The CPMG filter NMR experiment! What is it? When and why should we use it?

The CPMG filter is an NMR building block that can be incorporated within a 1D ¹H NMR pulse sequence with or without solvent suppression. As the name says, it acts as a filter, removing molecules with short T₂ relaxation values of a few milliseconds or less from the 1D NMR spectrum and observing only molecules with long T₂ relaxation values in the order of seconds.

Let’s remember that T₂ values are inversely proportional to the full-width at half-height (FWHH) of the NMR signal. To put this in perspective, an NMR peak that has a T₂ of 3 s would have a LW_1/2 in the range of 0.3 Hz, but another NMR peak with a T₂ of 1 ms would have a LW_1/2 in the range of 1000 Hz (Scheme 1).

Scheme 1: Simulated spectrum of a small molecule (long T₂ -> sharp signal) in the presence of a big molecule (short T₂ -> broad signal).

As described above, the presence of a molecule with a short T₂ has such a broad signal that prevents any quantitative analysis and, in some cases, even obscures the presence of analytes present in small concentrations. That’s the reason why in many applications it’s necessary to filter out the big molecules.

A real example would be a sample where a large molecule (protein, polymers, etc.) and a small molecule (active pharmaceutical ingredient, solvent, impurities, etc.) are present. In this blog, a sample of Bovine Serum Albumin (BSA) and histidine in D₂O was used (Figure 1) as an example to illustrate the application of a CPMG filter here combined with solvent suppression (WET).

Figure 1: A sample of BSA (3mM) and histidine (60mM) in D₂O was evaluated in the 100 MHz benchtop NMR instrument. Superimposed spectra: ¹H 1D spectrum and a set of CPMG-filter-WET with different filter duration (10, 50, 100, 150, 200, and 250 ms).

In this example, a CPMG filter of around 150 ms was enough to suppress the broad signals, allowing the analysis of the signals from the small molecules. In addition to the histidine signals, it was observed the presence of ethanol as an impurity from the purification of the BSA.

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