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Thursday, November 2, 2017

CPMG and Red Blood Cells

In a previous post it was shown how one could discriminate against broad NMR lines and emphasize sharp NMR lines by using a CPMG T2 filter.  This technique has tremendous power for getting detailed information from difficult samples.  Biological samples containing in-tact cells can be difficult because they contain semi-solid components with extremely short T2's and liquid components with dissolved compounds having much longer T2's.  The CPMG technique was used as early as 1988 to study red blood cells.*  The figure below shows 600 MHz 1H NMR data collected at uOttawa on a sample of in-tact red blood cells.  The sample, obtained from centrifuged human blood, was paste-like and contained no solvent.  The magnet was shimmed using proton gradient shimming and the data were collected without the deuterium lock.
The top panel of the figure shows the result obtained with a standard one-pulse measurement. Clearly, the spectrum shows very little detail and is dominated by the water resonance.  The bottom panel shows the CPMG spectrum obtained after 350 echos using an echo delay time (2τ = 640 µsec).  One can clearly see the suppression of all the broad signals (including the water signal) with short T2's leaving the incredibly detailed and high resolution spectrum of the soluble compounds dissolved in the liquid component of the red blood cells (amino acids, sugars etc...).  The water signal is almost completely suppressed.

Thank you to Madeleine Adam of Dr. Robert Ben's research group at uOttawa for providing the sample.

* D.L. Rabenstein, K.K. Mills and E.J. Strauss, Analytical Chemistry, 60(24), 1380 A, (1988).


Unknown said...

Thank you, Glenn.
This is a nice CPMG spectrum of RBCs! Just as note, even a single spin echo pulse sequence would allow to reveal small molecules (metabolites) in this kind of samples with intact cells [Brown et al. (1977) Human erythrocyte metabolism studies by 1H spin echo NMR. Febs. Lett. 82:12-16], but you do get some extra effects on the peak lineshapes/intensities due to H-H J-couplings...

Glenn Facey said...

Thank you for your comment. Indeed a single echo, can be an effective T2 filter and in fact, the authors of the paper I referenced in the blog post have used single spin echos as well. The problem, as you indicated, is J modulation during the longer delays required in a single spin echo, leading to spectra with phasing problems.


Steve said...

Very nice article.

I tried to follow the reference, but the citation isn't exactly correct.

The correct citation is Anal. Chem. 1988, 60(24), 1380A-1391A DOI: 10.1021/ac00175a713

The reference as-cited in the blog post goes to this article.

Hope this helps!


Glenn Facey said...

Thank you Steve!
I have updated the post to correct the error.


Unknown said...

Interesting, it would also be interesting to extend this by using Gareth Morris's new REST sequence ( that uses T2 times like the diffusion rates in a DOSY to separate signals) on a Blood cell to see much of the overall content could be resolved.!divAbstract

Glenn Facey said...

Hi Matt,
Thanks for the reference. I had not seen it. Yes, I think it would be a neat thing to try. Perhaps it could distinguish between the larger sugars and smaller amino acids.