Office of Research

Center for NMR Spectroscopy

Processing

The two most common operations for manipulating the spectrum display are for expanding regions of the spectrum and for changing the vertical scale. To learn how to change the display regions click the Display tab which brings up the following window:

The Display tab has a multitude of options which allow the spectroscopist to set a reference point in the spectrum, display an axis in either ppm or Hz, integrate the spectrum, peak pick the spectrum and expand spectral regions.

Taller, Shorter, Wider, Thinner

Since the latter is the most often performed operation we will begin with how to expand around peaks in the spectrum. If the spectrum is not already displayed click on the SPECTRUM button in the upper left hand corner. The spectrum should now be displayed with a single red cursor. This cursor can be grabbed and drug about using the Left mouse button. Putting the pointer on the left side of a region you wish to expand will set the cursor to this position. Moving the pointer to the rightmost edge of the desired expansion region and clicking the Right mouse button once bring up a second cursor. This effectively encloses the region for expansion as shown below:

Clicking the red Expand button causes only the enclosed region to be shown as below:

Clicking the blue Full button reverses the process. As with all things this process takes practice. When the two cursors are displayed (box mode) the left button drags the box around and the right button is used to expand the length of the box. To change the vertical scaling of a peak the Middle mouse button is used (on 3 button mice, on 2 button mice depress both button simultaneously) by eiter grabbing and dragging a peak up or down or by placing the pointer directly above the peak and clicking once.

To display a chemical shift axis on the spectrum click the Display Scale button with either the PPM or Hz radio buttons set to allow for PPM or Hz. At this point it is preferable to check / set the reference point of the spectrum to ensure it will be accurately displayed for peak picking.


Phasing your spectrum

The processed spectrum may require phasing to present the data in pure absorption mode. To access the phasing controls click the Display tab which brings up the window shown below:

The phasing controls are the series of button on the right side of the panel. By far the easiest way to phase the spectrum is to click the Auto button. Normally this works quite well unless there is a large peak close to the right edge. To manually phase the spectrum click the Manual button. This allows you to phase the spectrum by clicking on the peaks at the right hand edge with the left mouse button. This gives a "goal post" set of cursors which allow you to set the 0 order phase correction as shown below:

Carefully move towards the left by clicking on the next set of peaks and using the right mouse button to trim the 1rst order phase correction. The furthest peaks on the left should require little if any correction and the spectrum should look as follows:

Once the spectrum has been phased to your satisfaction the Done button is clicked to leave the phasing mode.

If the phasing process gets out of hand and you end up with a large 1st order phase correction you can reset the phase constants to 0 by pressing the Reset button.

The last common function to perform on 1D data is to peak pick the spectrum in order to get a record of the peak positions for determining the chemical shift and for determining J coupling constants.

Setting threshold

Begin by displaying an expanded region of the spectrum unless there are few peaks present. This is to minimize the clutter at the tops of the peaks. Once a suitable region is displayed the threshold for the peak pick must be set. To do this click the Threshold button in the Peaks cluster. A yellow line representing the minimum height for picking will appear as shown below:

The yellow line may be grabbed with the Left mouse button and positioned anywhere in the region. Once the bar is set you can click either the Peaks PPM or the Peaks Hz buttons to see the values on the spectrum as shown below:

For the most part the spectrum should be referenced properly IF you set the solvent parameter correctly at the start of the acquisition process. This is because the deuterium chemical shift of the solvent is used to calculate the position of where TMS should be in your proton spectrum (even if you don't have TMS). It is often a good idea to check the auto referencing and if needed to correct it for any discrepancies.

First find a peak you wish to set the reference to and expand around the peak so that the cursor can be positioned properly at the top of the peak as shown here

(To ensure the cursor finds the tallest point you can type nl followed by a return on the command line)

Now click on the Set Reference button in the Display panel which will bring up a prompt for a new reference to be input on the command line in PPm as shown here:

Enter a new number and hit return, now your spectrum is referenced to the new number.


Integrating the spectrum

One of the most common practices surrounding proton NMR spectra is integration of the peaks to determine the relative number of protons in each set of peaks. Whereas the overal process is simple the practice of getting accurate integrals is not always so. To integrate the spectrum we begin by going to the Display tab and switching the Integration radio button from Off to Partial which causes a green line to occur above the spectrum as shown here:

The task at hand now is to define the regions we wish to integrate so as to be able to compare them. To accomplish this we must click the Resets button in the Integration column. We begin by setting the pointer to the left side of a peak (such as the aromatic group) and clicking the Left mouse button . The green line to the left of the pointer is now a broken line. Moving to the right of the peak the process is repeated leaving just the region over the aromatic group as unbroken. The rest of the peaks are integrated in a similar fashion (note: it is best to work from left to right). The final integrated spectrum is shown below:

Clicking on the brown Refresh Display or on the SPECTRUM button will leave the integration routine. Setting the value of the integral to that of a known number of protons is the next step. Placing the cursor anywhere on one of the green integrals and pressing the Set Int Value button will bring up a prompt to input the new number of protons on the command line as shown here:

Note that the default is 100 which indicates the value of all defined integrals added together. If the default value is used than all integrals are expressed as percents of the whole. After hitting a return the new value is taken and the value for each defined region shown on the screen is output to the bottom window as shown here:

The integral values may be placed under the peaks on the spectrum by pressing the Show Int Values button. If the integration is not to your liking it can be erased by pressing the Clear Integral button which has the effect of removing the break points. Note that when an integral is shown on the screen the Middle mouse button controls the integral intensity and NOT the spectrum. To regain control of the spectral intensity it is neccessary to turn the integrals off temporarily.


Processing your data

All processing of 1D fids can be done via the panel marked Process as shown below:

On this panel are two main topics, the final size of the data and how it may be mathematically massaged to alter the spectral properties.

In the Fourier transform the computer must only deal with powers of 2 in terms of the final FT size. From the data acquisition panel you may have noted that the acquired number of points (parameter np in Varian) is not constrained by a power of two (np = acquisition time / (1/sw))

If the acquired number of points is not equal to a power of two the computer automatically adds a sufficient number of zeros to the end of the data to bring it up to the next power of two. Using the up and down arrows you may set the final FT size to whatever power of two you like. Note that setting the final FT size to less than the acquired number of points will truncate the data and produce what are known as sinc wiggles.

The other portion of the processing panel which is of great use is the apodization functions. Choose a function to multiply the data by by clicking the radio button (more than one can be clicked but be warned that the result may be unusual). After choosing a function click the interactive button to bring up a window like the one below:

You can adjust the function by using the left mouse button and clicking on the green line to the left to increase the function or towards the right to decrease it. Once you are satisfied with the result click the process button to apply the apodization function and the final FT size to your data.

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