Category Archives: techniques

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.

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.

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.

A very easy and quick NOESY to probe stereo chemistry

There is a much easier way to run NOESY than the old 1D NOE-difference and the 2D NOESY:

  1. first, run a proton spectrum.
  2. with the spectrum on screen, click Acquire, then click Options, then click Setup Selective 1D Experiments.
  3. Follow the menu and its prompts; select the peak(s) that you want to probe.
  4. Click Create Datasets, select Selective gradient NOESY:

selnoesy-setup5. the computer will ask you to confirm some experimental parameters. The default choices are usually good. For each of the peak area that you select, one experiment will be run, and it will be stored under the same sample name, in incremental experiment numbers.

Following is a proton spectrum (blue) of pamoic acid and two selective NOESY spectra. Each took less than 1 minute. You can immediately see which proton is spatially close to the targeted protons (the large negative peak).

pamacid-selnoesy

Setting up DOSY experiments

Related link: How to minimize DOSY artifacts due to convection

 

1. In Topspin, go to Help -> Manuals, find “DOSY” handout. Read and understand the principle.

2. Decide a suitable d20 (big delta) for your experiment. In edc window, click Experiment. in “…/user” directory, choose experiment DIFFUSION1D. check getprosol box.

3. lock; shim; atma; rga

4. Run a spectrum. This one is with gpz6 = 2 (default; means 2% of the gradient amp power is used).

5. Create a new experiment by edc and choose “Use current parameters“. Don’t do rga. type gpz6 and enter 95 (give the gradient amp 95% of the power). run the experiment.

5a. If the tall peaks have negative spikes on their sides that cannot be fixed by phase correction, type loopadj, then rerun the experiments.

6. compare the two spectra with 2% and 95% gradient powers. if the latter is less than 1% of of the former, you need to reduce d20. If the latter is greater than 10% of the former, you need to increase d20. d20 could range from 0.02 s to 0.5 s. every time you change d20, you need to rerun both experiments.

7. Once an optimal d20 is decided, create another new file by choosing DIFFUSION2D. change d20 to the value that you have determined. enter a suitable NS (should be multiple of 16). enter command dosy 2 95 16 q y y. This is a macro that will run a 2D DOSY expt. Meaning of the parameters: 2: beginning gradient amplitude = 2%; 95: ending gradient amplitude = 95%; 16: number of 1d slices = 16; q: increment is square rooted (alternatively, you could do l for linear increment); the first y: begin acquisition; the second y: do a rga before the run. For more details, please read the Bruker brochure mentioned in step 1.

8. when done, click ProcPars tab and change SI of F1 column to 64. In command line, type xf2 to Fourier transform (only in the 2nd dimension). Correct phase. Read Bruker manual to learn how to phase a 2D spectrum. Only phase-correct rows.

9. type setdiffparm. This sends the experimental parameters that you used to Dynamics Center for data fitting.

10. on main menu, click Analysis -> T1/T2 module -> Dynamics Center. Follow the flow in the new window. Instruction on using Dynamics Center can be found here.

Following is a DOSY spectrum of cyclosoprin-A in benzene-d6. Most peaks are in the upper row while there is only one peak in the lower row. Why?

dosy-cyclosporin

Kinetics Experiments on the Bruker 500

Following is a 31P kinetics spectrum run on the 500, collected by Zak Page. Over the course of the reaction, the decrease of the reactant peaks at 28 ppm, the increase of the product peaks at 1 ppm, and the first increase, then decrease of the intermediate signal at 27 ppm can be clearly seen. The time scale of the F1 dimension is erroneous (a software glitch).

kinetics-31P-ZakPage

 

19F with 1H Decoupling

(Unfortunately, this experiment cannot be run on the 500 cryoprobe)

Comparing 19F spectra with and without 1H decoupling can probe the interaction between 19F and neighboring 1H.

Here is a comparison. Sample: (CF3)2COOH in 90/10 H2O/D2O. Note that the 19F-1H J coupling is ca. 8 Hz even though they are four bonds away. (run on the old DPX300. Now gone)

F19-CPD