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Tuesday, March 11, 2014

Chemical Exchange Agents to Simplify NMR Spectra

One can simplify 1H NMR spectra by eliminating exchangeable proton signals.  This is most commonly done by adding a drop or two of D2O to the NMR sample.  An example of this can be seen in a previous post.  The deuterium from the D2O replaces the exchangeable protons (-OH, -NH, -NH2, -COOH) of the sample and their 1H signals disappear.  The disadvantage of this technique is the introduction of a strong HDO signal which may overlap with other signals in the spectrum and thereby hinder the interpretation.

An alternative of the "D2O shake" is to add a drop or two of concentrated trifluoroacetic acid (TFA) to the sample.  TFA has a single exchangeable proton at ~ 14 - 16 ppm.  The -COOH proton of the TFA exchanges with the exchangeable protons in the sample.  The exchange rate is usually fast enough on the NMR time scale such that the resultant spectrum has a single broad resonance representing all of the exchangeable protons at a chemical shift between the chemical shift of the pure TFA and that of the exchangeable protons in the sample (usually >10 ppm depending on the sample and the amount of TFA added).  The broad peak at a shift > 10 ppm is not likely to overlap with other resonances in the spectrum and therefore will not hinder the interpretation of the data.  An example of the use of TFA is shown in the figure below.


The bottom spectrum is that of sucrose dissolved in DMSO-d6.  One can observe all of the -OH protons in addition to all of the other sugar protons.  The middle spectrum is that of pure TFA in DMSO-d6.  The -COOH resonance appears at ~ 15.6 ppm.  The top spectrum is that of sucrose in DMSO-d6 with a drop of TFA added.  One can see that all of the -OH protons of the sugar (highlighted in yellow) have combined with the -COOH resonance of the TFA yielding a single broad resonance at ~ 13 ppm as a result of the exchange.  In addition to moving the -OH resonances out of the way, one can see simplifications to the other sugar protons as the result of loosing the J coupling between the -OH protons and the remaining sugar protons.

A comparison of the use of TFA compared to D2O as an exchange agent is shown in the figure below.


Both methods produce similar results except that the spectrum with added D2O has a large HDO peak (off-scale in the figure) which overlaps with other signals.

12 comments:

John Trant said...

This is a good idea. I don't know why I haven't thought of doing this before. I like using TFA as an NMR co-solvent for some particularly insoluble materials, but never thought of this effect. Excellent and educational as always Glenn!

Anonymous said...

Glenn, your posts are always very helpful.
I've got 2 questions:
1. Is it permitted to use TFA-deuterated instead of TFA?
2. Can I use TFA as "shake agent" with sample dissolved in Pyridine-d5? I suppose that chemical shift of pyridine will change noticeably with lowering pH.
Regards
Luke

Glenn Facey said...

Luke,

Thank you for the questions.
1. Yes, you can certainly use deuterated TFA . In my experience though the extra cost is not worth it unless you are interested in resonances at very hight chemical shifts.
2. Yes, you should be able to use TFA as an exchange agent for samples dissolved in pyridine. Even if it is not soluble a bit of shaking should facilitate the exchange.

Glenn

zaheer said...

In case of poly(L-glutamic acid) in TFA-d the peaks of NH-CO, NH2 and COOH dispear.Can some one explain it for me.

Zaheer Ahmad

saubanzaheer@gmail.com

Glenn Facey said...

Zaheer,

The deuterium from your TFA-d has exchanged with the exchangeable protons in your sample effectively deuterating them and making them invisible in the proton spectrum.

Glenn

Yuan Li said...

Glenn,

I used to run some samples with deuterated TFA, but found I could never shim it well. Neither manually nor with bruker automatic shim program. Do you have idea what could be the reason for that? TFA-d somehow is not in our regular solvent list, so I had to add this solvent and set up its lock parameters myself. Maybe it was not correctly locked?

Yuan

Glenn Facey said...

Yuan,
Thank you for the very good question. The chemical shift of the deuterium in TFA-d is extremely dependent on whether or not it is exchanging with any other exchangeable protons in the sample. It could be between ~16 ppm and ~5 ppm. This probably explains why TFA-d is not present in the standard lock solvent tables and will account for the possibility of having difficulty establishing the lock. Furthermore the deuterium resonance of TFA-d is very broad and insensitive to magnet homogeneity compared to a sharp deuterium resonance (such as that from acetone-d6). When you shim manually, you monitor the height of the deuterium resonance of the TFA-d in the lock channel. Since the 2H resonance is broad, its height is not very sensitive to magnet homogeneity. The gradient shimming routines are also less effective for broad resonances. This may be a good subject for a future BLOG post.

Glenn

Yuan Li said...

Glenn,

Thank you for the helpful information! I think you are right, the lock signal of TFA-d does not respond to the magnetic field changes very much. Based on my experience, the lock signal almost doesn't increase with changing shim values, though it will start to drop at some point.

Yuan

Mike Bernstein said...

Glenn,

You clearly have raised a good, interesting, and helpful point. Thanks, and keep them coming.

Here's my request: tricks to cause (hetero)aromatic proton peaks to shift and (sometimes) become easier to assign. TFA was the trigger for the suggestion!


Mike

Glenn Facey said...

Mike,

Thank you for the comment. With regard to your suggestion, take a look at this earlier post:

http://u-of-o-nmr-facility.blogspot.ca/2007/10/improve-your-chemical-shift-resolution.html

Cheers,
Glenn

Corey R. Johnson said...

Will glacial acetic acid work similarly?

Glenn Facey said...

Hi Corey,

Yes, glacial acetic acid would work in the same way as TFA however in using it you would introduce a huge methyl resonance from the acetic acid which may overwhelm the spectrum.

Glenn