Multiple Solvent Suppression

Some times it is difficult to completely get rid of the protonated solvent in your sample, which results in huge peaks that could overwhelm your solute signals. In such cases you can perform a solvent suppression technique. Many solvent suppression techniques are available. An easy and effective technique is called WET, which also has the ability to suppress multiple solvent peaks. Follow these steps to set it up:

  1. Lock and shim your sample. Good shimming will assure optimal solvent suppression. Collect a proton spectrum, which you will use to define the peak regions that you wish to suppress.
  2. With the proton spectrum on the screen, click Acquire in the main menu, Click Options, then select Setup Selective 1D Expts:

3. Click 1D Selective Experiment Setup. You will see an instruction of the expt setup. Read it to get a basic idea. Then click Close.

4. Click Define Regions. You are given the integration module for this task, though your job here is not integrating the peak areas, but rather to select the peak regions that you wish to suppress. I suggest that you select a narrow region around each solvent peak which covers most of the peak intensity, like shown below. Don’t worry too much about the tails. If you select a wide region, you will suppress solute peaks in the region. It is always a good idea to be a little conservative in the beginning. When done, click Save Region as…, and select “Save regions to ‘reg’“. Then click Save and Return.

5. Click Create Datasets, then select “Mult. Solvent Suppr./WET

6. It will ask you NS and first EXPNO (experiment number). Usually NS of 16 is good enough. For first EXPNO, give an experiment number that you have not used (let’s say 3).

7. A window shows up summarizing the peak position that you have selected for suppression. Click Cancel – you will need to do atma (to tune 13C, as the WET uses 13C decoupling to remove the 13C satellites of the solvent peaks) and customize some parameters before you run the expt.

8. Issue command “re 3“, which will read in experiment #3, which you just created. Perform atma which will tune both 13C and 1H. Then type d1 and change it to 6 or 10. Since the WET experiment uses 90 deg excitation, T1 relaxation for many solutes might not be complete for the default d1 of 3 s, so it is safer to use a longer d1.

9. Do rga, then zg.

In the following picture, the blue spectrum is a proton spectrum of a sample with protonated DMF. The red spectrum is a WET spectrum which have the three DMF signals suppressed.

Why don’t some protons have signals on 2D spectra?

This small molecule has good signals on 1H-15N HMBC:

However, upon incorporation into a polymer, the region expected to show correlation peaks is completely noise:

Why?

If you look at the 1D proton signals, plotted on top of the 2D spectra, the aromatic protons in the small molecule have sharp peaks, while those in the polymer have much broader peaks. The broadening is due to short T2. A simple though not the most rigorous way to estimate T2 is T2 = 1/(pi*Delta), while Delta is proton peak width in Hz.

Some 2D pulse sequences keep magnetizations on the xy plane for a long time (a few ms to > 100 ms). For protons with short T2, unfortunately, their signals cannot survive these pulse sequences. HMBC has the harshest requirements for T2.

While the no-show on 2D spectra is a bit disappointing, a short T2 does tell you important information about the dynamics of your molecules – it often indicates that they interact poorly with the solvent and are aggregating.

Should I wipe clean my NMR tube?

Dirt stains become little magnets once inserted into the big magnet and severely distorts the magnetic field homogeneity, and such distortions cannot be fixed by topshim or manual shimming. See the spectra below.

Red: Before wiping.

Blue: After wiping with KimWipe(r).

How to manual shim

For the new generation of NMR users who are not familiar with manual shimming:

  1. In Topspin, lock the sample
  2. Type lockdisp to display the lock window.
  3. type bsmsdisp to display the shim window.
  4. In the bsmsdisp window, find the buttons z and z2
  5. Click z. Click Step Size. Change it to 10
  6. Click the + or – button to adjust z value and watch if the lock line is going higher or lower. Adjust z such that lock line goes higher. Do it until the lock line does not go any higher.
  7. Click z2. Change step size to 10. Repeat step 6 for z2.
  8. Click z and repeat step 6.
  9. Repeat steps 6 – 8 until the lock level does not go any higher. you are done.

How To Calibrate 90deg pulse

Type edc and select the experiment H90CALIB. This is a single-scan, single-pulse 1H experiment. Check the getprosol box. Acquire a spectrum. This only takes 10 seconds. Correct phase using apk.

Create another file using edc, and this time select the “Use Current Parameter” option. This will copy all the acquisition and processing parameters from the current file to the new one, including receiver gain, phase correction amount etc. Type p1 to view the 90deg pulse length. It should be somewhere around 12 us (microseconds). Double this value and enter it into the box. E.g. if the current value is 12.5, change it to 25 (the default unit is us). Acquire a spectrum. Don’t correct phase or do rga because we want to keep all the parameters the same for an exact comparison of this one against the prior one.

Use Multiple Display to compare the above two spectra. If the second spectrum has much smaller intensity than the first but is positive, the flip angle is less than 180deg, and you need to slightly increase the p1 (e.g. from 25 to 25.6) and observe again. If the second spectrum is negative, the flip angle is more than 180deg, and you need to slightly cut p1. Repeat until the final spectrum has a pretty much zero intensity or half-positive, half-negative (which is quite common). You have found a good 180deg pulse length. Let’s say it is 23.2 us. This means that the 90deg length should be 11.6 us.

md-1d

In the picture above, the red spectrum uses a p1 that doubles that of the blue one. The sharp solvent peaks become essentially zero on the red spectrum, which means that its pulse length is a pretty good 180 degrees. There is some broad hump on the red spectrum, which might be background signal which you can ignore.

Correct Baseline for Pseudo-2D Data

Baseline correction is essential for T1, T2 and diffusion experiments as our target peaks are often broad and short, so any baseline imperfection can cause large errors.

Baseline correction is done by computer fitting of the baseline by a fifth order polynomial: y = A + Bx + Cx2 + Dx3 +Ex4 + Fx5. Since the baseline shapes cannot always be perfectly fit by such a polynomial and sometimes some peaks are so broad that the computer is having a hard time decide whether it is a peak or part of the baseline, we need to be aware of the limitations of the fitting algorithm and tweak strategy if the correction result is not good.

To check if your slices have good baselines, go to the Multiple Display mode. Make the peaks very tall so you can see the baseline well. When you do Scan Rows, zero intensity of each spectrum is at the middle line of the window. This means that a good baseline would not shift downward or upward if you adjust peak heights (remember: ten times zero is zero!). If, when you left click to make the peaks taller, the baseline sinks or rises, the baseline is not good and needs correction. Note that the vertical scale is erroneous – zero intensity of a slice is at the middle line of the window, while the 0 as indicated by the scale is at the bottom of the window.

While in the Multiple Display mode, you need to decide two factors for baseline correction: (1) a chemical shift range of spectrum in which you want baseline correction. Since it is often difficult to perfectly correct baseline across the entire spectrum, only correcting a smaller range often produces better result. For this, you will need to properly choose the left and right limit of the range that you want the baseline correction be done. Choose the limits such that tails of peaks do not extend there, i.e., there is only baseline or noise near there. In the figure below, I chose the limits to be 4.5 and 0 ppm. Write your choices down for the next step. (2) shape of the polynomial. I often find that lower orders of polynomial (linear, which means I only use the shape A + Bx; or quadratic, which means I only use the shape A + Bx + Cx2) do a better job than the ones that use all five orders, which often overdo the job.

Exit Multiple Display mode.

Click the tab ProcBars (on top row of the spectrum window). In Baseline Correction category, set ABSG to 1. This means that you allow the computer to fit the baseline with only a linear function (A + Bx), not the entire five orders of polynomial. I found this usually produces good result. Sometimes 2 is better. Experiment with it yourself. Set ABSF1 and ABSF2 to the left and right limits of your choice.

Click the Spectrum tab. On the command line, type abs2. This will perform an automatic baseline correction in the range of spectrum that you chose. Go back to Multiple Display and check the slices. It should look like the figure below. Note that the baseline of the spectrum between 4.5 and 0 ppm has been corrected. There are step changes at 4.5 and 0 ppm, which is because the spectrum within the window has been corrected for baseline and that outside the window still has very negative baseline. This is normal.

baseline

If you don’t like the correction result, just type xf2 again. This will erase all the baseline corrections that you have done. Start fresh from here.

The Multiple Display Function

The Multiple Display module can be accessed by clicking the Topspin icon that shows two spectra on top of each other. This module has two very useful functions: 1. compare a number of different 1D spectra; and 2. view each 1D slice of a 2D or pseudo-2D spectrum.

To compare two 1D spectra, let’s say spectrum A and B, (1) load A onto the display; (2) click Multiple Display button; (3) find B in the browser, and drag it into the Multiple Display window. See the picture below. You can highlight one of the spectrum in the lower left window (the one below the Browser), then use the buttons to move or rescale it.

md-1d

To view each slice of a 2D or pseudo-2D spectrum, first go into Multiple Display mode, then click the Scan Rows button (the highlighted button in the figure below). Then hover the mouse above the contour lines and you will see the slice. The first slice is at the very bottom while the last slice is at the top. You can use left click to make the peaks bigger, or click the mouse wheel to make them smaller. Making the peaks taller will help you better see if phase correction and/or baseline correction is needed.

scanrows

The picture below shows one slice (slice #2, as the info box on the upper left corner shows “Index = 2”) of a pseudo-2D file.

baseline

Phase Correction for Pseudo-2D data

A pseudo-2D file is very similar to a 2D file, with the only difference being that the former only does Fourier Transformation in the horizontal dimension (by command xf2) while the latter needs FT in both dimensions (by command xfb). The data in these files are arranged in a matrix, with rows and columns. In pseudo-2D data, we only do phase correction and baseline correction on rows.

You can phase correct a pseudo-2D file by clicking the menu Processing -> Phase Correction, or by clicking the icon with a little distorted peak picture. In the new window, you need to select 2-3 slices as “representatives” of all the slices. Right click on the bottom-most slice, which is the first slice (you can tell that by the message “row: … Index = 1” in the info window on the top left corner), select Add. You will see a red circle being marked for the slice. Move mouse to one of the middle slices, repeat the Add operation. Repeat again for the last slice. Then click the button “R” (which means phase correcting rows), and you will see a window showing the three slices that you picked, like this:

phasecorr

Now you can phase correct the pseudo-2D file just like the way you correct 1D files. Drag the button 0 and 1 up and down so that all the peaks on all the slices have positive intensity and the baseline are not distorted. Click the Save and Return button. Then click Return, to go back to the main menu.

Note that for Inversion Recovery experiments, the first few rows are negative.

Then use the Multiple Display function to visually make sure each slice indeed has good phase. You are done.

 

What to do if you have insufficient solvent

Normally, you should have 0.4-0.5ml of solvent, which is ca. 3-4cm tall in the NMR tube. If you have less than 3cm of sample, please follow these steps:

– instead of rsh shims.best, do rsh shims.short

– always lock after you do rsh

– You can try topshim, but watch if the lock line gets higher or lower after topshim. If it gets lower, topshim did not work, and you need to type rsh shims.short again to get back the better shim. Topshim does not always work for short samples.

– If topshim did not work, type bsmsdisp, and adjust z and z2 to get your lock level higher. z2 is the most important for short samples

– Shim is never perfect for short samples, so you need to be prepared to see low resolution on your spectrum. If every peak has the same tail, asymmetric lineshape, or fine splitting, it is mostly like a shimming problem.