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.

 

13C Integration Can Be Very Helpful If Used Wisely

Although 13C spectra are commonly known as not being quantitative, peak integrations can be helpful in many situations. All protonated carbons have similar integration values, as you can see in this example:

13cinteg-aroma

Figure 1 is aromatic area, in which all protonated carbons have integration values of ca. 1.0. All unprotonated carbons have values of 0.4-0.5. The tallest peak is solvent.

13cinteg-aliph

Figure 2 is aliphatic area, in which each carbon (all are protonated) has an area of 1.1, slightly bigger than the protonated aromatic ones, but quite close.

Integrations can also help to solve puzzles of missing peaks. In this molecule, seven aromatic carbon peaks are expected, but only six show up on Figure 1. Why?

13cinteg-aroma2

Let’s look at the 134.6ppm peak closely.  Integration of this is 1.4 – this means that this is not just one protonated carbon (area = 1), but contains two overlapping peaks – one protonated and one unprotonated, which would have a total area of ca. 1.4. This solves the mystery of the missing carbon.

 

Processing T1/T2, kinetics, and diffusion data using Dynamics Center

Topspin’s new feature Dynamics Center does everything the old T1/T2 module does, and with much more capability and much less buggy issues. It also processes diffusion data better than the dosy2d command.

To set up T1/T2/kinetics experiments, please visit this entry. An example of a kinetics experiment can be found here.

Click here for instructions on how to set up diffusion experiments.

The data from these experiments are in pseudo-2D format. Each pseudo-2D file consists of many 1D spectra, each of which is called a slice. To process these data, follow these steps:

First, you need to properly phase correct your spectra. When done, examine each slice of the pseudo-2D file in Multiple Display mode. Scan through each slice to see if phase correction is good.

Next, you need to perform baseline correction. This is especially imperative if you are tracking some small peaks when there are other much larger peaks present on the spectra, in which imperfect baseline will severely skew your results.

Then you can launch the Dynamics Center in Analysis -> T1/T2 module -> Dynamics Center. Or you can type dync at the command line. It opens up a new window “Dynamics Center”:
dyncenter

Click the option that you need (T1; T2; diffusion; etc). Then click each step within the option: Sample -> Data -> …  For the Data step, you will need to look for the “2rr” file that has been generated by your experiment, as shown in the above figure (unfortunately there are many layers of folders that you will have to dig through). The 2rr file stores the pseudo-2D data, which contains a number of 1D spectra.

Using peak areas is often more desired than using peak intensities, especially your target peaks are broad and ugly (which is actually often the case for interesting objects of relaxation and diffusion study!). In the Data step, after finding the 2rr file in the Spectra tab, click the Integrals tab, select “Use peak areas (user defined) integrals”.

dync-select-integral

After clicking OK, a spectrum will be displayed. You can define your interested peaks and peak integration limits here. There might be a number of peaks that the computer has picked for you that you don’t really care about. Move cursor near one of the peaks, right click, select Delete in a region. Then drag a box around all the computer-picked peaks that you want to get rid of. This will delete these selections.

dync-define-integral

Next, select the peaks that you are interested in. move the cursor near the peak, right click, then select Add peak integration area. A black bar will appear on the bottom of the peak (see the figure below). You can drag the bar around or resize it.  Repeat for all the peaks you are interested in. You could also right click near a peak and select “Resize All” option to change the width of the integral for all the peaks.

selectpeakarea

In Analysis, you can define the function that you want to fit, and various fitting parameters. For T1 experiment by Inversion Recovery, pick the following fitting function:

t1function

In View, you can define various options for display. Then you should get a window like this:

dyncenter2

You can get a decay curve for each peak you pick (lower left window; move your mouse to other defined peaks in the upper left window to see decay curves for other peaks in the lower left window), and a 2D spectrum (upper right window) with the vertical dimension displaying the dynamics information (T1, T2, diffusion coefficients, etc). For diffusion data, the upper right figure would be a DOSY spectrum (I think the Dynamics Center does a better job than the “dosy2d” command).

The Report function summarizes all the fitting results. You should pay particular attention to the “error” column, which is the standard deviation of your fitting. Usually, relative standard deviation should be < 5% to indicate a high-quality fit. High errors usually indicate either poor data processing (phase correction; baseline correction) or multiple dynamic components existing in your sample.

The Export function can export the fitting results to an Excel file.

The Help menu has a manual in which you can find the information on how to navigate this relatively easy software package.

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