Basic NMR experiments on Bruker WM/AC/AM

(C) Philip Toukach 1993, IOCh NMR
Ver. 3.25 (last revision: 2000 mar)

These are methodic materials for the Course of NMR Spectroscopy for students of Higher Chemical College, Russ. Acad. Scis.

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Content

1H 1D NMR experiment without solvent suppression (in details):

Preparing the hardware
Lock and resolution
Initialization
Acquisition
Selecting area
Spectrum editing
Plotting the result

Notes on solvent suppression

13C 1D NMR experiment

Homodecoupling:

The direct way
Difference mode
Difference mode with solvent suppression

Pulse length measurement

Chain of NMR experiments

Homonuclear COSY

Solvent suppression in COSY
COSY with Relayed Coherence Transfer
Double-Quantum Filtered COSY

Nuclear Overhauser Effect spectroscopy:

NOESY
ROESY (WM250)
Difference mode 1D NOE
Multiple 1D NOE

{1H, 13C} correlation (AM300):

Preparing the hardware
Acquisition
Processing

Attached Proton Test

Heteronuclear COSY

DISNMR commands:

Basic operations
System and file maintenance
Plot commands
2D acquisition and processing
Phase Editor

* AM, WM, AC abbreviations mean AM300, WM250 and AC200 NMR instruments.
* Knob names, commands, filenames and other keywords are given in capital case.
* 1,2,3 commands change the current program block (“job”).
* WR <filename> / RE <filename> command writes/reads the current block to/from disk.

1H 1D NMR EXPERIMENT without solvent suppression (in details)

A. Prepare the hardware

  1. First time restart ASPECT by START button (AC) or STOP-CLEAR-LOAD PC-CONT sequence (WM) or like that.
  2. Set up the probehead suitable for the 1H NMR experiment and connect the cables. LOCK cable must be connected to the 2H-socket in the probehead. Red one is the decoupler cable so the connection depends on the kind of experiment. The probehead may be adjusted by knobs ‘Tuning’ and ‘Matching’ while evaluating the result by the reflected signal (it must beminimized). For the probehead adjustment start acquisition by GS with parameters read from ADJ.2.
  3. Turn on the air pump.
  4. Turn the temperature unit on if the experiment temperature differs from 297K. The desired temperature is set by TE command or manually. To warm up the sample turn on the heater (each separator equals to 10 degrees up), to cold it down evaporate the liquid nitrogen. The resolution is usually better in hot samples due to their less viscosity. The better resolution you need the longer you are to wait until the sample temperature is stabilized (up to 20 min for precise experiments). Always stir the sample before the experiment by warming it up with the lighter.
  5. EDIT OPERATOR.ASC file in which specify your name (Opr.:), the solvent (Solv.:) and the preparator name (Prep.:). Each field is followed by comma-semicolon (;). Save the file by ESC-S.

B. Find the lock signal and tune the resolution

  1. RSH DUAL.SHIM - read shim-parameters (files with parameters for certain solvents and probeheads are DMSO.SHIM, D2OSEL.SHIM etc.). The lock display is turned on and off by Ctrl-L (AC, AM).
  2. Find the LOCK signal using LOCK POWER change button (AC, AM) or knob (WM) if necessary.
  3. Centre the signal on the screen gradually changing field by FIELD button/knob, then phase it by LOCK PHASE.
  4. Lock ON. If the LOCK LED does not light try to vary the FIELD slightly or slow down SWEEP RATE.
  5. Set LOCK power to get the lock level the highest but not so high that the solvent saturation begins. If the saturation happens decrease LOCK POWER until the lock level comes relatively stable.
  6. Tune X,Y, XZ, YZ gradients if necessary, always finish with X and Y. Wrong X- and Y-containing gradients result in side peaks (The SPIN RATE Hz far from the main signal) when spinning is on.
  7. Spin ON, if X and Y gradients are right LOCK level must increase. If the sample does not rotate it is either inserted bad, SPIN RATE is too low or GAS FLOW is too strong.
  8. Set LOCK GAIN to the optimal value.
  9. Tune Z and Z2 gradients to get the lock level the highest. Then tune Z3 and repeat with Z and Z2. The criterion of resolution is either lock level (the red line on the screen on AM and WM) or the FID square. The last is displayed in the second screen line each time the next scan is done after acquisition is started by GS command. The repeating step of the gradients tuning algoryhtm is the following (on the example of Z and Z2). Change Z2 anywhere, independently on how it affects the resolution, then try to get the best resolution with Z gradient only. If the result is better than it was before Z2 changing then you changed Z2 the right way, if worse then Z2 must be changed in opposite direction. After correcting Z2 repeat this step with less values of gradients change etc. Wrong Z and Z3 gradients result in symmetrical distortion of line form, e.g. wide base. Wrong Z2 and Z4 gradients result in unsymmetrical distortion (‘shoulders’).
  10. If you got a good resolution the shim-values may be stored by WSH <filename>.SHIM (AC, AM)

C. Initialization

  1. RJ <file> ; standard files are ACET.1, CDCL3.1, C13DUAL.1, etc. You may create your own files (by WJ command) with currently set parameters. The file for routine 1H experiments is H1U=D7. D7 is the device code for server hard disk. The default device is set with DU command and usually is D1. E2 is the network drive available from any spectrometer.
  2. RGA sets receiver gain automatically. In routine experiments this slow operation is not needed if the concentration does not vary much from sample to sample. If the Y-scaling is 1K the first FID point must be in the range of ± 2 gridcells (If its more after RGA then decrease pulse length with PW). Receiver gain may be set manually with RG command. If the solvent signal is much more intensive than all the rest spectrum it is better to increase RG. Too high RG results in a sine baseline.
  3. PJ <file> in job 2 - read processing parameters (if to process data in job 2).
  4. Check out the following parameters and set if needed.
  5. TD defines the length of a matrix to store the acquisition data (usually 8K or 16K). The less is TD the faster is the acquisition but the worse is the digital resolution (pt/Hz). SI is the matrix size for math operations (2n, usually 16K). If SI is greater than TD the end of matrix is zero-filled - sometimes it improves the spectrum outlook.
  6. RD is the relaxation delay. The less is the molecule the longer is the relaxation time (quarternary carbons in little molecules can relax for up to 10 sec.). For average molecules the best cycle length is 3 sec. Cycle is AQ (acquisition time)+RD.
  7. PW defines the pulse length in msec. For usual experiments the best is what corresponds to [1H-30°].
  8. NS is the number of scans to do (usually 8 or 16, must devide 8). -1 means to scan until stopped.

D. Begin scanning - ZG and wait until enough scans are done (if NS = -1).

ZG performs the zero filling (ZE), then the serie of scans (GO), each PW-FIXD-DE-AQ-RD.

E. Select the part of spectrum to acquisite

In usual experiments use standard spectrum width (SW) and offset (O1), so that these steps would not be required.

  1. TR 2 - transfer the scanned FID into job 2.
  2. If the title is specified PASC SEND may be used to store FID on a server before it is FT’d. Correct title is required (see G-4)
  3. FT - Fourier transformation.
  4. See below how to edit phase and assign the reference point (skipped now).
  5. Display the part of spectrum to acquisite in future. Remember that signals may reflect about the spectrum edges. In phase editor (EP) knob A moves the spectrum horizontally, knob B contracts and expands it, knobs C and D move the cursor horse or fine. Ctrl-R recalls the whole spectrum. R fixes the cursor, pressed twice gets the part of spectrum before the fixed and the current cursors.
  6. Ctrl-O called from the phase editor (EP) recalculates the spectrum width (SW) and offset (O1) (both in Hz).
  7. Ctrl-H - terminate aquisition in job 1.
  8. TR 1 - transfer aquisition parameters into job 1. To prevent the rest parameters set in job 1 from being overwritten O1 and SW may be set manually. If SW differs much from the standard value, respecify new values for TD and SI.
  9. ZG and wait until enough scans are done.

F. Edit the spectrum

  1. TR 2 - transfer scanned FID into job 2.
  2. Check out or set the parameters for pre-FT multiplication. For Gauss multiplication LB is the peak thinnering degree (usually from -0.3 to -2.0 Hz) and GB is the strength of Gauss correction (usually from 0.1 to 0.5). For exponential multiplication LB is the peak widening degree (usually from 0.5 to 4.0 Hz) and GB is not used.
  3. For thinner peaks perform Gauss multiplication by GM, for lower noise perform exponential multiplication by EM.
  4. FT - Fourier transformation; EF=EM+FT, GF=GM+FT.
  5. PK - phase correction initilization, useful if the phase was stored before (EP-A-<correcting>-M); EFP=EM+FT+PK, GFP=GM+FT+PK.
  6. EP - call the phase editor; commands separated with ‘;’ may form a sequence, e.g. FT;PK;EP.
  7. Correct the spectrum phase: automatical correction is started with P, the additive one - with A. Both subprograms find out the most intensive signal on the screen and then consider it the primary point. The phase of this signal must be corrected by knob C (Ctrl-C/D redirects the knobs C/D rotation). The phase of the other signals is corrected by knob D while moving and stretching the spectrum by knobs A and B. The further is the signal from the primary point the stronger knob D affects its phase. M exits the phase correction subprogram with memorizing the result, RET does without memorizing.
  8. Set the reference point if not set before: G and specify the current point shift either in ppm (P) or in Hz (H). ‘Current point’ for this operation is usually selected on the top of the solvent signal. If it can not be found use SR parameter for scale adjustment. Proton chemical shifts for most common solvents are: (DMSO-d6: 2.5 ppm, J 1.8 Hz), (Acetone-d6: 2.05 ppm, J 2.5 Hz, 2.225 ppm in water), (CDCl3 : 7.27 ppm).
  9. If the peak picking is planned set the minimal peak to pick by M in EP.
  10. In some situations is useful to set the certain signal height (in cm) by CY (then to enter the height of it and the width of plot area).
  11. RET exits EP.
  12. ABC or ABS to correct the baseline automatically.
  13. To correct the baseline manually: start EP and find the point in the centre of the noise, then press LINEFEED and specify the filename. Find an appropriate number of points and press LINEFEED at each. W ends the procedure of spline definition and asks for the type of correction: Spline (S) or Polinom (P).
  14. AZF integrates the spectrum and stores zero points in a file INTx.001. AZFE parameter is usually 20, AZFW - 40.
  15. To integrate the spectrum manually: start EP and press I to start the integration subprogram (L to read the integral from disk). Define all zero points by Z key. ‘+ ‘and ‘-’ keys scale the integral vertically, A-key calibrates the screen's integral (cursor Y-pos =max). Store zero points on disk by E which asks for the filename (RET means the default name INTx.001).

G. Plot the result

  1. Select the part of spectrum to plot and display it (Ctrl-R, then R twice or by knobs in EP), then press U to update the range (there is no visible reaction).
  2. After exiting EP select and adjust the plotting device by CA followed by dialogue (if not done before).
  3. DPO and answer the questions of following dialogue, e.g. offset=0.5, mark separation=0.1P. If the offset is greater than 2.5 the values of square are plotted with the integral curve.
  4. TI and specify title if not specified in DPO. The title must begin with a slash (/) followed by 4-letter directory name, then a blankspace and the name first 8 characters of which form the filename.
  5. CX and specify plot area width if to plot via PX(B)U.
  6. CY and specify plot area height if to plot via PX(B)U, if CY is 0 the current value of Y-scaling is used instead.
  7. MAXY and set the Y-limit. The peaks are truncated if their height overgoes it.
  8. Set the horizontal and vertical offsets of plot area by X0 and Y0 if needed.
  9. PEN and set the letter size and what pens to use for each item (for plotter only).
  10. Preview: redirect to display by DSPL = 1, back to plotting device by DSPL = 0.
  11. If the plotting device is printer redirect the output by TOPL.
  12. PX(U) plots the spectrum, PXI <filename> plots the integral, PXB(U) <filename> plots the spectrum with integral. Spectrum screen's part can be plotted by S command from EP.
  13. PASC <program name, e.g. BOX1HAM> plots the border and the list of parameters.
  14. If the plotter is used plotting starts immediately and may be terminated by Ctrl-P-T, otherwise the page is printed only after NP command is entered.
  15. For the automatic print use AU-programmes: LPL, LPLI (with AZF before) or LPLx (with ready integral), e.g. AU LPL.

The special notes on solvent suppression

  1. The following parameters must be set (via AS SSHD.AU):

    D1=1.0 (the length of solvent suppression),
    S1=30L (the strength of solvent suppression, may be increased up to 24L if the solvent is suppressed bad),
    D2=0.01, S2=63L (empty space),
    RD, PW, DE - as it is,
    DS=2 (dummy scans), NS=-1 (number of scans).

  2. FL edits the file FQLIST.001 containing the list of frequencies:

    1.) the solvent signal shift in Hz
    2.) the empty space, e.g. 7000 Hz
    3.) END (entered by ESC)

  3. Acquisition is started with AU SSHD.AU, RG must be controlled by the amplitude of the first point in the first scan.

    SSHD.AU contains the following:

    1 ZE          ; zero memory
2 D1 HG O2 S1 ; homodecoupling at the next frequency from FQLIST (solvent) during D1 with power S1
3 D1 HG O2 S2 ; homodecoupling at the next frequency from FQLIST (empty space) during D1 with power S2
4 GO=2        ; acquisition with decoupler on
5 EXIT        ; end of batch file
  4. Badly suppressed solvent signals may be removed manually via (EP-Ctrl/T-points move by 1 and 2-Ctrl/S)-sequence.

Water chemical shifts are useful to assign the reference point:

t, °C 20 30 40 50 60 70 80 90
t, K 293 303 313 323 333 343 353 363
d, ppm 4.83 4.74 4.64 4.54 4.44 4.35 4.25 4.15

* 19F experiment is the same as proton but file is F19W

13C 1D NMR EXPERIMENT special notes

  1. Standard parameters files are DUALC13.001, C13W, DMSO.013 etc.
  2. Decopuler mode must be set to CPD (AC) or CW (AM, WM).
  3. MOD must be 1 and acquisition starts with AU ZG.AU (for WM only, ZG on other spectrometers)
  4. RG is about 1600
  5. Exponential multiplication is usually used for the noise reduction.
  6. 13C chemical shifts for most common solvents are (DMSO-d6: 39.6 ppm), (Acetone-d6: 31.45 ppm), (CDCl3: 77.7 ppm)
  7. If no broad-band decoupling is required use the GATED.AU program:
    1 ZE     ; zero memory
2 D1 BB   ; broad band decoupling during D1 (about 1 sec) for NOE
3 GO=2 DO ; decoupler off during acquisition
4 EXIT    ; end of batch file
  8. Use the following batch programm if selective proton decoupling is required:
    1 ZE      ; zero memory
2 D1 BB ; broad band decoupling during D1 (about 1 sec) for NOE
3 D2 HD O2 S1 ; homodecoupling at O2 during D2 with power S1. It switches decoupler mode to HD.
4 GO=2 ; acquisition with decoupler on
5 EXIT ; end of batch file

Special notes on HOMODECOUPLING

A. The direct way

  1. Find the signal to homodecouple (it will block out the spin-spin interactions with it) and locate it with cursor (in EP).
  2. O2 M to memorize the decoupler frequency.
  3. Transfer parameters (O2) to the job where you are going to acquisite.
  4. DP and set the decoupler power, e.g. 20L (the highest power for proton homodecoupling is 2L).
  5. HD to start up the decoupler.
  6. RG is to be set so that at 1K Y-scaling first FID point would be in the range of ± 2 gridcells.
  7. All the rest is as in 1H experiment: start with ZG etc.
  8. Terminate acquisition with Ctrl-H, switch off the decoupler with PO.

B. The Difference Mode Homodecoupling

  1. DP is set as 22L (up to 25L for narrow peaks). The low power turns affected signal wide, so in difference spectrum the initial signal multiplicity is clearly seen on its phone.
  2. NS defines the number of scans in each serie. The serie is acquisiting the normal spectrum (NS scans), then acquisiting NS scans of decoupled spectrum which is substracted from the normal and the result is stored.
  3. The following parameters are set via AS DIF.AU: D1=5M, PW,RD,DE - as it is, DS=0.
  4. The frequency list contains:
  5. 1.) The empty space (near but anyway further than 50 Hz. Empty space is to be selected at a side where no changes are expected)
    2.) The signal to homodecouple (Hz)
  6. VC-List contains the only line with the number of series to perform (usually from 2 to 4).
  7. Start with AU DIF.AU. It contains the following:
    1 ZE              ; zero memory
2 D1 HD O2        ; homodecoupling at the next frequency from the FQLIST during D1
3 GO=2 DO         ; acquisition with decoupler on
4 NM              ; negotiate memory so that te result is substracted from the next serie data
5 LO TO 2 TIMES C ;loop to the next serie, the number of times from VCLIST 6 EXIT ; end of batch file

C. The solvent suppression in difference mode homodecoupling

  1. The following parameters are set via AS DIFSUP.AU:

    S1=30L (solvent suppression),
    S2=22-25L (homodecoupling) as DP if using DIF.AU,
    NE=the number of series to perform (usually from 2 to 4),
    NS=16 or 32 (scans per serie),
    D1=1.0 (the length of solvent suppression),
    D2=0.01 (the length of homodecoupling).

  2. Two frequency lists are required:
    FQLIST.001: 1.) The solvent signal (Hz)
           2.) The empty space (near but anyway further than 50 Hz. Empty space is to be selected at a side where no changes are expected)
    FQLIST.002: 1.) The solvent signal (Hz)
          2.) The signal to homodecouple (Hz)
  3. Start with AU DIFSUP.AU. It contains the following:
     1 ZE               ; zero memory
 2 RF FQLIST.001   ; set current FQLIST to FQLIST.001
   FL FQLIST       ; initialize FQLIST, to read water and suppressed signal frequencies then
 3 D1 HG O2 S1     ; homodecoupling at the next frequency from the FQLIST during D1 with power S1
 4 D1 HD O2 S2     ; homodecoupling at the next frequency from the FQLIST during D1 with power S2
 5 GO=3            ; acquisition with decoupler on
 6 NM              ; negotiate memory so that te result is substracted from the data obtained in line 10
 7 IF FQLIST       ; increase the FQLIST conter from 001 to 002FL FQLIST ; initialize FQLIST, to read water and empty space frequencies then
 8 D1 HG O2 S1     ; homodecoupling at the next frequency from the FQLIST during D1 with power S1
 9 D1 HD O2 S2     ; homodecoupling at the next frequency from the FQLIST during D1 with power S2
10 GO=8            ; acquisition with decoupler on
11 NM              ; negotiate memory so that te result is substracted from the next serie data
12 LO TO 2 TIMES X ;loop to the next serie, the number of times from NE
13 EXIT            ; end of batch file

PULSE LENGTH measurement, e.g. on the solvent signal:

  1. Locate cursor on a signal slightly moved from the screen center.
  2. Ctrl-O to set appropriate SW and O1.
  3. Decrease SI to economy time.
  4. NS=1,
    PW=2.
  5. ZG;FT;PK;EP etc. (first time store phase, phase at PW=1 and PW=[1H-90°] must not differ much. For exact measurement the phase is to be stored again when PW is about [1H-90°]).
  6. After PW reaches [1H-90°] set the intensity mode to absolute by AI=1.
  7. Sequentially increase PW (up to 50) and repeat step 5 until the signal disappears into a “wave” or better "upside down W". Decrease PW if PK makes the signal negative.
  8. Now PW indicates the [1H-180°] pulse. The lower is [1H-90°] which is the half of it the better are the spectra. The standard accuracy of PW measurement is 0.1.

*. When measuring pulse for ROESY it is needed to model the experiment conditions, i.e. (WM) to reconnect the decoupler cable to Transm F1, turn on the Spin Lock to lit The LED and turn on the decoupler (DO) with DP set to 6H (or 3H if [1H-90°] is too long, if so S1 in ROESY must be 3H too).

Organizing the CHAIN of NMR experiments:
(at the example of COSY-->COSYRCT-->COSYDQF)

  1. EDIT chain3.au (filenames vary from chain to chain)
    ;chain3.au
1 RJ PAR
2 AU COSYHG
3 IF PAR
4 RJ PAR
5 AU COSYRCT
6 IF PAR
7 RJ PAR
8 AU COSYDQF
9 EXIT

    ESC,X

  2. Define all the parameters for each experiment in a chain:

    AS COSYHG.AU & set params, then WJ PAR.001,
    AS COSYRCT.AU & set params, then WJ PAR.002,
    AS COSYDQF.AU & set params, then WJ PAR.003.

  3. Define filenames for each experiment:

    FN
    1) ....COS.SER
    2) ....RCT.SER
    3) ....DQF.SER
    ESC

  4. AS CHAIN3.AU, then start with AU.

* To read all the 2D-environment: RJ xx.PAR; RE xx.SER, to save - WJ xx.PAR; WR xx.SER.

H/H-COSY Experiment

  1. Acquisite an 1H spectrum to use as a projection and save it on disk (better D2).
  2. EP, select area for COSY, Ctrl-O to memorize SW and O1. To avoid reflections better to deal with all the spectrum but the more is SW the longer is the experiment. Always leave some empty space on the spectrum edges because the 2D-field edges are usually raised, lowered or overnoised.
  3. DU=D2 to set the default disk device to D2.
  4. ST2D, SI=... (begin with 1K) - repeat this operation until the digital resolution is better than 3 Hz/pt (the less is the value the better is the resolution).
  5. NE=SI/4, I2D=1 (X- and Y-scales equal), MC2=M (acquisite magnitude spectrum).
    WDW1=S, WDW2=S, SSB1=0, SSB2=0, ND0=1. SI1 or SW1 may not be equal to SI2 or SW2 etc.
  6. AS COSY.AU and specify the following parameters:

    D1=1,
    P1=[1H-90°],
    P2=[1H-90°] for usual COSY or =[1H-45°] for COSY-45. In usual COSY the Signal/Noise Ratio is maximal, so it is used for the low concentration samples. COSY-45 gives better resolution as the diagonal line is thinner. More than all the shape of COSY-45 cross-peaks is a parallelogram tilted to a side correlating with the JH-H-constant sign (2JH-H <0,3JH-H >0). P2 may be set as [1H-65°] or similar to get the compromise between COSY and COSY-45. D0=3U,
    D2=5M,
    RD=0,
    PW=0,
    DE as it is,
    NS depends on the experiment time (must devide 16 if P2=[1H-45°]),
    DS=2,
    IN as it is,

  7. TIME and confirm the AU-program name, then specify any filename, e.g. A.SER - as a result you see the experiment time (EXPT works faster but less accurately).
  8. AU <then specify something like A.SER>, EP to evaluate the receiver gain (RG is to be decreased if needed) - at Y-Scale set to 512 FID first point must be in the range of ± 3 gridcells.
  9. When RG is set start with AU and specify the filename (*.SER) for your data. Wait until the experiment ends. If the experiment is terminated before it is finished, reduce TD1 up to the number od series done.
  10. ST2D and check that WDW1 and WDW2 are S, SSB2 is 0 (SSB2 <>0 will not work in DISNMR871).
  11. XFB and wait until the Fourier transformation is done ('SMX 1' is displayed in the first line).

    * To economy time the next sequence may be performed:
    1.) WJ2D xxx.PAR - write experiment parameters after all of them are set correctly
    2.) AU, then specify the file name (xxx.SER) - start aquisition
    3.) In another job: RE xxx.SER, RJ2D xxx.PAR, XFT;EP2D - this will transform FID when enough of it is stored

  12. SYM simmetrizes the 2D-spectrum about its diagonal.
  13. EP2D In EP2D: Y+/- stretches 2D-spectrum vertically (in the third dimension) (AM,AC),

    Knobs C and D move the cursor,
    R gets row-projection (1D),
    C gets column-projection (1D),
    E starts EP for 1D-projection, e.g. to set the chemical shift,
    L cuts a block to display (press L twice when Row=Column to cut a square),
    X displays the cut area. The area frames are reset each time you plot the spectrum.
    ESC-X to exit EP2D.

  14. Chemical shift is assigned by memorizing SR in 1D 1H-spectrum and specifying this value for SR1 and SR2.
  15. The manual selecting of what part of 2D-spectrum to plot (in ppm) may be done with PLIM.
  16. The manual specification of Y-levels may be done via ILEV. These data are reset each time you plot the spectrum.
  17. CX and CY set the size of 2D-field.
  18. DPO sets plot parameters in the dialogue that follows. If using plotter it is better to rotate the spectrum 90° CCW.
    Better WYSIWYG via DSPL=1 (redirect output to display).
  19. Change the projection's Y-size if needed: RE <1D-spectrum>, PJ <1D-spectrum>, CY = <new value>, WR <1D-spectrum>.
  20. CPLP <projection file name (the ready-for-plot 1H-spectrum)> plots 2D-spectrum with one 1D-projection (after a short dialogue).
    CP2P <both projections file names> plots 2D-spectrum with two 1D-projections (after a short dialogue).

Special notes on solvent suppression in COSY

  1. The AU-program is COSYHG.AU (HG means H-gated).
  2. O2 = solvent signal offset in Hz.
  3. S1=30L, D3=5M, S2=30L (set via AS with the rest parameters).

COSY RCT Experiment special notes

  1. The AU-programs are COSYRCTG.AU (Relayed Coherence Transfer (RCT): The far H1-H3 interaction is seen via H2), COSRCT2G.AU (RCT2: adds HH1-H4) and COSRCT3G.AU (RCT3: adds H1-H5).
  2. If no solvent suppression is needed set S1 to 63L and O2 (Hz) to a place where there are no signals
  3. For COSY RCT everything as in COSY except:

    D2=0.032,
    P2=[1H-180°].

  4. For COSY RCT2 everything as in COSY except:

    D2=0.032,
    P2=[1H-180°],
    D3=0.032,
    NS and RG twice more than in COSY. RG has to be set accurately via EP at first two series of aquisition (detect the end of the first serie by dummy scans): the beginning of the second serie must not overscale ± 3 gridcells.

DQF COSY Experiment special notes

  1. The AU-program is COSYDQF.AU (Double Quantum Filter (DQF) locks out the diagonal signals to simplify the picture).
  2. NS and RG are twice more than in COSY. RG has to be set accurately via EP at first two series of aquisition (detect the end of the first serie by dummy scans): the beginning of the second serie must not overscale ± 3 gridcells.
  3. Everything as in COSY except:
    D0=5U,
    D2=0.2,
    D3=5U.

NOESY Experiment special notes

  1. The AU-program for NOESY with solvent suppression is NOESYHG.AU (acquisites Nuclear Overhauser Effects in 2D-form).
  2. To avoid reflections deal only with the whole spectrum. As NOESY is less sensitive than COSY it requires longer time (increase NS).
  3. All the rest is as in COSY plus:

    P3=[1H-90°],
    S3=30L,
    D9=<mixing time, from 0.1 sec. to 2.0 sec.>. Best for carbohydrates is 0.3,
    V9=<mixing time variation in percents> 6 for sugars if D9=0.3.

  4. Mathematical operations does not differ from COSY, except that SSB1 and SSB2 are set to 2 or 4 accordingly to XFB result.

ROESY Experiment on WM250 (with water suppression)

  1. Turn off the spinning after the resolution is tuned, then tune X,Y,YZ,XZ,XY,X2-Y2.
  2. Switch Spin Lock to lit the LED.
  3. Connect the (f2) cable to the (Transm f1) socket.
  4. Better to use the already-done file with ROESY experiment: RJ <name>.SMX (=Dx).
  5. II to initialize the interface.
  6. ST2D <name>.SER.
  7. Set parameters: NE=SI/4 (e.g. 256), ND0=2, MC2=W, WDW1=Q, WDW2=Q, SSB1=2, SSB2=2 and check everything via ST2D.
  8. Measure solvent (water) signal frequency and remember.
  9. Measure the frequency of the left edge of the spectrum part to deal with.
  10. Display this part and press Ctrl-O with the teleprinter turned on (to remember values of SW and O1).
  11. Compose frequency list (e.g. FQLIST.001) in a memory block and in a disk unit in which 2D-data will be stored:

    1.) The solvent signal frequency remembered on the step 8
    2.) O1 printed on the step 10
    3.) The frequency of the spectrum edge measured on the step 9
    4.) The same as 2.)
    5.) END (entered by ESC)

  12. SW and specify the value printed on the step 10; ST2D to control.
  13. I2D =0.5.
  14. Standard resolution is about 2 Hz/pt (F1) and 4 Hz/pt (F2). To improve the resolution SI may be increased (and so NE too).
  15. AS ROESYW.TE (TE is to turn off the temperature after the experiment ends) and set the following parameters:

    D1 =0.1 or less (the relaxation time in addition to P8),
    S2 =26H-30H (the water suppression strength - define experimentally),
    FRQ-List: the name of the frequency list composed on the step 11,
    P8 =1S (the time of water suppression),
    D2 =5M,
    S1 =6H (or 3H if [1H-90°-HD] is too long),
    P1 =[1H-90°-HD] as it was set in the read file,
    D0 =3U,
    P4 =4,
    D4 =54U,
    RD =0,
    PW =0,
    DE as it is,
    NS =depending on the experiment time (must divide 8),
    DS =2, NE as it is, IN as it is (not zero anyway),
    D7 =1S (the time for temperature switch back to normal),
    then set the final temperature (297° C) via TE.

  16. TIME ROESYW.AU=D2 <any filename> and then re-enter NS to have the time Ok.
  17. Check if everything is Ok:

    Set NS to 8 temporarily, AU, enter <filename>.SER.
    Set RG so that the FID first point does not overscale ± 2 gridcells (in EP) at the first scan in the second serie.
    In another job: RE <name>.SER <serie number>; WM2 (for this command SSB2 and WDW2 must be set to 2 and Q), FT.
    EP to evaluate the serie: the solvent must be suppressed well, signals must present and not on the spectrum edges (first serie looks like the normal spectrum), the spectrum from the second serie must differ from the one from the first.

  18. Restore NS got on the step 16, then AU, specify the <filename>.SER and wait until the experiment ends.
  19. ST2D <filename>.SER and set the correct ND0 (=2), MC2 (=W), WDW1,WDW2 (=Q), SSB1,SSB2 (=2), GB (=0).
  20. RE <filename>.SER 1 - read the first ROESY line, LB=1, EF, EP-P and equalize the phase as in the usual spectrum (P by H2O is bad due to its probable distortion). One edge may be strongly downer.
  21. TY to get the phase correction result, then specify two got values in PC0 (the first) and in PC1 (the second) to phase all the lines the same way.
  22. PZ - restore correction; XF2 and wait until the F2 Fourier transformation is done.
  23. PC0 =0, PC1 =0, XF1 and wait until the F1 Fourier transformation is done.
  24. The vertical phase is usually bad, so correct it in 24.-27.:
    EP2D to remember co-ordinates (Row=Col/2) of both downer left and upper right corners of 2D-field.
  25. RSC <filename>.SMX <value of co-ordinate of the downer left corner - better to select some positive diagonal peak near>, then EP-P and equalize the phase as usually (M), before exiting EP by RET set cursor onto a primary point (a peak) of the phase correction done.
  26. RSC <filename>.SMX <value of co-ordinate of the upper right corner - better to select some positive diagonal peak near >, PK, if after this the phase is equalized bad - EP, C (locate the cursor on a point to which it was moved by P on the previous step, this may require moving the cursor manually with knobs before C), A and correct phase only by knob D, then M.
  27. XF1P and wait until the the vertical phase correction is done. In 25-26 numbers of columns are meant as co-ordinates.
  28. If the horizontal phase is bad too, repeat the same set of operations as in 24-27 but for the horizontal phase: XF2P instead of XF1P, RSR instead of RSC and mean the numbers of rows by co-ordinates.
     
    *. Operations 24-27 and 28 are repeatable any number of times.
  29. EP2D to evaluate the result. All the cross-peaks are in the negative area. To switch the positive side to negative and back the keys '-' and '+' are used. Plotting resets the mode to positive, so before plotting the second time the mode must be set negative again (via EP2D).
  30. All the following is as with any 2D-experiment: CX and CY are to be set so that scalings on both axii would be equal, PLIM may be used for exact definition of a plot window in ppm etc.

NOEDIFF Experiment on WM250

  1. Stabilize temperature near 300 K.
  2. Turn off the spinning.
  3. Tune resolution using X- and Y-containing gradients if needed.
  4. In EP: a. Set the cursor to the empty place of a spectrum and press O2 L
            b. Set the cursor to the signal to homodecouple and press O2 L
  5. FL to check the frequency list (RET=Ok, ESC=cancel frequency).
  6. AS NOEDIFF.AU and set the following parameters:

    LO = number of frequencies in a list (confirm) (use VC command to change),
    D3 =5M,
    O2 - check again,
    S3 =<the decoupler power (e.g. 20-35 L)>,
    D1 =2S,
    RD =0,
    PW = [1H-45°],
    DE as it is,
    NS =<the number of scans in each serie> (8 or 16),
    DS =2 (dummy scans for relaxation between series),
    NE sets the number of series (e.g. 20).

  7. TIME NOEDIFF.AU to evaluate the experiment time.
  8. Start with AU, then specify filenames for FID (without extension which is added automatically .00n) and frequency list (confirm).
  9. Wait until the experiment ends.
  10. RE <name>.1.
  11. AI =1 to set intensity to absolute.
  12. To avoid the line form distortion set LB to 0.5.
  13. EF, EP etc.
  14. WR <spectrum name>.1 (e.g.)
  15. Operations 10-14 for the <name>.2.
  16. EP-D - enter the double display mode then specify the file name to show above the current data,
    EP-D-S - substract spectra,
    EP-D-M - memorize the result (here: of substraction).
  17. NM command flips the spectrum vertically.

NOEMULT Experiment on WM250

This experiment produces several FIDs, each with NOE by one signal plus one FID without NOE (it’s used then as a source to substract NOE spectra from). The exact temperature setting is important so it is needed to wait about 30 min. for thermostabilization after the sample is inserted and the desired temperature is set.

  1. AS NOEMULT.AU (or .TE if it is needed to stabilize the temperature after the experiment ends; if so D7=1, (TE)=<required temperature>) and set:

    VCLIST.001
       1.) The number of frequency lists used (equals the number of signals to deal with plus one for the empty space)
       2.) The number that devides the multiplicity of each signal to affect; it can be increased twice if to suppress the solvent. This number is selected so that the full time of one signal saturation becomes about 1 sec.; each component is saturated for the time specified in D2. E.g. it is 24 if there are triplets and doublets and water is suppressed.
    D3=0.1,
    FQLIST.001 - The frequency list containing the only line: the empty space (in Hz) at 50Hz beside the leftmost signal,
    S3=40-45L. The least value is 36L (the solvent is suppressed better, but the selectivity falls).
    D1=0.1,
    D5=5M,
    D2=<the time of each component saturation, e.g. 0.1>,
    RD=0,
    PW=3,
    DE=Ok,
    NS=16,
    DS=2,
    NE depends on the experiment time (evaluate via TIME, not EXPT).

  2. Edit several frequency lists (FQLIST.00n) - by one list for each signal, one entry for the offset (Hz) of each its component.
    If the solvent is suppressed frequency lists contain twice more lines: lines 1,3,5 etc. are the solvent offsets, e.g. it may be “ 1.)<H2O> 2.)<left component> 3.)<H2O> 4.)<right component> 5.)END “ for doublet.
  3. AU and specify two filenames: first is the name (without extension) to store FIDs (results in <name>.00n, better to make it shorter); the second is FQLIST.
  4. EP to evaluate the receiver gain. RG is to be set so that FID’s first point would be in the range of ± 2 gridcells at 1K Y-scaling.
  5. This experiment is interruptable so it is only needed to wait until enough data are stored. The processing may begin immediately:

    In another block RE <name>.001, LB=0.2, EF, then edit phase and obtain the spectrum again via EFP for control, then WR <name>.<001+N> (N is the number of frequency lists).
    Sequentially read all the FIDs (<name>.00n), EFP, then substract them from the stored ‘empty-space-spectrum’ (<name>.001+N), memorize and compare the result with the ‘empty-space-spectrum’.

{1H-13C} CORRELATION on AM300
(with water suppression & without JCH suppression)

A. Setting up the hardware

  1. Install the “BB-5 Inverse 1H-{X}” probehead (cables connecting: red cable - D-Lock; BB - BB, remaining - remaining)
  2. Insert SELECTIVE POWER AMPLIFIER (13C 75.48 MHz 80W CW) into the second slot from the right in BROADBAND MODULATOR B-BM1 (stands above) and insert the modulator cable into the probehead instead of BB.
  3. a. POWER
    b. Tune observation channel
    c. INTERN ON, set meter to ‘forward’, 3-5W is Ok. If REFL.>FORW. then it is needed to tune MATCH and TUNEING bars.
    d. Set meter to ‘refl.’ and minimize the wattage (W)
    e. INTERN COMP, EXTERN OFF

*. “BB-5 Inverse 1H-{X}” probehead tuneing: Set AQ to 0.0013 and run GS. The reflected signal (OBS REFL) must be low (one led lights) or zero. If it is not so, turn up the switch beside the back-panel m A-meter and minimize the mesurement data on it. The coarse tuneing is setting MATCH and TUNEING bars to values specified on the probehead card. To tune fine correct these bars position accordingly to the reflected signal measurement.

B. Acquisition

  1. Memorize the solvent signal offset in Hz (in proton spectrum).
  2. Select the proton spectrum part to deal with, display with blankspaces aside not less than 100 Hz (because the 13C satellites are acquisited).
  3. Ctrl-O and remember O1.
  4. Turn off the spinning and tune the resolution (lock may be ‘jumping’).
  5. RE <13C spectrum>, in EP display the appropriate spectrum part then locate the cursor to the screen center and press G 0 (set shift to zero).
  6. After exiting EP: SF and remember the exact value.
  7. EP again, display the part to deal with, then Ctrl-O and remember SW.
  8. DU=D2 (set the current disk unit to D2).
  9. Set the synthesizer frequency: SS and specify the value remembered on the step 6. It defines the 13C spectrum offset (O1). Then initialize interface: II.
  10. ST2D and set the parameters: ND0=4, MC2=W, NE=128 (for beginning), set SI (e.g. 1K) so that the resolution by F2 would be about 1.5-3 Hz/pt.
  11. SW1 =a half of a value remembered on the step 7. Resolution by F1 may be about 15-50 Hz/pt, if it’s not so change NE by 2.
  12. WDW1,WDW2 =Q,
    SSB1, SSB2 =2,
    REV=N.
  13. Remember the current values of SW and O1 (got on the step 3).
  14. AS BIRDPHPR.AU and set parameters:

    O1 FQLIST.001, this file must contain:
      1.) Solvent signal frequency in Hz remembered on the step 1
      2.) Spectrum center offset remembered on the step 3.
    D5 =0.5M,
    P3 =9.8 [13C 90°],
    P5 =0.5U,
    D0 =3U,
    P1 =6.5 [1H-90°], RD as it is,
    D2 =0.0032 (1/2JXH),
    PW=0,
    P2 =13.0 [1H-180°], NS depends on the experiment time (40-160, must divide 16),
    P4 =19.6 [13C 180°],
    DS=4,
    D4 =0.25 (see below),
    NE, IN as it is.

  15. EXPT and modify NS to get the experiment time Ok (NS must devide 16 anyway).
  16. Check SS; then ST2D to check everything.
  17. Set the optimal RG: AU A.SER, then EP and ensure that first point is in the range of ± 2 gridcells.
  18. Remember chemical shifts of some signals which position can be easily recognized to evaluate their ‘main’ intensity then wait at least 16 scans, Ctrl-H, WM2, FT, then edit phase as usually and check that only (!) satellites are acquisited. If main signals are too intensive modify D4 to get them less intensive (usually D4 is to low). The result of the second serie transformation must differ from one of the first.
  19. If the solvent is suppressed bad: P5 =1U.
  20. Start the experiment with AU (asks for the <filename.SER>) and wait until it ends.

C. Processing

  1. ST2D <filename>.SER and check everything: ND0 (=4), MC2 (=W), WDW1,WDW2 (=Q), SSB1,SSB2 (=2), NE.
  2. Set SF1 to the value of the 13C spectrometer frequency (75.47 for AM), set REV to N.
  3. RE <filename>.SER 1 - read the first ROESY line, WM2, FT, EP-P and equalize the phase as in the usual spectrum basing on the leftmost signal.
  4. TY to get the phase correction result, then specify two got values in PC0 (the first) and in PC1 (the second) to phase all the lines the same way.
  5. PZ - restore correction; XF2 and wait until the F2 Fourier transformation is done. Do not call ST2D after as it resets SF1 and REV.
  6. PC0 =0, PC1 =0, XF1 and wait until the F1 Fourier transformation is done.
  7. Set the cursor to some peak and call R-E from EP2D to evaluate the vertical phase. We usually get bad vertical phase, so correct it in 7.-10.:
  8. EP2D to remember co-ordinates of both downer left and upper right corners of 2D-field (find appropriate signals).
  9. RSC <filename>.SMX <value of co-ordinate of the downer left corner - better to select some positive diagonal peak near>, then EP-P and equalize the phase as usually (M), before exiting EP by RET set cursor onto a primary point (a peak) of the phase correction done.
  10. RSC <filename>.SMX <value of co-ordinate of the upper right corner - better to select some positive diagonal peak near >, PK, if after this the phase is equalized bad - EP, C (locate the cursor on a point to which it was moved by P on the previous step, this may require moving the cursor manually with knobs before C), A and correct phase only by knob D, then M.
  11. XF1P and wait until the the vertical phase correction is done. In 7-10 umbers of columns are meant as co-ordinates.
  12. After calling C-E from EP2D the satellites must be nearly equal to represent the good horizontal phase. If the horizontal phase is bad too, repeat the same set of operations as in 7-10 but for the horizontal phase: XF2P instead of XF1P, RSR instead of RSC and mean the numbers of rows by co-ordinates.
     
    *. Operations 7-10 and 12 are repeatable any number of times.
  13. All the following is as with any 2D-experiment: CX and CY are to be set so that scalings on both axii would be equal, PLIM may be used for exact definition of a plot window in ppm, EP2D-R(C)-E-G sets the chemical shift (remember that 1H shift must be set by the point between the satellites) etc.

APT Experiment

  1. Prepare everything as for usual 13C experiment.
  2. Set the following parameters (via AS JMODXH.AU):

    D1=from 0.7 to 1.0,
    S1=30H,
    D2=5M,
    S2=<good H-decoupling value for MOD-1 i.e. 16H (WM) or 24H (AM)>,
    P1=[13C-90°],
    VDLIST contains the only value of 1/JCH=0.07 (To get primary/tertiary carbons positive and secondary/quarternary - negative) or 1/2JCH=0.035 (to acquisite quarternary carbons only),
    P2=[13C-180°],
    RD=1U,
    PW=0,
    DS=2,
    NS=-1.

  3. Start with AU JMODXH.AU, then specify the filename and wait until enough data are stored.

{13C-1H} CORRELATION

  1. MOD=1, ND0=2,
    cables are to be connected as for 13C-spectrum acquisition.
  2. DU=D2 to set the current disk unit to D2.
  3. In another job: EP and display the part of 1H-spectrum to deal with, then Ctrl-O and remember spectrum offset (O1) and width (SW).
  4. In EP display the part of 13C-spectrum to deal with, then Ctrl-O.
  5. Evaluating via ST2D set SI=2K or 4K to get about 2-3 Hz/pt by F2 (the resolution by F1 may be about 5 Hz/pt)
  6. Set SW1 as the half of a proton spectrum width remembered on the step 3.
  7. Set O2 as the proton spectrum offset remembered on the step 5.
  8. SSB1=2, SSB2=2, WDW1=Q, WDW2=Q, MC2=M, then ST2D to check everything.
  9. AS XHCORRD.AU and specify the following parameters:

    D1=1, for polymers it may be decreased down to 0.7 to economy the experiment time,
    S1=0H (the strongest possible),
    P1=8.5 [1H-90°],
    D0=3U,
    D3=0.0032 (1/2JXH),
    P2=17.0 [1H-180°],
    P4=25.6 [13C-180°],
    P3=12.8 [13C-90°],
    D4=0.0016 (1/4JXH),
    S2=<a good value for CH-interaction suppression in 13C-experiment, displayed on the top line>, 16H (WM) or 24H (AM),
    RD=3U (for MOD=1),
    PW=0,
    DE, IN - as it is,
    NS= several hundred, enough to get 13C spectrum with signal/noise ratio over 2.
    DS=2,
    NE=64, 128 or 256. May be decreased to 2(n-1)+x (e.g. from 64 to 33-63) to economy time.

  10. TIME and modify NE to get the experiment time Ok.
  11. AU, specify <filename>.SER, evaluate RG (anyway must be not more than 3200).
  12. To ensure that everything is Ok: check SF1 (spectrometer 1H-frequency), in another job: RE <filename>.SER 1 after first NS scans, the result must be normal 13C-spectrum.
  13. Wait until the experiment ends.

DISNMR COMMANDS

BASIC COMMANDS

SYSTEM COMMANDS

PLOT COMMANDS

2D ACQUISITION and PROCESSING COMMANDS

Phase Editor COMMANDS