Advanced Baseline Correction

In some cases the automatic baseline correction command abs does not generate a desired baseline. This often happens when some of your peaks are broad and the correction algorithm has a hard time decide whether some data points in a certain spectral area is signal or baseline. In such cases, you can try an advanced baseline correction method: bas. Upon giving the command bas, you are offered a number of choices. I use the first and the third choices most often.

Choice 1, manual baseline correction. The correction is done by manual fitting of the baseline by a fifth order polynomial: y = A + Bx + Cx2 + Dx3 +Ex4 + Fx5. First, you display the range of the spectrum that you want to correct (note: you can baseline correct a small range of the spectrum instead of the entire spectrum). Second, You drag each of the five buttons A, B, C, D, and E to adjust the red baseline until it passes through the spectral baseline. I found that adjusting A, B, and C is often good enough. Finally, you click the “Return, Save regions” button, which will perform the correction. This option is very useful if you want to integrate a small peak riding on the shoulder of a big peak.

Choice 3, “Auto Correct Spectral Range…. only”. You need to decide two factors for this correction method: (1) a chemical shift range of spectrum for which you want baseline correction (ABSF1 and ABSF2). 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 there are at least 0.5 ppm wide of signal-free regions at both ends of your spectral window. This will help the correction algorithm to recognize the baseline. (2) Shape of the polynomial (ABSG). I often find that lower orders of polynomial (0, which means that I only correct a constant offset A; 1, which means I only use the shape A + Bx; or 2, 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.

Multi-Component Fitting of a Curve

We often encounter decay behaviors that cannot be accurately described by a single exponential curve, e.g. in T1 relaxation curves and in diffusion NMR data. In such cases, multi-component fitting of the curves could be attempted.

See the following diffusion NMR data:

There is clearly a systematic deviation between the experiment data (black dots) and the single-exponential fit (red straight line). The resulting diffusion coefficient (D) has a relative error bar of ca. 5%. Upon close visual examination, we can notice that the experimental data decays faster in the beginning and decays slower toward the end. So a two-component fit might produce better results.

The equation of the two-component fit function can be seen in the inset of the figure above. The fitting quality is clearly much better. The reduced chi-square is two orders of magnitude smaller. A1 and A2 are the amplitudes of the two components, while D1 and D2 are their diffusion coefficients. Comparison of the two figures shows that a two-component system is a much better representation of the sample.

How to run a 2H experiment

If you want to investigate the structure and behaviors of deuterium in your molecules, 2H NMR is a good method. The chemical shift of 2H is very similar to that of 1H as both nuclei experience the same electron environments. The resolution is also often as good as that for 1H.

Follow these steps:

  1. Type edc. In Experiment, choose H2. Check getprosol box. Create a new file for 2H experiment.
  2. Type rsh shims.best
  3. If your sample is dissolved in a deuterated solvent, do the regular lock and shim
  4. If your sample is not in a deuterated solvent, type ii. This will stop the locking mechanism from interfering with your 2H experiment. Often, your spectral resolution is already good enough so that you do not need to further shim. If you need high resolution, you will need to manually shim on the FID: Type gs (similar to zg but without accumulating data, used for real time adjustment of various parameters), then adjust z and z2 in bsmsdisp window to make the FID as long and thick as possible.
  5. Type atma
  6. Type rga
  7. Run your experiment. Adjust d1, ns, and lb as needed.

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.

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.
  10. Sometimes after you adjust, the lock signal goes out of the top of the window. When this happens, find the Auto – Gain button and click once. This will automatically adjust the lock signal gain and bring the lock signal back in view.

How To Calibrate 90deg pulse

The calibration of 90deg pulse is done by determining the length of the 180deg pulse then dividing it by 2 (as a 90deg pulse is half the length of the 180deg 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 (either apk or manually) 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.

Note: though your ultimate interest might be a small peak on the spectrum, during pulse calibration you need to watch the changes of the largest peak on the spectrum, which might be a solvent peak.

How to Overcome the Convection Evil in DOSY Experiments

Related link: Setting Up DOSY Experiments

Related link: Dynamics Center

Convection can be quite bad in low-viscosity solvents such as CDCl3 and acetone-d6. It yields a diffusion coefficient (D) that is artificially larger than the actual value and the apparent D could vary with varying d20. To minimize convection, please follow these suggestions:

  1. Use higher viscosity solvents if possible. D2O and DMSO have much less convection than CDCl3.
  2. Minimize sample height, which will minimize vertical temperature gradient and thus reduce convection. My recommendation is 2.5cm of sample height, which reduces convection while do not sacrifice spectral resolution too much (peak width of 1.5-2 Hz can be achieved).
  3. Recommend use 3mm tubes if you must use CDCl3 or acetone-d6 as solvents. Reducing cross section area of the liquid can drastically reduce convection.

 

A Fast Way of Nitrogen NMR

As 14N is a difficult nucleus to work with, 15N is usually used for nitrogen NMR. 15N has very low natural abundance (ca. 0.36%). In addition, 15N has  low gyromagnetic ratio, which makes 15N signal sensitivity a great challenge.

Due to the low gyromagnetic ratio of 15N, a 2D 1H-15N spectrum often takes less time than a 1D 15N spectrum. The most useful 2D 1H-15N technique is HMBC. It is fairly easy to run:

  1. In edc, click Experiment, and select HMBCGP_15N
  2. be sure to check “getprosol”
  3. run the experiment like any other NMR experiments: lock, shim, atma, rga, zg.
  4. When the experiment is running, you can type xfb to check the spectrum.

Following is a 1H-15N HMBC spectrum of 50mM natural abundance cyclosporin, which takes only 20 minutes on the 500. All eleven nitrogen peaks can be resolved, along with their 1H neighbors. Note: 1-bond NH pairs appear at doublets, while 2-bond and 3-bond NH pairs appear as singlets. Weaker peaks are likely 3-bond NH pairs.

hmbc-hn-cyclosporin

For Quantitative NMR Work: How to Estimate T1

You might have heard that in order for your integrations to be quantitative, T1 relaxation has to be complete. But how do you know it is complete? The measurement is easy. Set ds = 4 (ds: dummy scans), ns = a minimum number that you can get an OK signal (for proton, 4 is often good enough). Compare the integrations obtained with d1 = 1 s and 2 s. If they are identical, then T1 relaxation is complete at d1 = 1 s. If the latter is bigger, Repeat the comparison of the integrations obtained with d1 = 2 s and 5 s. If the latter is bigger, repeat the process until you have two d1 values which produce the same integrations. The lower of the two in your final round of comparison represents the d1 for which the T1 relaxation is complete.

This works for both the standard 1H and 13C techniques.

Please note that the above trick is not a strict measurement of T1 values. To accurately measure T1, please read this post.