Water signal displacement in 1H NMR spectroscopy using trifluoroacetic acid

In ¹H nuclear magnetic resonance (NMR) spectroscopy, it is common to observe overlapping between signals arising from different protons in the molecules being analyzed. While these protons might be chemically and magnetically inequivalent, thereby giving rise to unique and distinct signals, the relatively narrow chemical shift range inherent to ¹H NMR spectroscopy means that almost all proton signals will appear in a range between δ = -1 to +12 ppm.¹ Of course, exceptions exist, especially for paramagnetic species, but this range encompasses most signals.

Since signal dispersion in NMR spectroscopy is directly proportional to the magnetic field strength of the spectrometer, a common way of resolving overlapping signals is to use an instrument with a larger ¹H operating frequency. However, this is not always feasible, especially considering the large upfront and recurring costs required to purchase and maintain these traditional high-field NMR spectrometers. An alternative method to influence signal separation in ¹H NMR experiments is to use a reagent like trifluoroacetic acid.²

Trifluoroacetic acid is a very strong acid, easily capable of participating in hydrogen bonding with other functionalities bearing labile protons. When added to an NMR sample containing such labile protons, all signals arising from exchangeable protons will be displaced to an average chemical shift and appear as a single, broad signal. To illustrate this, a sample of diclofenac sodium (100 mM) in DMSO-d₆ was prepared, and its ¹H spectrum was collected (Figure 1, top). Then, 15 μL of trifluoroacetic acid were added to the sample, and its ¹H spectrum was re-collected using the same acquisition parameters (Figure 1, bottom).

Prior to the addition of trifluoroacetic acid, the methylene signal (δ = 3.4 ppm) overlaps significantly with the water signal (δ = 3.3 ppm). This makes accurate integration nearly impossible and makes it more difficult to extract chemical information from the spectrum. However, upon addition of trifluoroacetic acid, all labile protons in the spectrum are averaged to a single chemical shift (δ = 10 ppm). Importantly, the extent of the displacement of the water signal is related to the relative amounts of trifluoroacetic acid and water in solution, as previously reported.²

Note: trifluoroacetic acid is highly corrosive, harmful when inhaled, and causes severe skin burns. Caution must be exercised when handling and disposing of this strong acid.³

Figure 1. ¹H (61.4 MHz) NMR spectrum of diclofenac sodium in DMSO-d₆ with (bottom) and without (top) the addition of 15 μL of trifluoroacetic acid. The asterisks represent the residual solvent peaks for dimethyl sulfoxide. The chemical structure is included, and the corresponding signals are labelled, including the one arising from water prior to the addition of trifluoroacetic acid (δ = 3.3 ppm).

The next time you experience signals overlapping with a water signal, try adding a little trifluoroacetic acid to your sample! Please keep in mind that not all species are stable in the presence of such a strong acid. If you have any questions about the work presented herein or would like to learn more about how benchtop NMR spectroscopy can be included into your existing workflows, please don’t hesitate to contact us!

References

(1) Claridge, T. D. W. High-Resolution NMR Techniques in Organic Chemistry, 2nd ed.; Elsevier, 2009.
(2) Ross, S. A.; Lowe, G. Tetrahedron Lett. 2000, 41, 3225-3227.
(3) (a) Caution: trifluoroacetic acid is highly corrosive, harmful when inhaled, and causes severe skin burns. Caution must be exercised when handling and disposing of this strong acid.
(b) Trifluoroacetic acid safety data sheet: https://www.sigmaaldrich.com/CA/en/sds/SIGALD/T6508 (accessed May 2025).