console prompt) to turn on the display.

DIR to list all files in the directory: H to halt and C to continue. <ctrl> Q to stop.

SDIR **.* for sorted directory (sorted by alphabet; can be quite slow).

� A filename has up to eight characters, followed by up to four characters. The wild card * represents only groups of four characters for only blocks 1-4, 5-8 or for the suffix. DIR *.* will only show filenames having four characters or less. DIR aa*.* is invalid, since * cannot represent the 3rd through 6th characters. ? can be used for any single character (only available in ADAKOS, unfortunately not in DISR).

� D1 is the system disk. Use F1 for the floppy disk. The disk designation must follow the filename unless it is the default drive (D1). e.g.,

DIR **.*=F1 to list all files on the floppy. COP <input file>/<output file>=F1 to copy a file from disk onto a floppy. COP <input file>/=D2 to copy with the same filename. COP **.*=F1/=D1 to copy every file on a floppy to the hard disk. DEL <filename>=D1 to delete a file from disk. DISR91 to run DISNMR (RUN DISNMR on WP-console).

II. DISNMR A. Control Keys and System Commands

<ctrl> D toggles the grid ON and OFF on the display; <ctrl> L toggles between the lock display only, the data only, and both. <ctrl> H halts following next acquisition; active only in current job <ctrl> E halts immediately; does not save data; active only in currect job <ctrl> K halts all activity including acquisitions, plotting and printing; active in all jobs; use only

as a last resort (try <ctrl>H first, the <ctrl>E) <ctrl> X toggles between 1st and 2nd sessions <ctrl> Y toggles display from interleaved to separate quadtrature channels

� OUT <ret> for setting up output devices. COMMAND INTERPRETER? B (both). OUTPUT DEVICE? P (printer only).

AM/AC Spectrometer Software Page 29

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B. Files in DISNMR

� In DISNMR, any file except the current one can be deleted in the same manner as above. DIR is also available, but the wild character ? can't be used. The * wildcard can only represent characters 1-4 or 5-8, or the suffix:

DI *.* will list all files having 4 characters or less in prefix DI **.* will list all files DI cgf1*.* will list all files starting with cgf1 DI cgf*.* not allowed since * must start in position 1 or 5 of prefix DI cgf?*.* not allowed in DISNMR; ok in Adakos

� There are many types of files:

File Type Process 1D data files 2D data files Job files Shim files Automation

Common suffix **.001 **.SER **.1DJ **.2DJ

**.SHIM **.AU

Disk I/O WR, RE (.AU writes), RE WJ, RJ, PJ WSH, RSH EDIT, AU Directory DI:F (FIDs)

DI:S (spectra) DI **.SER DI **.SMX

DI:J DI:Z DI:A

� TI will allow entry of up to 80 freeform characters, stored with data file. Users should include a brief

sample description, and notebook number and page in the TI area.

� RE <filename> to read a file ("?" following any command opens a helpfile). One can also save or read files from an external 8" or 3-1/2" floppy disk (F1) as follows:

WR <filename>=F1 to save a file to floppy RE <filename>=F1 to read a file.

� COP and DEL commands work as in Adakos.

C. Common Acquisition Commands (WJ, RJ) AQ � acquisition time AS � automation setup CO � continue acquisition CPD � composite pulse decoupling

(Waltz-16 decoupling) DO � decoupler gated off DE � pre-scan delay DP � decoupler power (actually,

decoupler attenuation in dB) DS � # dummy scans GO � begin acquisition GS � GO setup mode HD � pulsed homonuclear single

frequency decoupling II � initialize interface NE � # experiments

NS � # scans O1 � transmitter offset frequency O2 � decoupler offset frequency PO � decoupler power off RD � relaxation delay RG � receiver gain (actually,

receiver attenuation in dB) RGA � automatic receiver gain

setting SF � spectrometer frequency SI � spectrum size SW � sweep width TD � time domain size ZE � zero data and initialize for

acquisition


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D. Common Pulse Sequence Commands and Parameters (WJ, RJ)

( ):D � ( ) operates on decoupler An � transmitter quadrature phase Bn � decoupler quadrature phase Dn � delay length for nth delay D0 is 2D t2 delay incremented by

IN (not the loop parameter) FL � input frequency list GO=j � perform GO, jump to line j when

finished; continues NS times

IF � increment file number IN=j � loop index jumps to line j;

continues NE times IN � time incr. in 2D set by SW1 O2 � read decoupler frequency,

increment list pointer Pn � pulse length for nth pulse Sn � decoupler power setting

E. Common Data Processing Commands (WJ, PJ)

AI � absolute intensity = 0 (default) autoscales = 1 absolute intensity AT � additive transfer of two spectra BC � apply linear baseline correction FT � Fourier transform PK � apply phase correction last

stored in EP mode SR � spectrum reference frequency

EM � exponential multiply chose LB ~ linewidth GM � gaussian multiply

chose LB ~ �linewidth chose GB ~ fraction AQ where S/N first = 0

F. Common EP Commands

^B � re-enable knobs A and B ^F � display current F1,F2 region ^O � set SW,O1 to expanded region ^R � display whole spectrum LINE FEED � baseline points selection mode A � add phase corrections B � phase on biggest peak C � phase on cursor selected peak D � dual display D � decrease separation I � increase separation S � subtract spectra E � toggle EP info. displays F � enter frequency limits F1,F2 G � set spectrum reference (SR) H � hard copy (print) cursor info. I � enter integration mode A � calibrate (normalize) integrals M � toggle knobs C,D from cursor mode

to slope and bias mode S � section plot of displayed integral X � full plot of displayed integral Z � zero integral; set starting-end points

of integrals

J � enter line fitting mode K � enter interactive baseline correction M � set minimum intensity for peak picking P � phase on biggest peak displayed O1 � printout absolute freq. of cursor M � will store frequency as O1 O2 � printout absolute frequency of cursor L � stores freq to specified freq list file M � will store frequency as O2

R � use twice to set freq limits for expanded display (^B to reset A,B knobs)

S � section plot T � enter cursor to T1PNTS file U � update region for plotting X � plot expanded region (PX) Y � change CY,CX within EP < � left shift one point (LS) > � right shift one point (RS) : � toggle 5 � peak peak and display 6 � move display down 1/8 screen 7 � move display up 1/8 screen 8 � display rms S/N 9 � toggle to dots/connected display

Plotting with DISNMR Page 31


III. Plotting with DISNMR A. Overview

Plotting on Aspect computers starts similarly to any computer system, with page and plotting regions. The plotting region depends on the particular plotting device; here we'll assume either an HP LaserJet printer, or an HP 7475 or 7550A plotter. Landscape plotting is default on both plotters. The spectral region plotted is set with the EP U (update) command. Plot parameters can be read in with the PJ command (these jobfiles may not be present; we use NUTS to plot nearly all spectra):

PJ A3PLOT.1DJ 11×17� landscape plotting PJ A4PLOT.1DJ 8.5×11� landscape plotting PJ A3STKPLT.1DJ 11×17� stack plotting with rotated (portait) spectra PJ A4STKPLT.1DJ 8.5×11� stack plotting with rotated (portait) spectra

The parameters and commands that control plotting (see figure above):

Parameter/ Command

Description

Landscape 11×17� A3

Landscape 8.5×11� A4

Portrait 11×17� A3

Portrait 8.5×11� A4

X0 X offset for origin 0 0 0 0 Y0 Y offset for origin 0-4 0-2 0-4 0-4 CX X-axis length of spectrum 35 24 24 16 CY Height of reference (usually

tallest) peak in spectrum 20 16 30 20

MAXY Maximum Y deviation plotted 22 18 32 22 DPO rotate?

define plotter options N

N

Y

Y


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B. Standard Plots

1. RE spectrum in 2. PJ plot jobfile (see above) 3. check DPO, CY, and MAXY 4. EP, get spectrum on screen as wanting to plot and enter U to update plot region 5. use X and S inside EP, or exit EP and use PX 6. remember to cap pens on pen plotters when finished

C. Stack Plots

1. Start by RE'ing bottom spectrum 2. PJ plot jobfile (see above) 3. check DPO (plot x-axis now, and rotate if want portrait stack), CY, and MAXY (title height) 4. check X0,Y0 for initial plot (use OP if using STACK.AU; manual plotting is recommended) 5. use EP and U to update plot region; exit EP 6. plot first, bottom spectrum with axis and title using PX 7. RE next spectrum 8. change DPO for no axis or title plotting 9. increment X0,Y0 10. plot using PX 11. goto 9 and continue for all spectra

12. remember to cap pens on pen plotters when finished

IV. Data Transfers A. UW Network

Windows NT - Backbone file server network for PC�s - completely transparent to users, data is uplinked to a Snap server NMR-Link - Bruker file transfer network; bidirectional - writes files in Bruker image format (widely read by NMR software) - uses PC AT�s to automatically send FASTRAN data to WinNT server FTP - File transfer protocol; generic file transfer between many platforms NFS - Sun�s Network File system (must use FTP in our facility to transfer)

(All FID files must be stored on AC spectrometer hard disk by using WR <filename> before the transfer.)

B. NMR-Link

• NMR-Link exports and imports Bruker binary (and with a non-default setup, ASCII) data. The two commands used in this facility are:

topc filename.ext to send files to the PC network

frompc filename.ext to receive files from the PC network

The above two commands are always issues while sitting at spectrometers or the datastation, with a PC-AT providing dedicated file servicing.

File Transfers & Backup Page 33

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• NMR-Link at an AC Spectrometer

When at a spectrometer, the commands are run from the 2nd session, toggle to/from with the <cntl>-X command.

C. FTP to/from NMRSnap

� Login to NMRSnap.chem.wisc.edu from any FTP-capable computer (see postings in lab for password).

� The only directory that has write priviledges is \\nmrsnap\DataArchives\temp

V. Formatting Floppy Disks � Insert a blank 3-1/2" floppy disk in the disk drive and turn the drive ON. MO to exit to the monitor

from DISNMR. DSP ON will toggle display to the monitor. Then, do as follows:

*ADAKET <ret> SELECT FUNCTION? F (to format a floppy disk) ERASE DATA? Y <ret> ...formatting begins and takes ~45 seconds... M (to exit to the monitor) DIR=F1 (to verify formatting) LOAD ADAKOS

� Reboot the computer by STOP-CLEAR-DISK. If DISNMR is not loaded automatically, type DISR91, followed by <ret>.


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6. 2nd Order Shimming and High Resolution Check-Out by cg fry: created 12/22/94 � updated 11/1/96

I. 2nd Order Lock Shimming (Match Tuning) on Z2 � Make sure the lock is not saturating. Check by watching that the lock signal increases consistently as

increase lock power. Once the lock signal drops or stays steady with increasing power, back off the power by at least 20%.

� Adjust lock level to one square down from the top of the display with LOCK GAIN. Optimize lock level with z-coarse, z-fine, z2-fine, and z-fine.

� Change z2-fine by 20 units in one direction. Optimize lock level again with z-fine. If newly optimized lock level is lower than the previous one, try changing z2-fine in the other direction.

� Keep changing z2-fine by 20 units in the same direction and optimizing lock level with z-fine, until a maximum has been found. Set z and z2 to this maximum setting.

� Lower lock level to one square below top of the display with LOCK GAIN.

II. High Resolution Check-Out [o-Dichlorobenzene (ODCB) with 3% TMS in d6-acetone is used as High-Res check-out standard.]

A. FID Shimming (Line Tuning)

� After 2nd order shimming (see Section I above), RJ ACETONE.1DJ (for d6-acetone); GS; <ctrl> H; FT; EP; <ctrl> R; BP; <ctrl> B; phase using C- and D-knobs; M (memorizes phasing); <ret> (exits EP); TR = 2 or 3 (if presently in Job 1); EP; find TMS peak, then R <move cursor> R to define a window with SW = 150-200 Hz in which TMS peak is positioned approximately half-square off the center; move the cursor to TMS peak; <ctrl> O (to set O1 and SW for TMS peak).

� <esc> to Acquisition Parameters display. Check SW = 150-200 Hz, SI = 16K (total data size for FT) and HZ/PT = 0.018-0.024 Hz. [i.e., HZ/PT = 2 SW / SI.]

� TD = 4K (total number of data points for FID) and check AQ = 15 sec. If AQ > 18 or AQ < 13 sec, then AQ = 15 to automatically set TD. [i.e., AQ = TD / 2 SW.] Remember to use SI > 2 TD for zero-filling!

� RD = 2-4 (sec); <esc> four times (returns to FID/Spectrum display); GS; increase Vertical Display to make FID signal fit the entire display; change z2-fine by 2 units and optimize FID signal by changing z-fine by 2 units at a time at each new z2 setting; keep optimizing until the maximal optimization is reached; <ctrl> H.

B. Window Set-Up and Acquisition

� Switch to Job 1 (the original spectrum display of ODCB); EP; R <move cursor> R to define a 150-200 Hz window which contains all the resonances for ODCB; move the cursor to the center of the display; <ctrl> O (to set O1 and SW for this region).

� Make certain that SW = 150-200 Hz, SI = 16K, HZ/PT = 0.018-0.024 Hz, AQ = 15 sec, and RD = 2-4 sec. AQ > 15 s will actually work better since (resolution) ~ 1/(2*AQ).

� NS = 8 (scans); ZG; WR <filename>; transfer FID with TOPC

� Or, if desired, store FID and work it up on the AC console as follows:

2nd Order Shimming and HR Check-Out Page 35

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� After storing FID with WR <filename>, FT; EP; <ctrl> R; BP; <ctrl> B; phase using C- and D-knobs; M; and check if you have split all the peaks into small doublets!

C. Lorentzian to Gaussian Line Shape Transformation (Resolution Enhancement)

� RE <filename> (to read FID for ODCB obtained as above); LB = -HZ/PT / GB (Note that LB is a negative number; GB is the fraction of the display occupied by the FID when Vertical Display is set to 512; e.g., if HZ/PT = 0.018 and GB = 0.5, then LB = -0.036); EF (or EM + FFT on data station); and, phase the spectrum. See also Primer chapter, section on Lorentzian and Gaussian apodization.


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7. 1H NMR on AM-500/ACs by cg fry: created 12/22/94 � updated 11/1/96

I. Sample In � Put the sample in by using LIFT and LIFT OFF keys.

LIFT: lifts the lock standard (or the sample) from the probe. LIFT OFF: lowers the sample into the probe. SPIN: starts spinning. SPIN RATE: to change the spin rate. Set this to ~15.

II. Shim Control Module (SCM): Locking on AM Spectrometers � <ctrl> L: toggles between ON and OFF of the lock signal and FID.

� <ctrl> D: toggles between ON and OFF of the grids on the display.

� Find the lock signal (hit LOCK if it is flashing) and center it on the screen by using FIELD.

SWEEPAMPLITUDE: 18:00 narrow range (8 o'clock in WP console) � use to lock

25:00 wide range (9 o'clock in WP console) � use to look for lock signal.

SWEEP RATE: to control the sweep rate on screen. Set it at ~13.

DUAL SWEEP: displays two traces of the sweep.

LOCK PHASE: if necessary, use it to adjust the phase of the lock signal.

DUAL SWEEP display sweep in both directions; very useful for adjusting lock phase.

Set LOCK GAIN to 105 and adjust LOCK POWER to fit the lock signal to the full screen (or at 35 for CDCl3).

LOCK POWER: 0-60 (operating range before lock: 10-40); start with 19-35 (35 for CDCl3). Make sure you are not saturating!

LOCK GAIN: 0-175 (operating range before lock: 90-120); start with 105.

� To lock, LOCK. If the lock level�the lock signal which is now leveled out and moving back and forth horizontally on the display�does not increase and go off the screen, then FIELD and turn the cursor one way or the other very slowly until the lock signal shoots up and off the screen. If this does not work, go back to the original FIELD value and increase the LOCK POWER a bit, and repeat. The LOCK POWER decreases by 10 units automatically when locked.

� After shimming, LOCK POWER should be: 0-5 for acetone and acetonitrile 2.5-7 for benzene 10-20 for CD2Cl2, CDCl3.

� Use WSH <filename> to store a shim file; RSH <filename> to read the shim settings back into SCM.

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III. Coaxial Shims � All shims can be drastically affected by the quality of the sample tubes. The stockroom 507 tubes

will work ok for routine 1H (expect to spend extra time with the shims, however), but 527 to 535 tubes are recommended for best, consistant shimming.

� Shimming can be done by starting at any set of z-z4 shim coils. Do not change z4! First, match tune z and z2 using the fine control. When match-tuning, change z2 by 20-30 units at a time. Next, match tune z3 (change z3 by 50-100 units at a time) with concurrent adjustment of z and z2.

� When shimming is done, lower the lock level down to one grid below the top of the screen by adjusting LOCK GAIN and <STD BY>.

Note: (STOP-CLEAR-�DISC if the console does not respond. This will reboot the computer.)

IV. Nonspinning Shims (Not necessary for routine 1H NMR; do if spinning sidebands get too large, ~>1%)

� SPIN OFF causes the lock level to drop, so increase LOCK GAIN. If this drop in lock level is one or two grids, this indicates that nonspinning shims are not far off from their optimum.

� Maximize the lock level by adjusting x, y, xz, yz, and higher orders in the following order:

x; y; xz; yz; x; y; xy; x2-y2; x; y; xz2; yz2; x; y.

Change x and y by 5-10 units, and xz and yz by 50-100 units.

There can be a significant interaction between xz and yz with x and y respectively (i.e., 2nd order shimming may be necessary; similar also for higher order shims that include z).

� SPIN and maximize the lock level by adjusting z and z2. <STD BY> to take a spectrum.

V. Taking 1H NMR Spectrum (The procedure is basically the same as in WP-270 case.)

� RJ CDCl3.1DJ (for CDCl3); SW = 5500; O1 = 8000 (-0.5 to 10.5 ppm); II (initialize interface); GS; RG (adjust if necessary); <ctrl> H; ZG; WR <filename>; FT; EP; . . . so on.

� EP (Expansion and Phase) mode:

<ctrl> R returns to the whole spectrum display.

BP to phase the spectrum with respect to the tallest peak in the current display.

<ctrl> B returns to the expanded spectrum display. C-knob for coarse phasing (use it to phase the tallest peak) D-knob for fine phasing. End with M (memorize).

G sets the reference.

: toggles between Hz and ppm.

E toggles between three sets of cursor information displays.

R �� R dual cursor mode; displays region defined by R <move cursor> R.

X updates and plots the current display.


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I integration. Z <move cursor> Z defines an integration region. + increases vertical scale twofold; - decrease twofold. C- and D-knobs phase. M toggles between ON and OFF of spectrum display. Plot integration using S. End with <ret>.

F horizontal expansion based on two frequency limits: F1 and F2. <ctrl> B returns to the previous spectral region.

N horizontal expansion based on Hz/cm.

U updates the current display as the fixed (plotting) region.

S section plot.

Y redefines CX and CY (plot x and y sizes in cm; CY=0 autoscales vertically).

V plots X-axis.

M to set minimum intensity for peak-picking.

<ctrl> O resets O1 and SW with respect to the cursor position in the current display.

D dual display.

<ret> exits to Command Interpreter.

VI. Plotting � DPO (digital plotter options) and answer to the following:

DRAW X-AXIS? YOFFSET? -2.5 (will print the integration on plot; use -1 if you don't want this.)

MARK SEPARATION? 0.1P (or what you would prefer to use) MARK CM = -0.2

PEAK PICKING ON PLOT (P = PPM; H = HZ; N = NO)? any

DRAW Y-AXIS? N PARAMETERS? N (or Y) ROTATE? N

TITLE? Y (or N) .....> (put a title or a message up to 80 characters; <ctrl> L moves to the new line)

CENTER TITLE? N (or Y)

CX = 36 (or some other number <36. CX is the limit of the horizontal axis of the plot in cm.)

CY = 20 .........(or some other number <21. CY is the height of the tallest peak in the plot in cm. 0 autoscales the vertical axis.)

� Note that DPO, CX and CY can be set before or after the crude spectrum is processed in EP subroutine.

� PX to plot the spectrum. <ctrl> PT terminates the plotting.

VII. FID Shimming (Line Tuning) � Use for unlocked (nondeuterated solvents) samples. When taking a routine 1H NMR spectrum, line

tuning is usually not required. Line tuning often achieves the best resolutions, however. Line tuning is required for High-Res check-out.

� Line tuning procedure is basically same as in 2nd Order Shimming Chapter. However, the following tips will make line tuning easier and quicker:

� Change z2 by 20 (not 2) units first, then optimize the transient FID signal (in GS mode) by changing z by 2 units at a time. Repeat with smaller change in z2. Also, use AQ = 15 sec to set SW and TD.

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VIII.Baseline Correction � Not necessary when taking a routine 1H NMR spectrum.

� K, then 0 for the zeroth order correction. Align the left hand of the baseline with the cursor, then type 0 again for higher order corrections (A-knob for first-order, B-knob for second-order, etc.). End with M.

IX. Linefitting � Not necessary when taking a routine 1H NMR spectrum.

� Obtain one-peak expansion by R <move cursor> R command, then type J. Answer to the following options:

FUNCTION: LORENZIAN

CONVERGENCE LIMIT: 0.8 (or try some other number) <ret>


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8. 13C NMR on AM-500/ACs by cg fry: created 12/9/94 � updated 5/2/96

I. Sample In � After putting the sample into the probe, turn the air valve to VT reserve (air - routine heteronuclear;

N2 - low temperature NMR) and cap the probe. Adjust AIR FLOW to ~5 if necessary (this cools the decoupler).

AIR FLOW: 0-15 range (instead of 0-5 on WP-270).

� Lock as usual before probe tuning (shim after tuning the probe).

II. Probe Tuning (not needed on AC spectrometers)

� This is an electrical adjustment of the probe, which can affect the shims but is not part of the shimming process.

� Make certain that there is a 2dB attenuator placed on the POWER OUT channel! This is important in creating pulse sequences with correct pulse widths.

RJ C13.SET; II; ZG (not GS) to create the rapid frequency pulse (~200 pulses/sec) for the probe tuning.

BB Preamp: 2, D and 3 for 13C (125MHz); see plate on preamp for appropriate settting.

Initial Setting: tuning slide (z) at 7995.5; matching slide (z2) at 992 for 13C; see card on probe for correct settings.

� Minimize the value in LOAD MATCHING METER on the side of the console by match-tuning the two slides under the probe. When match-tuning, adjust only the last digit on the slides by 0.5 unit at a time. Note that the change in the tuning slide acts in opposite direction to the change in the matching slide.

� For 13C, the final LOAD MATCHING METER reading should be approximately 0.02mA. For other sensitive nuclei, it should be <0.05mA.

� <ctrl> H to halt acquisition and to go on to decoupler tuning.

III. Decoupler Tuning (not needed on AC spectrometers)

(The principle is same as in probe tuning; however, the decoupler operates on the 1H at 500MHz. The objective of this tuning is to minimize the reflected power.)

� Set the decoupler power (DP) approximately 10 times higher than the power actually used for decoupling. The DP value represents an attenuation; thus if you want to run with DP=16, set DP=6 for the next step. Note that DP=0 is the decoupler at full power, which can damage the probe!

� RJ C13ACET.1DJ (for acetone-d6; CPD; II; DP = 10H; then, go under the probe and find two screws M (matching) and T (tuning) for the decoupler. Carefully adjust T (longer screw) first and, if necessary, M until the intensity of DEC REFLECTED light on the switch panel decreases and finally goes off.

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� The switch panel has three positions: (1) DEC FORWARD - used to verify the decoupler output; (2) DEC REFLECTED - initially this red light is on, but want it "off" ultimately; and (3) OBS REFLECTED - verifies output on the X transmitter (e.g., 13C output).

IV. Shimming (Exactly same procedure as in 1H NMR, but with only one difference: somewhat higher lock power is required for the heteronuclear case because two independent coils�heteronuclear observe coil and decoupler 1H coil�are used instead of one.)

� DP = 20H

� Match-tune z, z2, and z3. Do not change z4. Do not use Autolock or Autoshim.

Autolock: usually lock power is increased (used for long runs).

Autoshim: used for overnight runs. (hit z, then, Autoshim to activate.)

� <STD BY> when shimming is done.

V. Taking 13C NMR Spectrum � RJ C13ACET.1DJ; SW = 28900; O1 = 13350 (-10 to 220 ppm); O2 = 7500 (for CDCl3; O2, not O1,

varies from solvent to solvent!); CPD (composite pulse decoupling sequence); NS = -1 (infinite number of scans, or set to number if known to give sufficient sensitivity); II; ZG.

� From time to time, transfer the acquired FIDs to another Job and process it to see if enough FIDs have been collected. This can be done in the following manner:

� TR = 3 (if FIDs are being collected in Job 1 or Job 2); LB = 2; EF; EP.

� Repeat this until satisfactory sensitivity is obtained. Then, <ctrl> H; WR <filename>.

VI. DEPT � While the normal 13C spectrum is being acquired in Job 1, switch to Job 2 for setting up the DEPT

experiment.

� RJ DEPTACET.1DJ; SW = 28900; O1 = 13350 (-10 to 220 ppm); O2 = 7500 (for CDCl3; O2, not O1, varies from solvent to solvent!); do not II if the data are being accumulated in another Job

� AS DEPT.AU � parameters for DEPT.AU microprogram:

D1 = 3 (sec) S1 = 0H (max. decoupler power) P1 = 12.5 (90° pulse in µsec in decoupler channel; see pulsewidth sheet for newest calibration) D2 = 3.3 to 3.6M (1/2J) P2 = 25 (2×P1) P3 = 9 (90° pulse in µsec in observe channel; see pulsewidth sheet for newest calibration)

P0 = 18.7 for DEPT-135: 1.5×P1 = 135° pulse; CH and CH3�s are positive, CH2�s are negative. 12.5 for DEPT-90; CH positive, CH2 is zero (tuning experiment), CH3 close to zero.

P4 = 18 (2×P3) S2 = 20H (decoupler power) RD = PW = 0 DE (set automatically by the instrument) NS = -1


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DS = 0 P9 = 110.0 (see pulsewidth sheet for newest calibration of 90° at DP power)

� Then, II; ZE; AU to run the DEPT microprogram.

� Check the acquisition from time to time as in the normal case as shown above. Then, <ctrl> H; WR <filename>.

Caution: Before you leave, make certain that you have turned OFF the decoupler! If you have not, use PO to turn it off.

VII. Data Processing � Process the FIDs of the normal spectrum in the following manner: Set LB = 2 (Hz); EF; DPO

(digital plotter options; to set the plot parameters); CX (the width of the spectrum in cm); CY (the height of the tallest peak in the spectrum in cm; 0 autoscales); EP (phase the spectrum, put it in memory and plot); <ret>; MI (minimum intensity in cm; try 1 cm first); activate the printer; PP (peak pick; prints the peak positions on the line printer and/or on the display); SR (spectral reference: the numeric value of O1 for 0 ppm).

[CX = 20; CY = 13 for a thesis-size spectrum.]

� Process the DEPT FIDs in the same manner as above, i.e., set LB = 2; EF; DPO; CX; CY; SR (set this equal to the previous numerical value to align both spectra); EP (phase, save, and plot using S to put the DEPT spectrum just above the previous normal spectrum).

� To improve the apparent S/N ratio, one can use PS (power spectrum) command. This will square every data point in the spectrum so that all peaks (and noise!) will be positive. Also note that there may be some broadening at the base of peaks if a dilute sample is used.

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9. AM/AC QuickGuide for X-Nucleus Experiments by cg fry: created 05/24/95 � updated 20.Aug.2002

^L - toggles DISNMR display AI=0 is normal setting; if =1 can get strange display effects

I. Probe Changes (not needed on AC spectrometers)

1. Check underneath magnet to determine what probe is installed: � BB probes have three rf cables attached: BB, H1, H2(lock) � 1H probes have only two rf cables: H1, H2(lock) � The probe diameter is written on the front plate of the probe where the cables are attached. If the probe needs changing, find the replacement probe and proceed as follows: 2. Turn heater (on console) off 3. Turn Variac off if not performing low-temp VT 4. Turn lock off, stop spin, and remove sample 5. Unhook: all rf cables thermocouple hookup (Omega junction) heater air connector remove probe (note rotational position in magnet before removing) 6. insert new probe into magnet with same rotational position 7. insert sample (if 10mm probe, use 10mm position on sample gauge) on AM-500, remember to move air switch back to spin position indication of sample spinning (press spin on SCM) is easiest method of assuring that sample is seated

correctly 8. If BB, make sure 2 dB attenuator is in-line at connector above reflection meter on side of console Remove attenuator for 1H work

II. Probe Tuning (not needed on AC spectrometers)

1. Read in correct tuning job file: RJ nuc###.SET ;e.g., RJ XE129.SET II ;initializes hardware interface ZG Adjust tune and match to minimize reflection on meter on side of console 2. Check preamp setting are correct for your nucleus


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III. Acquire Data 1. Lock with appropriate lock power; then adjust only lock gain!!

� If running unlocked, press SWEEP OFF (light should be on) on SCM

2. Shim Z and Z2. Start with Z and maximize the lock signal. Then shim Z2 the same way. Return to Z and remaximize. Repeat these steps if the Z2 correction was substantial.

X, Y (these two while not spinning) and Z3 and Z4 may have to be adjusted if you are using 507 tubes (stockroom tubes) on the 500s, or poor quality tubes/poor solutions (i.e., suspensions) on the 300s.

� If running unlocked, shim on the 1H fid (RJ CDCL3.1DJ, then GS); set TD such that you do not see baseline, and shim for large integrated number (shown on 2nd line at top). Make sure RD is long enough: if you stop acquisition, then restart GS, the number should stay constant, if it decreases, increase RD.

� If running unlocked, note field setting on SCM, and always return to this setting

3. Read in X-nucleus job file: RJ C13ACET.1DJ ;see the info. sheets posted on the spectrometers then ZG ;adjust RG if 1st scan larger than 2 divisions total use TR to transfer to another region EF to transform (LB controls EM multiplication) EP to phase, expand, etc. IV. Transfer Data Use WR filename.ext to store data on spectrometer ^X switch to second session topc filename.ext ^X switch back to DISNMR

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10. Saving Group-Specific Jobfiles, Shim Files, etc. on AM/AC Spectrometers

by cg fry: created 11/16/95 � updated 3/16/96

I. Formatting 8" Floppies with ADAKOS 1. Exit DISNMR with command: 1. MO<ret> 2. Place commands on screen: * DSP ON<ret> 3. Power up floppy drive 4. Run floppy utility: * ADAKET<ret> 5. To format floppy, use: + F<ret> 6. Wait for format to complete, then: reboot ASPECT using STOP CLEAR DISK buttons

II. Create or Modify .EXE File A .EXE file will perform the series of copy commands needed to perform the backup. 1. To create or edit the .EXE file, use the following command from inside of DISNMR: EDIT CGF.EXE<ret> � Type in commands, looking something like the following: COPY LOWTEMP.1DJ/=F1:Y COPY LOWTEMP.SHIM/=F1:Y COPY HIGHTEMP.1DJ/=F1:Y COPY HIGHTEMP.SHIM/=F1:Y COPY VTOVERNG.AU/=F1:Y COPY PLOTIT.AU/=F1:Y � Use the arrow keys to move around, and the Del key to delete charaters � The only other key sequences needed is <ESC>K to remove current line. 2. Use <ESC>S to exit and save changes Use <CNTL>Q to quit without saving changes

III. Run Backup to Floppy 1. Make sure floppy is inserted and floppy drive is powered up 2. Exit to ADAKOS: MO<ret> DSP ON<ret> run .EXE: EXE CGF.EXE<ret>


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11. Variable Temperature Measurments by cg fry: created 11/11/94 � updated 4/27/2001

I. Variable Temperature on Phoenix (Bruker AC-250) A. Initial Equipment Checks

� Note that the actual sample temperature can be 20 K or more different from the VT set point!

� See the calibration curve on the board in the spectrometer room to gain an appreciation for how divergent set points and actual sample temperatures can be.

� NEVER go closer than 5°C�actual temperature�to a solvent�s boiling point. � Never go closer than 25°C�set point�to a solvent�s boiling point without performing a

temperature calibration immediately prior to the measurement in question. � Do not exceed the range of temperatures for the Nalorac DB250-5B probe: -150°C ≤ TE ≤ +120°C � It is the user�s responsibility to ensure that VT experiments are perfromed safely!

� Use the ceramic spin collar for experiments 35°C ≤ TE ≤ 0°C. � Be careful with this spinner, which costs ~$500!

� Use N2 gas for all VT experiments. � Switch the wall gas from air to N2.

� Use the LN2 tank usually stored next to the magnet for TE ≤ 305K (do not use dry ice/ethanol/acetone baths; the LN2 tank works and costs as little or less). � After filling the tank, make certain the o-ring and ring-clamp are securely tighened. � hook the black hose in place of the green hose onto the probe � make sure the wall gas is switched from air to N2

(you must perform the following checks to insure equipment will not be damaged) � make sure heater and thermocouple (TC) are inserted correctly into probe � after checking above, turn on Controller � turn on heating tape variac (in corner next to magnet) if running ≤ 210K, or ≤ 250K for >1h � turn on the LN2 heater (not the control heater); � changes in LN2 heater setting (table below) optimize the heater inside the LN2 tank to maximize

the hold time for the tank; use the following as initial settings:

LN2 heater setting Temps. available ~ hold time 20 ≥ 220 K > 12 h? 40 ≥ 190 K > 9 h? 60 ≥ 150 K > 6 h?

(please give lab director feedback how settings and holdtimes work)

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B. Changing set-point

(perform only from AC-console; the arrows on the controller will change the setpoint, but then the setpoint for your experiment will not match the stored TE)

� Enter TE <ret> type in new temperature in Kelvin, then <ret> � Enter PASC TESET <ret> � SP on controller should flash then change � wait until temp fails below set point, then turn on main heater

C. Taking data

� Wait ≥ 10 minutes for the temperature to equilibrate (longer with larger temp excursions). � For quantitative data, you must retune the probe at each temperature. Probe tuning may change for 30

or more minutes following a temperature change. � For X-nucleus work, the decoupler must be retuned (including non-quantitative data) after any change

in temp > 20°. � Plan your experiments so a minumum of 30 min is included at the end to allow the magnet and probe

to re-equilibrate to ambient temperatures for the next user. It is your responsibility to stop experiments early enough so you do not interfere with the next users scheduled time.

D. Finishing up

� turn off heater � turn off LN2 heater � replace LN2 hose with green hose with wall N2; do not force the connector�if you do and it is

frozen, you will snap off the ball-joint connector � wait until temp ≥ 250 K � turn off VT controller power � switch back to air from N2 � turn off Variac controlled heating tape

E. Self-tune (if control is erratic)

� turn heater off and wait for temperature to stabilize (if the sample might freeze, leave heater on throughout; self-tune will not be quite as good)

� hold down button until Pr comes up on display

� press until St is on display � press s and t buttons simultaneously � turn on main heater � SP will blink for about 1 minute, then A-T will blink until the self-tune is finished � temperature control should now work well


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II. 1H NMR Chemical Shift Thermometers

A. Low Temperature: methanol (with 0.03% concentrated HCl)

temperature range: 175�330K (�100 to 55°C)

[ ] ( ) ( )283.2346.290.403 MMmeas KT δδ ∆−∆−=

where ∆δM is the chemical shift difference (in ppm) between the methyl and hydroxyl peaks. Refer to A.L. Van Geet, Anal. Chem., 42, 679, 1970.

B. High Temperature: ethylene glycol (neat)

temperature range: 310�440K (35 to 165°C)

[ ] ( )EGmeas KT δ∆−= 24.1028.465

where ∆δEG is the chemical shift difference (in ppm) between the methylene and hydroxyl peaks. Refer to M.L. Kaplan, F.A. Bovey and H.N. Cheng, Anal. Chem., 47, 1703, 1975.

III. Automated VT Runs There are two sets of .AU routines: Phoenix VTAC.AU VTXAC.AU

1. Neither of these routines will provide quantitative data, since the probe will detune with changes in temperture (if you need quantitative data, you will have to retune the probe between each change in temperature). In addition, do not attempt to run VTXAC.AU over a range of temp >20°C, due to probe detuning. Tune the probe at the mid-point temperature for all experiments using these sequences.

2. Setup and shim as normal

3. Enter: AS VTAC.AU for VT 1H or coupled X-nucleus on Phoenix (AC-250) AS VTXAC.AU for VT X-nucleus with 1H decoupling on Phoenix (AC-250)

4. The only parameters to change are:

D1 = (enter in seconds)wait time following temp change to come to equilibration (usually > 600 secs)

VD normally use default VDLIST.001 (i.e. just type return) enter list of temperatures in K end by typing EN

NE = # of temperatures in VDLIST

for VTX: D5 = 5M (enter as shown, or 0.005 since we want 5 milliseconds)

5. If you are running unlocked, see me about other routines that will not try to autoshim.

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IV. Example Automation Routine Listing VTXAC.AU ; Measure X w 1H-decoupled spectra at temps specified in VT list ; Autoshim between temps ; C.G. FRY - 25 OCT 94 1 ZE 2 TE ;read in 1st temp from VT list 3 PASC TESET ;set temp in B-VT 2000 unit 4 D1 CPD ;hold time for VT stabilization (>5 min) TU4 ;autoshim Z1 and Z2 (takes about 2 mins) 5 ZE 6 GO=6 CPD ;uses RD and PW from normal job file 7 WR #1 ;1st temp .001, then increments by 1 8 IF #1 9 IN=2 ;loop back NE times for other temps 10 D5 DO ;D5=5M to turn decoupler off 11 EXIT ;read in normal job file, e.g. CDCL3.1DJ ;enter AS VTAC.AU ;type in VT list, use EN<ret> to end list ;make D1 long enough for good temp stabilization ;NE must = # temps in VT list ;D5=5M (type in M following the 5, to set to milliseconds) ;enter filename WITHOUT extension


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12. HOMODEC on AM/AC Spectrometers by cg fry: created 12/20/94 � updated 3/22/96

I. Setting Up � Obtain a normal 1H NMR spectrum. Save file using WR command.

� Choose all irradiation frequencies that you want to irradiate, e.g., you have two such frequencies (vide infra).

� Frequencies can be selected by moving the cursor in EP mode to a desired position in the spectrum, followed by O2 and L. On the first frequency, the spectrometer will offer FQLIST.001 as a default list name: you may hit <ret> to accept this name, or enter a new name of your own. All other frequencies, require just O2 and L. Repeat this until you have chosen all the irradiation frequencies.

� <ret> to exit EP to Command Interpreter.

� AS HOMODEC.AU <ret>

D1 = 1 (sec) O2 FQLIST.001 1 = (first irradition frequency) <ret> 2 = (second irradiation frequency) <ret> ...and so on, if you have more irradiation frequencies. 3 = END

(Note that this frequency list is automatically set if you have chosen the frequencies with O2 and L commands in EP subroutine.)

S3 = 20L (decoupler power; use 20L to 10L; the lower the number is, the higher the power) RD = 2.0 (0 can be used, but better if 1 or 2 sec is used) PW = 2.5 (whatever is used in job file) DE = (dead time: set automatically by the spectrometer) NS = 8 (number of scans) DS = 2 (number of dummy scans; 0 works fine in most cases) NE = 2 (number of experiments = number of irradiation frequencies)

II. Taking 1H NMR Decoupling Spectrum � EXPT does not work!! Calculate the experimental time as (RD+AQ)*(NS+DS)*NE*(~1.2). The factor 1.2 estimates the amount of disk access time spent. Use a larger number like 1.5 if

RD+AQ<2 s, or 1.0 if RD+AQ>10 s. If the experiment is too long, adjust NS, DS, AQ or RD accordingly.

� ZE; AU <ret>

INPUT FREQ. LIST FILENAME #1: FQLIST <ret> INPUT FID FILENAME #2: KL (any filename with no extension) <ret>

� FIDs are now collected and stored automatically into FILENAME #2's with extensions 1 through the total number of frequencies (i.e., KL.1 and KL.2).

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III. Processing FIDs � Work up the first irradiation spectrum as follows:

LB = 0; RE KL.1 (to read FILENAME #2.1); EF (do EM plus FT); EP; <ctrl> R; BP; <ctrl> B; phase the spectrum with C- and D-knobs; M; <ret>.

� Save the resulting irradiation spectrum, e.g., WR SKL.1.

� Process other irradiation spectra using EFP (EM + FT + PK) as follows:

RE KL.2 (to read FILENAME #2.2); EFP; WR SKL.2 (the second irradiation spectrum; any filename can be used).

Or set NE = # files, and use AU FT.AU to automatically process all the files.

� To plot these spectra, set DPO, CX, and CY. Then, PX. In WP console, USR FASTU to send the stored spectrum to the PC data stations.

� PO (decoupler power off) before leaving the spectrometer.

IV. Listing of Automation Routine ; HOMODEC.AU ; HOMONUCLEAR DECOUPLING USING ONE FREQ. LIST AND ONE POWER SETTING. 1 FL #1 / INPUT FREQ. LIST ; READ IN FREQ. LIST AND INITIALIZE POINTER 2 ZE 3 D1 HD O2 S3 ; TURN ON HOMODEC. WITH POWER S3 AND SET ; O2 FREQ. FROM CURRENT FL LIST, INCREMENT ; FREQ. POINTER. 4 GO = 4 ; ACQUIRE DATA 5 WR #2 ; INPUT FID ; STORE FID 6 IF #2 ; INCREMENT FILE EXTENSION 7 IN = 2 ; LOOP FOR NEXT EXPERIMENT 8 D1 DO EXIT ; EXIT WITH DEC. OFF ; PROGRAM REQUESTS A FILENAME FOR FREQ. LIST AND FID STORAGE ; D1 = 1 SEC TO SET FREQ. ; S3 = DESIRED DEC. POWER ; NE DEFINES NUMBER OF ITEMS IN FL LIST = NO. OF EXPERIMENTS ; USE RD AS DESIRED AND AT LEAST 2 DUMMY SCANS.


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13. 1H Spin-Lattice Relaxation, T1 by cg fry: created 12/11/93 � revised 4/1/96

I. Discussion Spin-lattice relaxation is nominally an exponential process, and becomes important for any quantitative, exchange, or cross-relaxation type of experiment such as NOE. The relaxation from zero magnetization and from an inversion pulse are both shown in the figure below.

Often a quick determination of the T1 is sufficient; for most liquids, use the rapid, inversion-recovery method in Section II below. This method involves finding τn for the 1H of interest. For more accurate determinations, use the procedure described in Section IV.

II. Rapid Determination of T1 by Inversion Recovery Null Method

(good for any sample�e.g. any liquid�but is semi-quantitative at best)

� Obtain a reasonable quality 1H spectrum. Locate protons of interest for visual observation.

� 180° and 90° pulse lengths must be reasonably accurate. If your sample is highly polar (e.g., ionic water solution), you must calibrate beforehand. Otherwise, if tuned correctly, prior calibrations can be used.

� Decrease SI and TD (keep SI=TD) to 8k if possible.

� Set AI=1 (answer Y to REINITIALIZATION?), NS=1.

� Reacquire fid with ZG, EF, and phase manually in EP.

� AS T1NULL.AU

P2=16 µs (180° pulse length; see calibration sheet on wall)

VD VDLIST.001 1 0.01 s (to start with) 2 EN (or [)

RD = 0 PW = 8 µs (90° pulse width) DE (set by spectrometer)

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NS = 1 DS = 0

� NE = 1

� AU <ret> (or equivalently AU T1NULL.AU<ret>)

� VD=0.01 should give large negative peaks. Lengthen VD until proton peaks of interest are nulled (slightly shorter VD should give negative peaks; slightly longer should give positive peaks). For the nulled VDn, T1 ≅ VDn/ln2 = VDn*1.44.

� Find VDn for other protons of interest.

III. Rapid, approximate measurement of T1 by Repetition-Rate method

(only for samples where T2 << T1, e.g. solids [but not for nominal liquids; see previous section])

1. if present, RJ H1REPRAT.005 or .010 (for 5mm or 10mm probes, respectively), or:

set NS = 1, DS = 0, RD = 0, SW = 20ppm (10000 on AM500, 7200 on AM360), SI = 16K, AQ = 0.1s, and PW = 90° pulse length (see calibration sheet next to spectrometer; or recalibrate 90° - see section xxx)

2. set O1 close to, or on peak of most interest (use EP, set cursor on peak and enter O1 then M)

3. check RG (I like to reduce TD = 1k to see beginning of FID clearly, with RD set to compensate for shorter AQ), then wait at least 5T1;

4. set absolute intensity mode by typing AI, answer Y if queried REINITIALIZE?

5. ZG, then EF, go into EP, and P; after good phasing type M and <RET> out of EP

6. adjust vertical scaling so 2<(height of peak of interest)<4 divisions; note height and scaling

7. set DS = 8, and RD so that RD+AQ ~ T1/2

8. ZG, then EFP

9. adjust RD until height of peak of interest is half height noted in step 6 (i.e. same height with scaling doubled)

10. T1 ≅ (AQ+RD)/ln2

IV. Quantitative measurement of T1 by Inversion Recovery Method

A. Comments

� A reasonable estimate of T1 must be known to correctly setup: use the Inversion Recovery Null Method (Section II).

� 180° and 90° pulse lengths must be accurate. If your sample is highly polar (e.g., ionic water solution), you must calibrate beforehand. Otherwise, if tuned correctly, prior calibrations can be used.

� For precise measurements, use D1 = 10 T1; D1 = 5 T1 will provide reasonable values if experimental times get long. The number of VD values can also be decreased to lessen experimental times ≅ NS*NE*(2*D1)*(second # in VC list)

B. Acquisition Set-up


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1. setup normally as for high-resolution 1H spectra; make sure RG is set correctly (not too big!), and NS = 8

2. set-up two lists used by the automation routine: a VC (variable counter) list, and a VD (variable delay) list

i) enter the variable delay editor by typing: VD<RET>

� use the default name VDLIST.001, or enter a unique name you can recall later � enter the delays; Table I giving a reasonable grouping � enter a number followed by S, M or U for seconds, milliseconds, or microseconds � end the list by entering ESC

Table I. A reasonable grouping of VD delays for Inversion-Recovery T1 sequence, where T1

r is the T1 estimate (usually from repetition-rate experiment.

0.01T1r 0.5T1

r 10T1

r 10T1r

2T1r 5T1

r 0.3T1

r 0.1T1r

10T1r 0.01T1

r 0.7T1

r 1.4T1r

1.0T1r 10T1

r

ii) enter the variable counter list editor by typing: VC<RET>

� use the default name VCLIST.001, or enter a unique name you can recall later � the first line must contain the number of delays in the VD list = NE � the second line contains the number of complete cycles of NS and VD to do; for 1H T1 = 1 � end the list by entering ESC

3. start automation setup for the inversion-recovery sequence: AS INVREC.AU

i) change values for the VC list, or enter <CNTL>-L to list

ii) D1 = 10T1r

iii) P2 = 2*PW, where PW = 90° pulse length (see calibration sheet next to spectrometer, or recalibrate using procedure in section xxx)

iv) RD = 0, DE set by computer, NS = 8 (or multiple of), DS = 0

v) NE = number of delays in VD list

4. experiment time ≅ NS*NE*(2*D1)*(second # in VC list)

5. start automation routine: AU<RET><RET>; enter unique filename with no suffix

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C. T1 Analysis

1. Transfer data to data station using Lightnet (can use wildcard), or enter other session

2. check NE = size VD list

3. set AI = 1, Y if asks REINITIALIZE?

4. RE filename.002 (assuming .002 has longest VD), do EF, then EP, P, and when good phase, M

5. do all FT's by running automation routine: AU FT.AU

enter filename of T1 set, then new name of FT'd spectra (e.g., T1SET and T1SETF)

6. if want plot, go through DPO (Y to both axes, N to parameter list, Y is want TI), CX=CY=10 to 20, MAXY = 10 to 20

7. go into EP, set cursor on top of peak of interest and enter T, then <RET> to accept T1PTS as filename

go to next peak of interest and enter T (no <RET>); repeat for all peaks of interest

8. enter T1 routine: T1<RET>

i) enter PD, N to integrals list, N to VD list, Y to EP list

ii) when see display, enter CT1 to calculate T1

iii) enter PLTD to plot (can change TI and DPO prior to plot)

iv) enter NXTP to go to next peak

v) enter QUIT to exit T1 routine


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14. 1D NOE Measurements by cg fry: created 12/10/94 � updated 3/23/2001

I. Discussion Measurements of NOE�s are nontrivial, but the experiment is so powerful as an assignment aid as to make the learning curve more than worthwhile. Sanders & Hunter contains an excellent discussion and many examples of applications of NOE�s in their text.

The measurement of NOE�s via difference experiments should no longer be attempted, except possibly in some very special circumstances. The NOESY-1D experiment as implemented on the Varian equipment in our laboratory is a significantly (by a lot!) better experiment than NOEDIFF, and NOESY-1D should always be the first choice of experiments. DO NOT ATTEMPT NOEDIFF or NOEMULT unless you have clear reasons for doing so.

General comments about the theory:

� NOE�s in general are positive for small molecular weights, and go negative for high molecular weights. The crossover molecular weight in very approximate terms is MWc o≈ 106 ν , where νo is the 1H frequency in MHz. Thus, for compounds of MW = 1000 to 5000, NOE�s may be very small or completely absent. The experiments should be tried on the smallest (Phoenix) and largest (Brutus) fields available for this MW range.

� For small MW compounds, NOESY experiments are often not successful. 1D NOE experiments can be performed, or 2D ROESY experiments.

� For high MW compounds, spin-diffusion can significantly interfere with NOE�s; keeping the saturation time (D2) small will help avoid the spin-diffusion problems.

� In general, for small MW saturation of the faster relaxing proton(s) will lead to larger NOE�s at the close-by proton(s) than the opposite experiment. Saturation of methyls and methylenes is often best in a small molecule. Experimental measurement of the various T1 values is necessary (see below), and spin geometry can strongly effect interpretations (see next).

� Three spin systems will often give plus-minus NOE signatures (useful for assignments!). E.g., saturating a proton close to a methylene will often give a large positive NOE to the closest proton on the methylene, which then induces a smaller negative NOE on its gem partner (see Sanders & Hunter, pgs. 169ff).

� Chemical exchange will appear in all types of NOE experiments, usually looking much like a negative NOE. Temperature dependent experiments will separate exchange from NOE phenomena.

� Observation of a true NOE can be confirmed using NOE buildup, where the increase in NOE intensity should track proportionally with length of the saturation pulse (D2 in the experiments discussed here) up to roughly T1. See the last chapter of this guide for measurements of T1.

General guidelines for performing NOE measurements:

� Measuring T1 for the protons of interest is highly recommended. Correct setup and interpretation of NOE experiments relies on reasonable knowledge of T1 values. See Chap. 11 for T1 experiments.

� Do not ignore assignments that can be made directly from the T1 measurements. Saunders and Hunter give a number of examples in their discussion about NOE and relaxation (see Figs. 6.14, 6.15, 6.20, 6.21).

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� Saturate both sides of a through-space pair, if at all possible. Make certain, in any case, to saturate the faster relaxing proton in the pair; this should lead to larger, more easily assigned NOE�s.

� For determining NOE�s for a small number of clearly resolved spectral peaks, use the most straightforward experiment, NOEDIFF.AU, as discussed in section III. For many compounds, NOEMULT.AU is preferred. Use NOESY-1D first, before trying the significantly inferior NOEDIFF or NOEMULT experiments.

� For determining NOE�s for a small number of closely spaced multiplets, use NOEMULT.AU, as discussed in section IV.

� For complex spectra, or when decoupler �spill-over� into close-by multiplets cannot be avoided, use the 2D NOESY or ROESY experiment.

� Use of a degassed sample is highly recommended for obtaining accurate NOE's. Qualitative stereochemical information can often be obtained by bubbling N2 gas through the sample just prior to performing the experiment.

� Saturation pulses must not spill over into nearby protons, so care should be given to the choice of saturation power, S3, in both NOEDIFF.AU and NOEMULT.AU.

� Uneven saturation of a multiplet must be rigorously avoided, or selective population transfers (SPT) will be observed to the nuclei coupled to the multiplet. The resulting SPT�s, which have an appearance as shown below for a simple doublet, can be as large or larger than the desired NOE�s, and confuse interpretation. NOEMULT.AU helps avoid SPT�s by spreading the decoupler more evenly over the multiplet than the simpler NOEDIFF.AU saturation scheme.

� Frequency jitter artifacts, giving dispersive difference line shapes as shown above, can be reduced by

increasing the number of scans. NS = 8 should always be used; use NE to increase the total number of scans. Frequency jitters, caused by floor vibrations, and changes in air pressure and temperature will be worse with increasing field, and during daytime hours. Use temperature control if available.

� Use the largest line broadening possible while still preserving chemical information (LB = 1 to 10) to help reduce frequency jitter artifacts.

II. Critical Parameters for NOEDIFF.AU D1 = relaxation time ~ 2-4 T1 [want D1+AQ ≥ 3 T1]

D2 = saturation time ~ T1 for low MW 50 � 200 ms for higher MW (over 5000) [4 to 5 D2 values from 0 to T1 seconds will provide buildup curve]

S3 = saturation power ~ 20L to 60L (lower number is higher power; too much power will spill over saturation into nearby proton, avoid this!; too low power will give incomplete saturation)


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III. Setting Up For Well-Resolved Spectra: NOEDIFF.AU � NOEMULT.AU described in the next section is usually preferred; see comments above.

� Obtain a normal 1H NMR spectrum. Save using WR command.

� In EP subroutine, set the window using R <move cursor> R and <ctrl> O if you wish to obtain NOE

for only that region of the spectrum (use the normal 1H spectrum if folding interferes). Retake the 1d spectrum in this case and save using WR command.

� Choose a control frequency which is far from all the resonances (i.e. saturation pulse will stay away from all 1H resonances). This frequency will be used to record a reference spectrum. Then, find all irradiation frequencies that you want to saturate, i.e. all protons that are close to moiety of interest.

� Select frequencies by: - moving the cursor in EP to desired position in the spectrum (for NOEDIFF.AU to middle of

multiplet; for NOEMULT to top of multiplet peak)

- type O2 then L. For the 1st frequency, spectrometer asks for frequency list name. Press <ret> to keep FQLIST.001, or enter any name with .001 as suffix. For rest of frequencies, position the cursor and type O2 then L. Repeat this until you have chosen the control and all the irradiation frequencies.

� <ret> to exit EP to Command Interpreter.

� AS NOEDIFF.AU <ret> ; automation setup

LO VCLIST.001 1 = 3 (the total number of frequencies you chose including control; 3 in this example) <ret> 2 = EN (can also type [ ) D3 = O.1 (sec) O2 FQLIST.001 (or your own freq list name chosen in EP) 1 = (control frequency) <ret> 2 = (first irradiation frequency) <ret> 3 = (second irradiation frequency) <ret> ...and so on, if you have more irradiation frequencies. 4 = END S3 = 40L (decoupler power; 20L-63L range; the lower the number is, the higher the power) D1 = 2.0 (2-4 T1 for 1H) D2 = 1.0 (T1 for 1H) RD = 0


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PW = (same as 1H job file uses) DE = (set automatically by the spectrometer) NS = 8 (use NE to increase number of scans to give desired S/N) DS = 2-4 (number of dummy scans) NE = 1 (number of loops through FL)

� Larger values of NS, DS, and/or NE will increase the S/N ratio and also the total operating time.

� To make a change to VCLIST or FQLIST, enter VC <ret> or FL <ret>, respectively.

IV. Critical Parameters for NOEMULT.AU D1 = relaxation time ~ 2-5 T1

D2 = saturation time at each O2 ~ 100 ms too long (> 0.1 T1) will allow relaxation within multiplets and lead to reappearance of SPT�s too short (?) will cause frequency modulations and large artifacts

n2 = number in second line of VCLIST; must be ≥ largest number of O2 values used for one multiplet; VC2⋅(D2+D5) = total saturation length ~ T1 for small MW or 50-200 ms for large MW

S3 = saturation power ~ 30L to 60L (use lower power than in NOEDIFF; too much power will spill over saturation into nearby proton, avoid this!; too low power will give incomplete saturation)

V. Setting Up Best-Case Spectra: NOEMULT.AU Set-up is the same as for NOEDIFF.AU except for the two sets of lists: VCLIST and the FQLIST, and S3.

� VCLIST contains the number of multiplets to be irradiated as in NOEDIFF in the first line, but now should also contain the total number frequencies to be irradiated on a single multiplet in the second line. Suppose you have chosen six O2 points to irradiate over for the most complex multiplet. The second line, containing n2, must be ≥ 6; e.g., if D2 = 0.2 s and T1 = 5 s, then set n2 = 25.

� FQLIST now is a series of lists, FQLIST.001, FQLIST.002, ..., FQLIST.00n, where n is the number of multiplets to be irradiated plus one (for the control spectrum). n is the first number in VCLIST.

Each FQLIST is made by entering EP and making the first (always the most complex multiplet), then exiting EP, and re-entering EP for the next list (type in the new name FQLIST.002, for example, following the first O2 and L). Repeat entering EP until all lists are made.


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FQLIST.00n should be the single frequency used for the control spectrum (i.e. on flat baseline)

FQLIST.001 must be the longest list and read in before starting AU (do this by typing FL).

� S3 should be smaller than used in NOEDIFF, if you have run this first. If you see SPT�s, reduce S3.

VI. Acquiring NOE Difference Spectra � The command EXPT does not work; calculate the experiment time manually as:

EXPT NS DS D D AQ NE N f= + ⋅ + + ⋅ ⋅( ) ( )1 2

where Nf is the number of frequencies. If EXPT is too long, adjust NS, DS, or NE accordingly.

� AU NOEDIFF.AU <ret> or AU NOEMULT.AU <ret>

DEFINE FID FILENAME #1: KL (any filename with no extension) <ret>

DEFINE FREQ. LIST FILENAME #2: FQLIST <ret>

� FIDs are now collected and stored automatically into FILENAME #1's with extensions 1 through the total number of frequencies (i.e., KL.1, KL.2, and KL.3).

VII. Processing FIDs � Work up the control spectrum first as follows:

� AI = 1 (absolute intensity); RE KL.1 (to read FILENAME #1.1); LB = 1 (usually set to 1-10 Hz); EF (do EM plus FT); EP; P; phase the spectrum with C- and D-knobs; M; <ret>.

� Set NE = # freqs (# files). Run AU FT.AU to transform all the data:

DEFINE FID FILENAME #1: KL (filename without extension of FID�s) <ret>

DEFINE FID FILENAME #2: KLFT (any filename with no extension for FT�d spectra) <ret>

VIII.Obtaining NOE Difference Spectrum A. via AT (additive Transfer):

� DC = -1 (data constant multiplied to 1st spectrum).

� AT KLFT.ref,KLFT.i1/KLDF.i1 (the 1st difference spectrum is stored as KLDF.i1);

AT KLFT.ref,KLFT.i2/KLDF.i2 (the 2nd difference spectrum is stored as KLDF.i2).

� AT A,B/C is equivalent to [spectrum A x DC] + spectrum B = spectrum C. The result of this operation is automatically stored into spectrum C.

B. via dual display:

� RE KLFT.ref (read reference spectrum); EP; D (dual display); provide a name of irradiation spectrum, e.g., KLFT.i1; S (to subtract reference spectrum from irradiation spectrum); M (to memorize); <ret>; WR KLDF.i1 (to save the resulting difference spectrum into a file named KLDF.i1); repeat until all irradiation spectra are processed.

� Plot difference spectra using DPO, CX, CY, and PX commands as before, or use STACK.AU to for a stack plot.

� Important: Set AI = 0 and PO (decoupler power off) before leaving the spectrometer.


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IX. Listing of Automation Routines ; NOEDIFF.AU ; NOE DIFFERENCE SPECTROSCOPY ; USING ONE FREQ. LIST TO DEFINE A SERIES OF IRRADIATION POINTS ; (ON-RESONANCE) AND ONE CONTROL (OFF-RESONANCE). ; THE INDIVIDUAL FIDS ARE STORED. ; FOR LONG-TERM AVERAGING THE ROUTINE CYCLES THROUGH THE ; FREQ. LIST AND FIDS SEVERAL TIMES. ; ALSO CAN BE USED FOR PSEUDO-INDOR. 1 ZE 2 WR #1 / DEFINE FID ; PREPARE A SET OF ZEROED FILES ON DISK 3 IF #1 4 LO TO 2 TIMES C ; C = NO. OF FIDS TO BE STORED FL #2 / DEFINE FREQ. LIST ; READ IN DESIRED FREQ. LIST 5 RF #1.001 ; RESET FILE EXTENSION TO .001, BEGIN CYCLE 6 RE #1 ; READ CURRENT FID FILE 7 D3 O2 S3 ; SET DEC. FREQ. O2 FROM CURRENT FL LIST 8 D1 DO ; RELAX. TIME WITH DEC. GATED OFF 9 D2 HG ; IRRAD. TIME (CA. T1) USING POWER S3 10 GO = 8 DO ; ACQUIRE DATA WITH DEC. OFF, LOOP TO 8 11 WR #1 ; STORE CURRENT ACCUMULATED FID 12 IF #1 ; INCREMENT FID EXTENSION 13 LO TO 6 TIMES C ; LOOP TO 6 FOR EACH FREQ. IN FL LIST 14 IN = 5 ; LOOP FOR ANOTHER CYCLE ; NE = NUMBER OF CYCLES THROUGH LIST 15 EXIT ; PROGRAM REQUESTS FILENAME #1 FOR FIDS, #2 FOR FREQ. LIST. ; A FREQ. LIST MUST BE DEFINED WHICH CONTAINS ONE O2 ; ENTRY FOR EACH DESIRED IRRAD. POINT PLUS ONE OFF-RES. CONTROL ; VALUE FOR O2 WHICH SHOULD BE WITHIN THE SW REGION (E.G., AT ONE ; EDGE OF THE SPECTRUM). THE NUMBER OF FREQ. IN THE LIST MUST BE ; DEFINED BY AN ENTRY IN A 'VC' LIST, WHICH ALSO DEFINES THE ; NUMBER OF FIDS TO BE STORED. ; NS DEFINES THE NO. OF TRANSIENTS PER CYCLE FOR EACH O2 VALUE ; AND SHOULD BE A MULTIPLE OF 8. ; NE DEFINES THE NO. OF CYCLES TO BE MADE THROUGH COMPLETE LIST. ; TOTAL TRANSIENTS PER FID = NE X NS. ; USE 2-4 DUMMY SCANS FOR STEADY-STATE! ; RD = 0 ; D3 = 0.1 SEC TO SET O2 ; D1 + AQ = 2-4 X T1 FOR TRUNCATED NOE APPLICATIONS WHERE NO SECONDARY ; OR STEADY-STATE EFFECTS (SPIN-DIFFUSION) ARE DESIRED. ; D2 = CA. T1 FOR SMALL MOLECULES (EXTREME NARROWING LIMIT) ; D2 = 50-200 MSEC FOR LARGE MOLECULES (CROSS-RELAXATION). ; S3 DEFINES DEC. POWER TYPICALLY 35-55L DEPENDING ON REQUIRED ; IRRAD. BANDWIDTH.


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; NOEMULT.AU ; NOE DIFFERENCE SPECTROSCOPY USING A SERIES OF FREQ. LISTS TO DEFINE ; MULTIPLE IRRADIATION POINTS FOR EACH ON-RESONANCE SITE AND ONE CONTROL ; (OFF-RESONANCE). THE INDIVIDUAL FIDS ARE STORED FOR LONG-TERM AVERAGING. ; THE ROUTINE CYCLES THROUGH THE FREQ. LIST AND FIDS SEVERAL TIMES. ; THIS TECHNIQUE ALLOWS USE OF LOWER POWER AND AVOIDS INDOR EFFECTS. ; D.NEUHAUS, J.MAGN.RES. 53, 109 (1983) ; M.KINNS & J.K.M.SANDERS, J.MAGN.RES. 56, 518 (1984) 1 ZE 2 WR #1 /DEFINE FID ;PREPARE A SET OF ZEROED FILES ON DISK 3 IF #1 4 LO TO 2 TIMES C ;C= NO. OF FIDS TO BE STORED 5 RF #1.001 ;RESET FILE EXTENSION TO .001, BEGIN CYCLE RF #2.001 /DEFINE FREQ. LIST 6 RE #1 ;READ CURRENT FID FILE FL #2 ;READ CURRENT FREQ. LIST VC ;SELECT SECOND 'C' FROM LIST 7 D3 O2 S3 ;SET DEC. FREQ. O2 FROM CURRENT FL LIST 8 D1 DO ;RELAX. TIME WITH DEC. GATED OFF 9 D5 HG O2 ;TIME TO SET O2 VALUE (5 MSEC) D2 LO TO 9 TIMES C ;IRRAD. C*(D2+D5) SEC 10 GO=8 DO ;ACQUIRE DATA WITH DEC. OFF, LOOP TO 8 11 WR #1 ;STORE CURRENT ACCUMULATED FID 12 IF #1 ;INCREMENT FID EXTENSION IF #2 ;INCREMENT FREQ. LIST EXTENSION VC ;SELECT FIRST 'C' IN LIST 13 LO TO 6 TIMES C ;LOOP TO 6 FOR EACH FREQ. IN FL LIST 14 IN=5 ;LOOP FOR ANOTHER CYCLE ;NE=NUMBER OF CYCLES THROUGH LIST 15 EXIT ;PROGRAM REQUESTS FILENAME #1 FOR FIDS, #2 FOR FL LISTS ;A FREQ. LIST MUST BE DEFINED WHICH CONTAINS THE O2 VALUES FOR EACH IRRAD. ;POINT IN A MULTIPLET. THE LAST LIST CONTAINS ONE OFF-RES. CONTROL VALUE FOR O2 ;WHICH SHOULD BE WITHIN THE SW REGION (E.G. AT ONE EDGE OF THE SPECTRUM). THE ;NUMBER OF DIFFERENT LISTS MUST BE DEFINED BY THE FIRST ENTRY IN A 'VC' LIST, THE ;SECOND ENTRY DEFINES THE NUMBER OF LOOPS FOR IRRADIATION. ;NB: LONGEST FL LIST SHOULD BE THE FIRST ONE AND IN MEMORY BEFORE STARTING AU!! ;NS DEFINES THE NO. OF TRANSIENTS PER CYCLE FOR EACH FID ; AND SHOULD BE A MULTIPLE OF 8. ;NE DEFINES THE NO. OF CYCLES TO BE MADE THROUGH COMPLETE SET ; OF LISTS. TOTAL TRANSIENTS PER FID=NE*NS ;USE 2-4 DUMMY SCANS FOR STEADY-STATE! ;RD=0 ;D3 = 0.1 SEC TO SET O2 ;D1+AQ = 2-4*T1 FOR TRUNCATED NOE APPLICATIONS WHERE NO SECONDARY ; OR STEADY-STATE EFFECTS (SPIN-DIFFUSION) ARE DESIRED. ;SET D2+D5 AND VC COUNTER FOR 'LO TO 9' TO GIVE TOTAL DESIRED ; IRRAD. TIME, WHEREBY MINIMUM VALUE FOR D5 IS CA. 5 MSEC. ;S3 DEFINES DEC. POWER TYPICALLY 40-60L DEPENDING ON REQUIRED IRRAD. BANDWIDTH.

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15. HOMONUCLEAR COSY by cg fry: created 12/27/94 � updated 11/26/00

In COrrelational SpectroscopY (COSY), a �fast� 2D spectrum is usually obtained first. This initial set of data takes ~15 min on UWChemMRF Bruker equipment, and has a final size after transformation of 256×256 words. The data is N-type, taken as a magnitude set. Use COSY-45 unless sensitivity is an issue. Be watchful of symmetrization traps (see Derome) with this type of data. If you decide a higher resolution COSY data set is then needed, phase sensitive DQ-COSY is recommonded (rather than HR-COSY).

I. Quick Summary for Acquiring and Plotting COSY Spectra [section II manual setup is recommended; enough can go wrong with 2D datasets that becoming

familiar with the setup in detail has considerable value]

A. Obtain a high-resolution spectrum

� use normal 1D jobfile, and set reference using EP-G

� write down SR

� optimize SW using EP-<cntl>O

� retake spectrum with new SW; set SR and CY (=15 for 8.5×11, 19 for 11×17) and WR filename.001

� write down DW and O1, then TR [to another job]

B. Setup 2D paramters:

� turn the spinner off and touch up non-axial shims X Y XY XZ YZ ...

� enter: PJ COSYFAST.1DJ ;use COSYHR.xDJ for high-res

RJ2D COSYFAST.2DJ

� enter aquistion parameters from 1d job:

DW = DW(1H 1d)

O1 = O1(1H 1d)

� start 2D processing screen: ST2D

� setup F1 SW (SW1): I2D <ret> 1 <ret>

C. Check 2D parameters

� make sure SI2 = SI = 2*SI1

TD2 = TD = 4*TD1 = 4*NE

HZ/PT2/HZ/PT1 = 1

SW1 = 1/2 × SW2 = 1/2 × SW (setting I2D=1 will do this)

MC2 = M

ND0 = 1


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� type IN <ret> , and make sure IN = 2*DW *10-6 [IN in sec, DW in µsec]

� check that P1 = 90° 1H pulse width

� make sure exp is a reasonable length using: texp = NE*(DS+NS)*(D1+AQ)

� enter: AU COSY.AU

FILENAME #1: filename.SER

D. Process 2D file in DISNMR (see NUTS Cheat Sheet for NUTS processing)

1. goto step 5 if you have just taken the data; if coming back later proceed with step 2

2. goto job1, RE filename.001

PJ filename.001

3. check that SR and CY are set as in step A above (if not, correct them and

WR <ret> <ret> Y to rewrite spectrum

4. goto job2, then PJ COSYFAST.1DJ ;use COSYHR.xDJ for high-res

RJ2D COSYFAST.2DJ

5. enter: RE filename.SER <ret> ST2D <ret> XFB <ret> SYM <ret>

or if already XFB: RE filename.SMX <ret> ST2D <ret>

6. check that SR=SR2=SR1 and CY are set as in step A above (if not, correct them)

7. enter: AP2D (AC�s) or EP2D (AM�s and DataStation)

� set contour level, and expand region if desired

enter <ESC>-X to exit

8. enter: CP2P <ret> FILENAME IN F1: filename.001 FILENAME IN F2: filename.001 NO. OF PENS: 1 (or 2 if have red pen) NO. OF CONTOUR LEVELS: 4 (1-7 levels) OUTLINE BOX: Y GRID: N


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II. Detailed Explanation of COSY Set Up A. Take high-resolution1D profile spectrum

1. Take a regular 1H NMR spectrum. While in EP, set the reference using the G command (do this here because TMS and often the solvent peaks are not included in the COSY). Write down the value of SR; set CY=15 if plotting to 8.5×11 or CY=19 for 11×17 paper; save the file with the WR command. This spectrum is best for profile plotting as no folded peaks are involved.

Note SR; save spectrum

2. To increase the digital resolution, set the window as small as possible leaving ~10% of the SW as baseline on either side. Ignore TMS and solvent peaks, but do not fold compound peaks unless you are certain you can avoid folding into regions of interest. Commands useful for adjusting the spectral window are: R <move cursor> R and then <ctrl> O which sets SW and O1.

3. Take another 1H spectrum with these settings. Note on paper DW and O1 (along with SR). Transform and phase the spectrum to insure that the new sweep width parameters are set OK. If desired, set CY=15 if plotting to 8.5×11 or CY=19 for 11×17 paper, and save the spectrum with the WR command.

Note DW and O1

B. Setup 2D parameters

0. turn the spinner off and touch up the non-axial shims X Y XY XZ YZ...

1. <ESC> to Acquisition Parameters display.

2. Two sets of parameters are useful. Normally, acquire a fast COSY set first. If more resolution is then found to be needed, a simple modification of the fast COSY will provide a high resolution HR-COSY dataset; phase-sensitive DQ-COSY (see next Chapter) is recommended, however, rather than HR-COSY. Be aware that HR-COSY and DQ-COSY take much more time to acquire (minimum ~4 h; overnight runs are common).

3. Parameters can be entered either by accepting jobfile standard parameters, or by manual entry (manual entry is recommended; see section 3b):

(a) Using job file parameters:

Fast COSY HR-COSY

enter: RJ COSYFAST.1DJ RJ COSYHR.1DJ PJ COSYFAST.1DJ PJ COSYHR.1DJ RJ2D COSYFAST.2DJ RJ2D COSYHR.1DJ

and re-enter sample-dependent acquisition parameters:

SF = SF(1H 1d)

DW = DW(1H 1d)

O1 = O1(1H 1d)

ST2D

I2D <ret> 1 <ret> , to make sure HZ/PT2/HZ/PT1 = 1 (ie. SW1 = 1/2 SW2.)

and skip table below, or


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(b) Manual entry setup: follow the table below

When to enter Parameter Fast COSY HR-COSY Before ST2D SI 512W [to 2K] 2K Before ST2D TD 512W [to 2K] 2K Before ST2D NE(=TD1) 128 [or 256] 512 to 1024 Before ST2D ND0 1 1 Before ST2D MC2 M M Enter ST2D now After ST2D SW2 =SW =SW After ST2D SW1 (setting I2D=1 is

easier) =SW/2 =SW/2

After ST2D SI2(=SI) 512W [to 2K] 2K Aer ST2D SI1(=SI/2) 256W [to 1K] 1K After ST2D TD1(=NE<=SI1/2) 128W [or 256W] 512W to 1K After ST2D HZ/PT 4-20 0.5-4 After ST2D WDW1=WDW2 S S After ST2D SSB1=SSB2 0 0 After ST2D SR1=SR2 SR SR Before ST2D REV N N

Important: TD1 = 1/2 x SI1. Zero-filling is reommended in F1 to increase digital resolution. SI1 = 1/2 x SI2. Necessary to form square matrix for SYMmetrization. SW1 = 1/2 x SW2. Full sweep width in F1 is actually +/- SW1. SW1/NE ≤ 24. Increase NE if necessary, and associated parameters TD1, SI1, TD2, SI2.

4. Make sure that:

(a) SI at top of screen, says W after 512 for Fast COSY, and TD in both F2 and F1 dimensions equal the above values. F2 is the data acquisition dimension; F1 is formed by the second Fourier transform set of points down F2 columns.

(b) I2D=1, to make sure HZ/PT2/HZ/PT1 = 1 (ie. SW1 = 1/2 SW2.) (c) SI×NE is the size of subprogram matrix file which will be created at the beginning of the COSY

acquisition. Make sure that there is enough space on disk before starting COSY; DIR COSY.AU will show the largest contiguous space (the .SER file has to go into this) following the listing.

5. AS COSY.AU <ret> [job file entry, all should be ok, but check; manual entry changes are needed]

D1 = 1S (sec; actually set to 1-3 T1 for 1H; use ~1T1 for fast-COSY, 2T1 for HR-COSY) P1 = 90° 1H pulse in µsec; observe transmitter; see pulsewidth sheet for lastest calibrations) D0 = 3 U (initial delay) P2 = 45° pulse in µsec = P1/2; if no close couplings, can use 90° pulse length for improved S/N RD = PW = 0 DE = (dead time; automatically set by the spectrometer) NS = 4 or 16 (4, 8, or 16; use 4 for fast-COSY, 16 would minimize artifacts, but 4 would still be

common for HR-COSY) DS = 2 (use 2 or 4 dummy scans) NE = set as discussed above with NE=TD1, SW1/NE ≤ 12 IN = increment of D0; in essense DW in F1 dimension; should be set from ST2D make sure IN = 2*DW (where, however, IN is given in seconds, DW in µs)


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III. Acquiring COSY Spectra A. The command EXPT is not accurate!! Calculate the experimental time as follows:

texp = NE*(DS+NS)*(D1+AQ)*1.3

If the experiment is too long, adjust NE (TD, SI, TD2, SI2, and SI1, accordingly), NS, or D1.

B. You may want to save the 2D parameters in a job file using WJ2D (I often use .2DJ extension). All parameters except NE (TD1 is saved, though, and NE=TD1) will be saved. Many of the 2D parameters will not be saved with the .SER file.

C. ZE; AU <ret>

FILENAME #1: <filename>.SER <ret> ...Provide a filename with the extension .SER (do not deviate from .SER; it is required!!)

D. After first scan, check FID and if necessary, adjust receiver gain. <ctrl> E; ZE; AU to restart COSY.

III. Processing 2D Data in DISNMR (see NUTS Cheat Sheet for NUTS processing)

(follow the next steps exactly to get correct projection plots)

A. Setup 1D projection file

1. Go to job1 and PJ COSYSMAL.1DJ [for 8.5×11; use COSYLARG.1DJ for 11×17]

then read spectrum file using

RE <filename>.001 (or unique number [no characters allowed!] extension)

2. Either set reference in EP, or type in SR from step I.A.1. above.

3. Check CY=15 (8.5×11) or 19 (11×17), and then enter WR <ret> <ret> Y to re-write spectrum

B. Setup and plot .SER 2D files

[see next section if you have already processed .SER and have .SMX to plot]

1. Enter PJ COSYFAST.1DJ <ret>

RJ2D COSYFAST.2DJ <ret>

RE <filename>.SER <ret>

[change COSYFAST.1DJ to COSYFT17.1DJ if you are plotting to 11×17 paper, and change to your own .J2D file if you saved one]

2. Check all parameters, especially SI, TD, and window functions for both F2 and F1.

3. Check that NE = TD1

4. <esc> to spectrum/FID display.

5. XFB (window multiplication and Fourier transform in both F2 and F1 dimensions)

This transformation takes a while on AM�s (~2-10 min fast-COSY; 20-40 min HR-COSY), but is much faster on AC�s (~ factor of 10). Progress is indicated by a number in the top portion of the display, decrementing from SI to 1. Final FID is displayed at completion.

6. SYM (symmetrizing function to reduce noise)

SYM is not applicable to heteronuclear COSY!


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Progress is indicated by a number in the top portion of the display, incrementing from 1 to 7, 2 to 7, so on, until it stops at completion.

7. set references: SR <ret> [value from I.A.1. or III.A.2.]

SR1 = SR

SR2 = SR

8. AP2D on AC�s, EP2D on AM�s and Datastation (2D contour plot and projection display)

+ or - to move the starting contour level up or down; adjust it to exclude artifacts/baseline noise. C- and D-knobs to move the cursor through contour display. C to go to a particular column. R to go to a particular row. I to increment the column or row number. D to decrement the column or row number. O toggles the data display on and off. L,...,L to do contour expansion; <ctrl> R to return to contour display. <ctrl> Q to update and to restart plotting updated display. <esc> X to exit AP2D/EP2D to Command Interpreter.

9. CP2P <ret> FILENAME IN F1: <filename>.001 FILENAME IN F2: <filename>.001 NO. OF PENS: 1 (use 2 if have red pen and want all but lowest contour red) NO. OF CONTOUR LEVELS: 4 (1-7 levels). (1 is lowest level; 7 highest) OUTLINE BOX: Y

Note: Set DSPL=1 to check plot on display; set back to DSPL=0 to plot on plotter.

C. Plot .SMX 2D files

1. Go through section III.A. if you haven't already, Setup 1D projection file

2. Enter PJ COSYFAST.1DJ <ret>

RJ2D COSYFAST.2DJ <ret>

RE <filename>.SMX <ret>

[change COSYFAST.1DJ to COSYFT17.1DJ if you are plotting to 11×17 paper, and change to your own .J2D file if you saved one]

3. <esc> to spectrum/FID display.

4. set references: SR <ret> [value from I.A.1. or III.A.2.]

SR1 = SR

SR2 = SR

5. AP2D on AC�s, EP2D on AM�s and Datastation (2D contour plot and projection display)

+ or - to move the starting contour level up or down; adjust it to exclude artifacts/baseline noise. C- and D-knobs to move the cursor through contour display. C to go to a particular column. R to go to a particular row. I to increment the column or row number. D to decrement the column or row number.


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O toggles the data display on and off. L,...,L to do contour expansion; <ctrl> R to return to contour display. <ctrl> Q to update and to restart plotting updated display. <esc> X to exit AP2D/EP2D to Command Interpreter.

6. CP2P <ret> FILENAME IN F1: <filename>.001 FILENAME IN F2: <filename>.001 NO. OF PENS: 1 (use 2 if have red pen and want all but lowest contour red) NO. OF CONTOUR LEVELS: 4 (1-7 levels). (1 is lowest level; 7 highest) OUTLINE BOX: Y

Note: Set DSPL=1 to check plot on display; set back to DSPL=0 to plot on plotter.


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IV. Listing of Automation Routine ; COSY.AU ; HOMONUCLEAR SHIFT-CORRELATED 2-D NMR (JEENER) ; W. P. AUE, E. BARTHOLDI, R. R. ERNST, J. CHEM. PHYS. 64, 2229 (1976) ; K. NAGAYAMA ET AL, J. MAGN. RES. 40, 321 (1980) ; D1 - 90 - D0 - 90 OR 45 - FID ; SYMMETRIC MATRIX WITH SHIFTS AND COUPLINGS IN F1, F2 ; OFF-DIAGONAL PEAKS CORRELATE SPINS WHICH SHARE A ; SCALAR COUPLING J. 1 ZE 2 D1 ; RELAXATION 3 P1 PH1 ; 90 DEG EXCITATION PULSE 4 D0 ; EVOLUTION OF SHIFTS AND COUPLINGS 5 P2 PH2 ; MIXING PULSE, 90 OR 45 DEG 6 GO = 2 ; ACQUIRE FID 7 WR #1 ; STORE FID 8 IF #1 ; INCREMENT FILE NUMBER 9 IN = 1 ; INCREMENT D0 AND LOOP FOR NEXT EXPER. 10 EXIT PH1 = A0 A0 A0 A0 A1 A1 A1 A1 ; PHASE PROGRAMS CANCEL AXIAL A2 A2 A2 A2 A3 A3 A3 A3 ; PEAKS (SCANS 1-2), SELECT N-TYPE ; PEAKS (SCANS 3-4), SUPPRESS F2 PH2 = A0 A2 A1 A3 A1 A3 A2 A0 ; QUAD IMAGES (SCANS 5-8), AND CANCEL A1 A3 A2 A0 A2 A0 A3 A1 ; ARTEFACTS FROM P1 (SCANS 9-16).

; PROGRAM REQUESTS FILENAME WITH .SER EXTENSION ; NE DEFINES NUMBER OF FIDS = TD1 ; USE QP, NS = 4, 8, OR 16 (COMPLETE PHASE CYCLE) ; DS = 2 OR 4 ; RD = PW = 0 ; D1 = 1-5 X T1 ; P1 = 90 DEG ; P2 = 90 DEG FOR MAX. SENSITIVITY ; = 45 DEG FOR MINIMAL DIAGONAL (GOOD FOR TIGHT AB SYSTEMS) ; AND 'TILTED' CORREL. PEAKS (SIGNS OF COUPLINGS). ; D0 = 3E-6 INITIAL DELAY ; IN = 0.5 / SW1 = 2 X DW ; ND0 = 1 ; I2D = 1, SW1 = SW / 2 ; CHOOSE SW AND SI SO THAT HZ/PT = CA. 2-6 HZ ; TYPICALLY USE TD = SI, NO ZERO-FILLING IN F2 ; NE = SI / 4, ZERO-FILL IN F1 ; MATRIX CAN BE SYMMETRIZED ABOUT DIAGONAL.

X-Nucleus Decoupling Page 71

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16. X-Nucleus Decoupling on Athena (AC-300a) by cg fry: created 02/23/95 � updated 02/23/95

I. Discussion There may often be times when X-nucleus coupling interferes with assignments in 1H spectra, or

where selective or broadband decoupling of the X-nucleus would help with 1H assignments, especially for 1H-X correlation. Broadband and selective decoupling of X-nuclei (specifically 31P and 19F�13C is also feasible, but not likely except for an enriched compound) can now be performed on Athena (AC-300a). Decoupling an X-nucleus while observing 1H is often referred to in NMR literature as an �inverse� or �indirect� experiment. �Inverse� refers to the opposite sense of the experiment: normally one decouples 1H and observes X. Here the inverse is performed. �Indirect� specifically refers to performing 1H-X heterocorrelation experiments (HETCOR, or XHCORR type), where 1H coupled modulations are observed through the X nucleus. The indirect experiment instead observed X coupled modulations on the 1H; observing 1H gives ~10 better sensitivity.

Athena can do selective 1D 1H-X correlation spectroscopy: e.g., selectively decouple on 31P, and observe which 1H is affected.

II. Critical Parameters�Broadband Decoupling

INVH1G.AU

O1 � center of X-nucleus spectrum

O2 � center of 1H spectrum (use O1 from jobfile for normal 1H for your solvent)

P* � X-nucleus pulses (lots of them!!); if experiment doesn't seem to be decoupling well, use INVP90DE.AU to calibrate X-nucleus 90° pulse width, and see NMR Director

S1 � = 0H

D1 � = repetition delay

D5 � = AQ/2 don't change if using normal 1H SW

L2 � number of GARP loops don't change if using normal 1H SW

III. Acquisition of Inverse Spectra


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A. Initial Measurements

� take an X-nucleus spectrum as normal

� write down frequency for center of nuclei of interest (use EP, center arrow, enter O1 and write down value shown)

B. Hardware Changes for Inverse Experiments

1. switch Pneumatic unit from COMPUTER CONTROL to nucleus to be decoupled.

2. switch mode plug inside right panel (looking from front) from NORMAL to INVERSE.

3. attach unlabeled/red-ring BNC cable (normally attached at TRANSMITTER position on preamp box) directly to BB BNC cable on probe using a straight-through BNC connector

4. move yellow BNC cable labeled DECOUPLER from DECOUPLER IN position to TRANSMITTER position on preamp box

5. insert 5 dB attenuation to EXTERNAL ATTENUATOR BNC connection on X-nucleus transmitter (located at far-right�looking from behind�in upper panel on back of console

C. Software Setup for Broadband Decoupling

� for 31P decoupling, enter RJ INVP31.1DJ RC INVP31.CON

for 19F decoupling, enter RJ INVF19.1DJ

� change O1 to value noted above as center frequency for X-nuclei wanted to decouple

� change O2 to O1 value used for 1H jobfile for solvent used (e.g., O1 from ACETONE.1DJ)

� enter AU INVH1G.AU

D. Switching Hardware Back to Normal Detection

All steps must be correctly done, or normal detection will not work!!!

1. switch Pneumatic unit back to COMPUTER CONTROL

2. switch mode plug back to NORMAL

3. move yellow BNC cable labeled DECOUPLER back to DECOUPLER IN position on preamp box

4. re-attach unlabeled/red-ring BNC cable to TRANSMITTER position on preamp box, and probe BB BNC cable back to X-nucleus filter box

5. remove 5 dB attenuation from EXTERNAL ATTENUATOR BNC connection; make sure small BNC is connected through both side of EXTERNAL ATTENUATOR connection.

Heteronuclear Correlation Page 73

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17. Heteronuclear Correlation on AM Consoles by cg fry: created 2/22/94 � revised 7/31/95

I. Discussion Heteronuclear correlation maps interactions from an X nucleus (e.g., 13C) to 1H. The nominal experiment, referred to here as the HETCOR experiment, is implemented on the Bruker spectrometers, and uses X-nucleus detection with 1H decoupling. The inverse experiment (i.e., HMQC), currently only possible on the Unity 500, uses 1H detection with X-nucleus decoupling, and has much improved sensitivity over the standard HETCOR experiment. HETCOR will work ok down to 20 mg material in a 5 mm tube; more dilute samples should be run on the Unity with the inverse experiment.

The HETCOR experiment is relatively straightforward, requiring as a minimum running standard 1H and 13C spectra with optimized sweep widths as setup. It is important to have correct pulse width calibrations for both 1H and 13C transmitters, however, for correct optimized use of the the experiment. An understanding of filter bandwidths and folding is helpful to properly set the sweep widths.

II. Critical Parameters

D3 � = 1

2JCH ~ 3.3 ms change if have unusual JCH couplings

D4 � = 1

4JCH

P1 � 1H 90° pulse width; see calibration sheet next to spectrometer, or recalibrate before experiment

P3 � 13C 90° pulse width; see calibration sheet

S1 � 0H for full 1H power

S2 � same decoupling power used for standard 13C spectrum (typically 20H)

IN � increment for 2D; make sure

III. Acquiring 2D HETCOR Spectra


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1. Use job 1: Obtain a routine 1H spectrum. Save this spectrum using WR, as nameH.001 where name is a seven letter or less name used for all HETCOR data for this sample. Do not use alphanumerics in the suffix for this file, to stay compatible with the DISNMR plotting routine CP2P. Note SR after setting the reference (using, e.g., EP-G command).

Note SR(1H) and save 1H spectrum as nameH.001

2. Use EP and CNTL-O command to set SW and O1 for region of interest. Take a full (e.g., SI=16 or 32K) 1H spectrum with new settings, being careful that foldovers do not interfere with important 1H resonances.

Note O1(1H), and DW(1H)

3. Use job 2: Obtain a routine 13C spectrum using CPD (i.e. Waltz-16) decoupling with O2 set to the O1 value used in the 1H spectrum. Set the reference, and write down the value for SR.

Note SR(13C)

4. Now obtain DEPT-45 to remove quats. Use EP and CNTL-O command to set SW and O1 to include only protonated 13C plus ~10%. Take full (e.g., SI=32 or 64K) 13C spectrum with new settings. Make sure you have sufficient S/N to observe all carbons of interest; S/N~3 is minimal, S/N~8 should be ok (i.e. decrease/increase NS until S/N~8). Save a good S/N 13C Dept-45 spectrum as nameC.001

Note DW(13C) and O1(13C) and save 13C Dept-45 spectrum as nameC.001

5. In job 3: enter PJ XHCORR.1DJ

RJ2D XHCORR.2DJ

6. Return to job 2, and transfer carbon parameters to job 3: TR <ret> 3 <ret>

8. Set SI=2K (larger or smaller depending on 13C SW; 4k for SW > 150 ppm, 1k < 70 ppm) and retake the 13C Dept-45 spectrum. Make sure you have sufficient S/N to observe all carbons of interest; S/N~3 is minimal, S/N~8 should be OK (i.e. decrease/increase NS until S/N~8).

7. Enter the following parameters

NE=128 (increase if want better 1H resolution, and have sufficient spectrometer time)

ND0=2

IN=[DW(1H); see step 2]; enter IN as number followed by U; e.g.,

IN <ret> 120U for DW(1H)=120

8. Enter

ST2D

and make sure that:

TD1=NE=128 (or if enough time, multiple of 128)

SI1=TD1×2

TD2=SI2=TD=SI=2K

SW2=SW

SW1= (SW from 1H spectrum)/2

Heteronuclear Correlation Page 75

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SF1= 500.13 AM-500 Brutus 299.87 Athena 250.13 Phoenix

O11= (O1 from 1H spectrum)

SR=(SR from 13C step 3)

SR2=SR

SR1=(SR from 1H step 1)

9. Set up automation routine (only typical values shown below; most should be ok from 13C DEPT):

AS XHCORR.AU (or XHCORRD.AU to remove JHH)

D1=1 sec S1=0H P1=1H 90° in µs D0=3E-6 P4=13C 180° in µs D3=0.5 JXH ~ 3.3 ms P3=13C 90° in µs D4=0.25/JXH~1.65µs S2=20H (as used for 13C) RD=0 PW=0 DE set by computer NS=# scans used for 13C (e.g. 8) DS=2 P9=1H 90° at S2 power NE=TD1 IN=0.25/SW1

10. Save job parameters using command: WJ2D name.J2D WJ name.J1D

11. Do not use EXPT!! Check experimental time as where the factor 1.2 is approximate to account for disk I/O. If texp is too long, reschedule more time later, or decrease NS (keep as multiple of 8).

12. Start acquisition: AU

use filename: name.SER you must give .SER suffix!

IV. HETCOR 2D Processing [If processing directly after taking data�if 1H in job1, 13C Dept-45 in job2, XHCORR in job3�goto

step IV.4]

1. Goto job1: RE nameH.001

Reference 1H spectrum and set CY=14 (18 for 11×17 plots); WR <ret> <ret> to rewrite data

2. Goto job2: RE nameC.001

Reference Dept-45 spectrum (use written down SR if needed), set CY=14 and WR <ret><ret>

3. Goto job3: PJ name.J1D

RJ2D name.J2D

Check that SR=SR2=SR(13C) and SR1=SR(1H)

4. Enter: XFB

and be patient. 2k×256w will take ~8 min on DataStation.


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5. Enter: EP2D (DataStation/AM�s) or AP2D (AC�s)

Set contour levels appropriately, and ESC-X

6. Enter: CP2P FILENAME IN F1: nameH.001 FILENAME IN F2: nameC.001 NO. OF PENS: 1 (or 2 if have red pen) NO. OF CONTOUR LEVELS: 4 (1-7 levels) OUTLINE BOX: Y GRID: N or Y

7. Enter: NP to feed in new paper to plotter or printer

NOESY on AM/AC Spectrometers Page 77

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18. NOESY by cg fry: created 1/08/96 � updated 1/08/96

In NOESY, a �magnitude� 2D spectrum is often obtained, but �phase sensitive� data are nearly always preferred. Sensitivity and resolution are both improved with the phase sensitive data set. For quick runs on simple compounds, magnitude data can be successful, and processing is easier with this type of data set. NOESY data requires some approximate understanding of the T1 times involved for the protons of interest (see the chapter for T1 measurements). Measuring T1 usually takes only 15-30 mins, and can preceed the NOESY setup and acquisition. The data acquisition times for NOESY vary widely, from 1 h to multiple days (only on the Unity 500). You should not attempt obtaining NOESY data unless you are willing to invest the time needed to correctly setup and interpret the data!

I. Quick Summary for Acquiring and Plotting NOESY Spectra

[see section II for more detailed discussion]

A. Obtain a high-resolution spectrum

� use normal 1D jobfile, and set reference using EP-G

� write down SR

� optimize SW using EP-<cntl>O

� retake spectrum with new SW; set SR and CY (=15 for 8.5×11, 19 for 11×17) and WR filename.001

� write down DW , then TR [to another job]

B. Setup 2D paramters:

C. Check 2D parameters

D. Process 2D file

1. goto step 5 if you have just taken the data; if coming back later proceed with step 2

2. goto job1, RE filename.001

PJ filename.001

3. check that SR and CY are set as in step A above (if not, correct them and

WR <ret> <ret> Y to rewrite spectrum

5. enter: RE filename.SER <ret> ST2D <ret> XFB <ret> SYM <ret>

or if already XFB: RE filename.SMX <ret> ST2D <ret>

6. check that SR=SR2=SR1 and CY are set as in step A above (if not, correct them)

7. enter: AP2D (AC�s) or EP2D (AM�s and DataStation)

� set contour level, and expand region if desired


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enter <ESC>-X to exit

8. enter: CP2P <ret> FILENAME IN F1: filename.001 FILENAME IN F2: filename.001 NO. OF PENS: 1 (or 2 if have red pen) NO. OF CONTOUR LEVELS: 4 (1-7 levels) OUTLINE BOX: Y GRID: N

Polarization Experiments (INEPT and DEPT) Page 79

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19. Polarization Transfer Experiments (INEPT and DEPT) by cg fry: created 05/08/96 � updated 03/23/2001

I. General Discussion Summarizing Derome (see Chap. 6 for very good discussion, p. 129ff): Polarization transfer (PT) experiments can offer sensitivity enhancements of:

Polarization Transfer � X

Iγγ

(1)

NOE � X

Iγγ

21+ (2)

where X is the nucleus being observed (e.g. 13C or 29Si), and I is the enhancing nucleus (usually 1H, but could also be 19F or 31P). PT enchancements are 4 for 13C and ~10 for 15N, compared to the NOE enhancements of 3 for 13C, and �4 for 15N. Thus, PT improvements are most dramatic as γ decreases. The following generalizations can be followed:

� Polarization transfer is always recommended for nuclei having negative γ values, 29Si, 15N, and 103Rh being three examples. NOE enhancement for these nuclei could result in 0 signal.

� Polarization transfer is recommended for X nuclei having long relaxation times, as PT experiments have recycle delays determined by the relaxation of the I nuclei (typically 1H or 31P).

� In general, DEPT is preferred over INEPT experiments. DEPT removes distortions that occur in INEPT spectra when many different J-coupling are present. One-bond carbon-hydrogen J-couplings in typical organic compounds fit this discription: aliphatic JCH = 110-130Hz, whereas aromatic JCH = 160-180 Hz. Selection of a compromise JCH ~ 145 Hz presents errors of 20% or more from that actual value. DEPT does much better under these circumstances than INEPT, and is recommended for 13C spectra for standard multiplicity analysis.

� For all these experiments, delays will be dependent on JXI. The better the coupling is known, the better the experiment will work. Make every attempt to measure the couplings from the isotope splittings in the 1H spectrum, or obtain good literature values. Lacking both, be prepared to run a series of experiments using different JXI values to find the optimum parameters.

� For coupled spectra, DEPT+ is the preferred experiment (use either DEPTC.AU or DEPTPP.AU).

� For small JXI couplings, a compromise between signal loss from T2 (inverse natural line width)�especially for low-temp or high MW samples�and PT must be made. INEPT often is the preferred sequence in these cases, running total delays half that of DEPT. In some cases (mainly when (T1)X is not too large, and T2 is very small), INVGATE may be the preferred experiment.


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II. Setting Up INEPT Experiments A. Critical Parameters

� The most important parameter is JXI (e.g., JCH); DEPT is recommended for compounds having multiple different J-couplings, i.e. for typical organic compounds where 13C is being observed.

� D2 = echo period involving two D2 delays creates 1H antiphase state

� D3 = ∆2

12

1 1360

11 1=

=

− −πJ n J n

sin ( ) sin (deg)rad o

where n = # I nuclei

example: suppose have triethylsilyl, and want to detect 29Si, and J2Si-C-H = 3.1 Hz,

then D2 = 1/(4×3.1 Hz) = 81 msec

and D3 = secm17Hz1.3360

47.199

1sinHz1.3360

1o

o1

o=

×=

×−

� D1 = repetition delay; should > 3 T1(1H); is a primary advantage over NOE-based acquisition where repetition delay relies on T1(X)

� all pulse widths, P1 thru P4, must be reasonably correct (within 10% or so)

B. Coupled Experiments

[DEPT is recommended for coupled polarization transfer experiments]

� Use Bruker�s INEPTP.AU automation routine. Setup is identical to INEPTRD.AU.

Polarization Experiments (INEPT and DEPT) Page 81

UWChemMRF

III. Setting Up DEPT Experiments A. Critical Parameters

� Most spectrometers have DEPT jobfiles present, so a RJ DEPTD2O.1DJ will setup all standard parameters (D2O, ACET, C6D6 simply changes the decoupler to be centered� ~ 4.5 ppm �in 1H spectrum for different solvents).

� The most important is JXI (e.g., JCH):

� D2 = 1

2J [ use JC-H = 150 if olefinic present, 130 otherwise]

� θ pulse $ P0 = sin−

1 1

n = P (o

190

11sin deg)−

n

for the triethyl-silyl example above (JSi-H = 3.1 Hz),

D2 = 161 msec,

P0 = 19.4790

P1 = 0.216 P1o

o×

� D1 = repetition delay; should > 3 T1(1H); is a primary advantage over NOE-based acquisition where repetition delay relies on T1(X)

� all pulse widths, P0 thru P4, must be reasonably correct (within 10% or so)

B. Coupled Experiments

� Use Bruker�s DEPTC.AU automation routine. Setup is identical to DEPT.AU.

� Could try DEPTPP.AU if confident of pulse widths, and see distortion on multiplets; DEPTPP.AU has only additional delay, D3 = D2

Bruker AC/AM NMR User’s Guidecic/nmr/Guides/BUG/bug.pdf · 2002-08-20 · UWChemMR Facility Page...

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Transcript of Bruker AC/AM NMR User’s Guidecic/nmr/Guides/BUG/bug.pdf · 2002-08-20 · UWChemMR Facility Page...