2D NMR Data Processing with nmrPipe

nmrPipe is a widely used, free, powerful NMR data processing software. nmrDraw is the packaged graphic user interface for data processing and display. nmrPipe is a Unix pipe command based program for streaming FID and data vectors during processing of a multi-dimensional spectrum. The data pipes feed results from previous commands to following commands, mostly in memory, to give the final processed data. The program can process data from 1D to 4D. This document limits the discussion to 2D data only.

Currently, the program runs under the Unix environment, including Linux, and under Mac and Windows XP operating systems. The programs have been installed on NMR500, NMR600, and the data workstations. For more information on software availability and references, visit author Frank Delaglio's website at NIH and his private website.

While learning the whole programming package takes time, I have written two programs (pipe2d or snap2d) that automatically generate scripts and do the processing for known types of routine 2D experiments. You do have to adjust phase parameters for phase-sensitive spectrum after the initial processing. Please report problems you may encounter using these programs.

The following details the procedure using pipe2d or snap2d. For details of nmrPipe and nmrDraw program, see the following pages.

• nmrPipe
• nmrDraw

See Section 3 for plotting with X and Y projections.

In the following, all commands must be entered in a Linux terminal window. To open a terminal, right-button click inside the Desktop, select Open Terminal.

1: Procedure (after experiment finishes and data saved)

See section below for taking snapshot during a 2D experiment run.

Reference the direct 1H dimension before saving data

Make sure that before you save your data, reference the 1H spectrum of the direct dimension carefully in vnmrJ. The pipe2d program uses the 1H reference of the direct dimension to reference the indirect dimension, regardless of the type of nucleus (1H, 13C, 15N, etc.) along the 2nd dimension.

If you use the experiment setup procedure given in the instructions, the reference should be carried over from the 1D 1H spectrum done before the 2D experiment. No additional action is needed.

Run pipe2d

The pipe2d program must be launched from within a .fid folder (we'll use noesy2d.fid as an example below) that contains Varian's data files. Enter the following commands:

• cd noesy2d.fid
• pipe2d

Enter the index number of the template experiment that matches the type of experiment you have. If you simply hit the RETURN/ENTER key, by default the program uses 0 (the 1st template experiment). The default value is correct most of the times. Next, the program creates fid.com (data conversion) and nmrproc.com (Fourier Transform) under the .fid folder and executes the files to create the processed spectrum (test.ft2). Once done, the program automatically launches nmrDraw. Press r with cursor in main window to draw contours. Both scripts can be executed manually, after manual editing of certain parameters, from the terminal window, by typing either fid.com or nmrproc.com. Data conversion usually only needs to be done once; processing may need to be re-run and fine tuned more times.

Adjust phases

There is no need for phase adjustment if the data is in absolute-value (or magnitude) mode such as in a typical gCOSY, HMBC. The phase parameters for both dimensions may need to be adjusted if the spectrum is phase-sensitive. These phase parameters are contained in nmrproc.com.

• pipe2d launches nmrDraw after data processing. If not, from within the .fid folder, type nmrDraw& to launch nmrDraw
• Put cursor inside the drawing area and press r to draw the contours. Click the +/- buttons next to Factor to adjust contour level to see all peaks. Press "-" (lower the contour threshold) and r to redraw contours until baseline noise starts to show. Then, press "+" to increase the contour level a bit so that baseline noise is barely visible and the positive/negative tails of peaks are visible. That's where you want to stay to make the rest of the adjustments.
• Check phase of the peaks along both X and Y axes. Out-of-phase peaks show uneven shoulders, bases, and positive/negative tails. If phases need correction, do the following:
• Right button click Mouse->1D Horizontal to bring out a horizontal line. Hold the left button to drag the line across peaks. To increase the vertical scale of the 1D slice, hold the middle button and click inside the grey area OUTSIDE the main graphic area (black), and drag the mouse up or down. To move the slice position in the window up or down, click and hold the right button and drag in the grey area.
• To make phase adjustment, toggle Phasing button to ON. Drag the P0 (zero order phase) bars (left bar: coarse; right bar: fine) until the base of the peak(s) is even and symmetric with the peak either pointing up or down.
• Click Mouse->1D Vertical to adjust phase for peaks along Y axis.
• If peaks across X or Y axis appear to need different amount of zero order phase correction, it means a linear (1st order) phase correction is required. The linear phase change shows as a linear increase or decrease of phase distortion along X or Y axis. Do the following:
• Bring out a horizontal or vertical slice. First, pick a slice that cuts a strong peak near the left edge of spectrum along X, or the bottom edge along Y. Set the Pivot point to this peak by clicking the Pivot +/- button. Make the yellow arrow point to the center of the peak.
• Adjust P0 so that this peak is phased perfectly.
• Then, move the slice to cut peaks at the other edge of the spectrum (right edge for X and top edge for Y axis). This should show out of phase peaks. Adjust P1 so that these peaks are phased nicely.
• Once P1 adjustment is done, select a slice across the center of the spectrum to check middle peaks. Their phases should have been mostly corrected automatically.
• Press M to show nmrproc,com. Add the P0 and P1 values shown next to P0/P1 bars to the nmrproc.com script for that dimension. The first line with nmrPipe -fn -P0 XXX-P1 XXX is for X-axis and the second is for Y-axis. Add or subtract the P0 and P1phases to those already there. Click Save, Execute and Done. Press r to redraw contours to check phases again. Re-adjust P0 and P1 again if needed.

2: Procedure (snapshot during experiment with snap2d)

Follow the same procedure above for data processing after experiment finishes, except the following:

• Make a temporary folder under your home directory and go inside that folder. All temporary raw and processed data will be contained in this folder. AS an example, we name the folder as temp.fid.
• cd (change to home directory)
• mkdir temp.fid (create temp.fid folder)
• cd temp.fid (change directory to inside temp.fid)
• Then, type:
• snap2d

• The program asks you to enter:

• Current vnmrJ experiment number being run. This number is displayed in the upper left corner of the spectrum window with name like exp1 or exp2, etc. Only enter the number after exp.
• Template experiment index number from the list (Default is 0 if you simply hit Enter/Return key)
• Current accumulated FID number. This number is updated at the bottom of the vnmrJ window as the experiment runs.

Once the information is provided, the program creates and runs the scripts and launches nmrDraw. Press r with cursor in nmrDraw window to draw contours.

You can stop the experiment (with aa or click Abort Acquisition button) anytime if you feel the data quality is adequate from the snapshot. Note that if a 2D data collection aborts before the last FID is collected, the data matrix is only filled partially. There appears to be no saved parameter in the parameter file procpar that indicates the index number of the last FID collected. nmrPipe gives an error if the full 2D matrix is to be processed. snap2D asks user input for the correct size of finished FIDs. But if you do not know this value after data collection, you can reduce the Y-axis points (-yN and -yT fields) via try-and-error and re-run the data conversion until the error message disppears.

After you finish with the experiment, to make a copy of the snapshot data folder as your final data folder, type:

• cd .. (the two dots indicate move up one folder)
• cp -r temp.fid my_final_data.fid

Alternatively, you can rename the temp folder to the final data folder name rather than copy it to a new folder:

• mv temp.fid my_final_data.fid

3: Plot 2D spectrum with X and Y projections

To generate a plot with the X and Y projection displayed along the edges, type the following commands:

• cd noesy2d.fid (change directory to inside the .fid folder)
• plot1D2D.tcl
• ps2pdf roi.ps

By default, the plot1D2D.tcl program takes test.ft2 as input, automatically set contour threshold and plot the whole region of the spectrum with projections along the sides. The default output postscript file is: roi.ps. The ps2pdf command turns the .ps file into a pdf file (roi.pdf). Both can be edited with Adobe Illustrator or other programs without the loss of digital resolution.

To plot only a region of the spectrum or use a different contour threshold (find the value next to First: in nmrDraw display), modify the following commands:

• plot1D2D.tcl -x1 2.0 -xn 5.0 -y1 2.0 -yn 5.0 (plot region from 2 to 5ppm along both X and Y)
• plot1D2D.tcl -hi 50000.0 -x1 2.0 -xn 5.0 -y1 3.0 -yn 8.0 (plot with a threshold of 50000, from 2 to 5ppm along X and 3 to 8ppm along Y)
• plot1D2D.tcl -out expand.ps-x1 2.0-xn 5.0 (plot from 2 to 5ppm along X and all along Y, with output file named as expand.ps)

If any of the options starting with a dash ("-") is not specified, the default value or name is used. See the entire list of the options from Frank Delaglio's website here.

4: Reference PPM adjustment

pipe2d and snap2d automatically set reference PPM for the indirect dimension according to IUPAC unified chemical shift method. The method relies on the presence of accurate reference parameters of direct 1H dimension saved (from vnmrJ) in the parameter file procpar. If for some reason, you forget to keep proper referencing before saving the data, use one of the several methods below to correct the reference PPM during processing.

• If you know the PPM value of a 1H signal in the spectrum
• The best way is to modify fid.com and re-do the data conversion. Open fid.com with a text editor (nedit fid.com&), and add or subtract the offset PPM value to -xCAR or -yCAR field to give the correct PPM value for the reference peak. Save the file. Then, type fid.com, followed by nmrproc.com. Check the correction in nmrDraw.
• Alternatively, from nmrDraw, right button click File->Calibrate Axis. Select the axis you want to adjust (either X or Y). Enter a new reference PPM value for that axis. You should add or subtract the offset between the your current peak PPM and the correct PPM value to the reference value field. Then, click Apply, and Save. Note that this method changes the reference parameters in the processed data (test.ft2) only, but not in the converted raw data (test.fid). Therefore, if you re-process the data with nmrproc.com, the reference correction is overwritten.
• If you do not know anything about the PPM value of the compound, the best is to re-collect a 1D 1H spectrum under the same condition, with TMS in your sample or less precisely use the lock (or residual solvent peak or simply enter setref) to get the reference right. It is possible to back calculate the PPM values based on the saved parameters (lock position), tof and dof offset etc., but the uncertainty could be substantial.

H. Zhou updated Oct 2010