NUTS is a Windows95 program that processes the raw data collected by PNMR into NMR spectra. It is used to import data, perform transformations, phase the spectrum, integrate sample peaks, and generate graphical and tabular reports of these data. NUTS has a lot of functionality, and consequently contains a lot of commands, but for this basic proton NMR only a few are required.
Most new operator time is spent in learning software, consider download nuts95.zip from the Acorn web site and install it a Windows95 computer. This demonstration version of the NUTS program has an excellent help system and allows plenty of practice processing data before using the NMR Laboratory. The demonstration comes with two example data files, or process local data collected on the Anasazi NMR computer. The demonstration version will not print the spectrum properly. There are certain other limitations, but it is a free software self-teaching tool.
The NUTS program runs under Windows95, usually with a white background. It contains menus of commands that can be accessed using either the keyboard or the mouse. Keyboard commands are entered as two keystrokes, such as A1 or ZZ, and are not followed by the 'Enter' key. Most of the common commands can also be selected from the program menus by using the mouse.
This is the NUTS boot-up window
Note the 'right-arrow' command line cursor at the bottom left of the white window background. Enter one of the two-keystroke commands and the letters appear at the command line cursor. Immediately after the second letter is pressed, NUTS executes the command -- Do not press Enter or use an incorrect command it will not work. If NUTS won't perform the command, look for a question mark to appear above the cursor or a 'Not a Valid Command' message in the status bar below the cursor. In either case, press 'Enter' a couple of times to clear the pending commands and then try the two-keystroke command again.
BC -- Baseline Correct. Applies baseline flattening algorithm and improves integration accuracy.
LB -- Line Broadening. Used with EM.
EM -- Exponential Multiply. Applies exponential smoothing function to FID using LB factor. Can improve S/N (signal to noise) ratio.
FT -- Fourier Transform the FID.
PH -- PHase manual adjustment. Hold left mouse button and move mouse left or right. Press 'Enter' to exit Phase mode.
PS -- Phase Same. Phases spectrum using current values.
AP -- Automatically Phase the spectrum.
ID -- Integrate Display. Allows manual peak integration and numerical value adjustments.
AI -- Automatic Integration of the peaks in the spectrum.
SZ -- Set Zero. Sets the offset of the peak in a Zoom region to zero.
O -- Adjust peak Offset. Hold the left mouse button and position vertical line on peak, then press 'o'. Enter offset value.
PP -- Peak Picking. Displays peak tick marks and offsets on spectrum. Used with MH and RM.
MH -- Minimum Height. The smallest peak height allowed for Peak Picking.
RM -- Multiples of RMS. Adjusts discrimination of peaks with small splittings. Use PP after changing RM.
ZO -- Zoom mode. Hold left mouse button and sweep across peak from baseline to baseline. Right-click highlighted region to Zoom. Right-click to return to previous view. Press 'Enter' to exit Zoom mode.
Note that there may or may not already be a spectrum displayed in the window. If a spectrum is displayed, it is from the previous acquisition. To automatically process any new data, use Ctrl-F1. The macro will read data from pnmr.fid and attempt to process it. A dialogue box will appear.
Fill in the 'Comment', 'User', 'Date', and 'Pulse Experiment' fields. NUTS will complete the rest of the fields from the data acquisition parameters. Click 'UH and Exit' to continue and a printer prompt appears with a printer dialog box. Click 'OK' to print the spectrum.
If the spectrum processing is not optimized, make manual adjustments of the processing parameters.
Manual Data Processing:
1) To read in new data, press 'zz' but don't press 'Enter'.
Notice -- If the Enter key is accidently pressed, read the following paragraph.
Commands are entered automatically at the bottom of the NUTS window. Most NUTS commands consist of only two characters so it tries to recognize them without pressing 'Enter'. If 'Enter' is accidentally pressed, repeat, e.g. 'Enter' a couple more times to clear the command display at the bottom left of the screen. It will probably display question marks, and this is OK. Now enter the command again 'zz' bit do not to press 'Enter'.
Now a FID appears in the NUTS window.
2) Apply a Baseline Correction by pressing 'bc'. The Baseline Correction algorithm helps flatten the baseline of the spectrum and is required for accurate peak integrations.
3) Apply an Exponential Multiplication factor if necessary.
Exponential Multiply (em) uses an exponential function to improve the signal to noise ratio of the spectrum, and may not always be required. The effects of Exponential Multiply will not be visible until after the spectrum is transformed, first Fourier transformation and probably phase the spectrum, Repeat the process for each Line Broadening change.
To use Exponential Multiply, first specify the Line Broadening factor. Press 'lb' and enter a value (in Hz, maybe 3 to start) then click 'OK'. Now press 'em' to apply the function.
Increasing LB also results in loss of detail in the transformed spectrum, so choosing an optimum value takes a few tries. If the transformed spectrum shows large broad peaks with loss of detail in the multiplets, use a lower LB factor.
Try processing the spectrum with an LB value of zero first. If the resulting spectrum is noisy, cycle through Steps 1-5 and increase LB until the noise level is acceptable.
4) To Fourier transform the FID, press 'ft'. Signal peaks appear in the NUTS window. Peaks may be positive or negative.
5) Adjust the Phase of the spectrum. Press 'ph' and the mouse cursor will change to a crosshair. Hold down the left mouse button and move the cursor left and right until the peak amplitudes are maximized. Press 'Enter' to quit the Phase mode.
6) If the peaks are off scale, press Alt_V_F to set the scaling relative to the largest peak. The phasing may need to be adjusted, after adjusting the spectrum scale. Scaling can also be adjusted using the scroll bar at the right of the window.
7) Identify the TMS or internal reference peak. Double-click the left mouse button to switch to the Zoom mode and the mouse cursor will change to a large cross hair with 'ZO' in the lower right corner. Position the cross hair on the spectrum baseline a little to the left of the TMS peak. Hold down the left mouse button and sweep across the TMS peak from left to right and release the mouse button when the cursor is a little to the right of the TMS peak. The TMS peak will now be highlighted in a red box. Position the cross hair inside the red box and click the right mouse button.
The display will switch to a zoomed view of only the highlighted region, which should be the TMS peak. Press 'Enter' to quit ZOOM mode. Now press 'sz' to set the TMS peak to zero PPM. The scale at the bottom of the spectrum reflect any change.
Click the right mouse button again to return to the full spectrum view.
8) To integrate the peaks, press 'id'. Integrals and numerical values will appear on the peaks and a scroll bar appears at the left of the window. Use the scroll bar to adjust the vertical size of the integrals. Adjust the size of the integrals for clarity.
If the integrations are poor then integrate the peaks manually. First, press Alt_E_A to clear all broken integrals. The integral display will switch to a single continuous integral. Now click the left mouse button once and the cursor will change to a single red vertical line. Position the cursor on the baseline a little to the left of the peak or multiplet to be integrated. Click the left mouse button once (a green line indicates the starting point), then move the cursor to a spot on the baseline a little to the right of the peak or multiplet. Click the left mouse button once to complete the integration. The integral will appear on the peak.
Continue integrating all peaks of interest and use the scroll bar to adjust them to proper vertical height.
If there is a mistake, press Alt_E_L to delete the last integral, or position the red vertical cursor on the integral and press 'd', or press Alt_E_A to clear all integrals and begin again.
Once the integrations are finished, set a numerical value for one of the integrals. NUTS will use this value as a reference for scaling the numerical values of all the other integrals. Click the left mouse button once to get the red vertical line cursor and position it directly on the reference integral. Now press 'v' and enter a value in the 'Current Relative Value' field and click 'OK'. The assigned value will appear next to this integral and scaled values will appear next to all other integrated peaks.
If the reference value is changed for any peak or multiplet then NUTS will always scale the values of all other integrated peaks.
Finish peak integration then, click the 'File' menu and choose 'Exit ID'.
If the integrals is not visible after exiting, then toggle them on and off by clicking the 'View' menu and choosing 'Show Integrals'.
9) Apply Peak Picking by pressing 'pp'.
Peak Picking displays tick marks and relative offsets of all peaks which meet the selection parameters Minimum Height (MH) and Multiples of RMS (RM).
To set the MH parameter, click and hold the left mouse button and position the horizontal line at the top of the smallest peak, then press 'm'. The peak listing will be updated automatically using the new Minimum Height.
RM is used to discriminate between adjacent peaks in multiplets or add/subtract small 'riders' on large peaks. Press 'rm' and then enter a value in the RM field, then click 'OK'. Press 'pp' again to apply the change.
If the tick marks and offsets after Peak picking overlay the peaks, then change the scaling of the spectrum with the scroll bar at the right of the spectrum window.
10) Enter the experimental Data Acquisition Parameters. Click the 'View' menu and choose 'Spectral Parameters'. Fill in the 'Comment', 'User', 'Date', and 'Pulse Experiment' fields and NUTS will complete the rest of the fields with current data acquisition parameters. Click 'UH and Exit' to continue.
11) Add a parameter listing to the spectrum by pressing Alt_V_P. The list appears in the top left of the spectrum.
12) Make sure that the integrals are visible on the spectrum. Press Alt_V_I to show the integrals if they are not visible.
13) Print the spectrum. Click the 'File' menu and choose 'Printer Dialog'. Click 'OK' to print the spectrum. The printer is setup to print in landscape mode, but it can be changed by using the 'Printer Setup' option.
