In the average case one can simply dissolve an analyte in an appropriate deuterated solvent and acquire a simple 1D spectrum to obtain all the required structural information. However, sometimes doing so may not provide you with all of the information you need! It is not uncommon to encounter labile proton peaks in functional groups such as alcohols, amines, amides, and carboxylic acids. These resonances move around the spectrum depending on temperature, pH and solvent choice and can overlap or interfere with the other static signals from the protons attached to the carbon backbone of the organic molecule. Such overlap can convolute a spectrum to varying degrees from affecting integration to completely drowning out a peak. This is vital information that we can obtain in a very simple fashion. At the start of the year, Matt Zamora discussed how a spectrum of acetic acid in D2O differs from the spectrum of acetic acid in CDCl3 (read it here!) because of the exchangeable nature of the carboxylic acid protons. Additionally, labile protons have a tendency to produce peaks that are substantially broader than protons that are not labile. In this blog entry, I will discuss using what is known as a D2O quench/shake to exchange out labile protons and the valuable information that can be gained.
First, we will look a relatively simple example that highlights instances where you would want to confirm the presence of a labile proton signal. In the spectrum of 4-hydroxypropiophenone (figure 1) you can see that the signal for the hydroxy proton at 10.19 ppm comes in as a nice sharp, well-resolved singlet. Adding D2O to the NMR tube and re-acquiring the 1D 1H spectrum shows complete disappearance of the signal at 10.19 ppm and confirms that assignment to the hydroxy proton.
Note here that while the –OH resonance completely disappears, a water peak shows up at ~3.8 ppm. This results from: a) residual H2O and HOD in the D2O bottle; and more importantly b) the exchanged out hydroxy protons (-OH) that now result in a greater amount of H2O and HOD in the NMR tube.
With the principle under our belt, let’s look at a more complicated molecule - ampicillin. Ampicillin is a commonly used antibiotic in the β-lactam family for the treatment of bacterial infections like streptococcus pneumoniae and meningitis. In looking at the 1H NMR spectrum of ampicillin before and after adding D2O (figure 2), similar to the 4-hydroxypropiophenone example above, the broad peaks at ~6.5 ppm and ~8.8 ppm corresponding to the amide and the carboxylic acid protons become quenched and result in a significantly cleaner baseline and better resolution in the remaining backbone peaks – perhaps most noticeable in the AB quartet at 5.34 ppm.
For a final example we will look at Epinephrine, more commonly known as adrenaline, is a molecule naturally generated by the human body, also used medicinally in the treatment of cardiac arrest and anaphylaxis. Really evident in this spectrum are the hydroxy and amino protons covering a large range from ~3-5 ppm (figure 3) and heavily overlapping with the triplet signal from the α-hydroxy proton. Again here, the D2O quench flattens out the baseline and allows clear integration of the triplet centered at 4.39 ppm to be resolved.
As you can see from the above examples, labile protons in alcohol, amine, amide, and carboxylic acid functional groups can sometimes be fairly broad and interfere with desired signals in a given spectrum. Doping your NMR sample with D2O easily exchanges out these protons for deuterium, effectively making these resonances NMR silent and resulting in the disappearance of labile proton peaks. With labile proton peaks removed from a spectrum not only can one identify the peaks belonging to exchangeable protons but also uncover valuable information that may be hidden due to overlap with these signals. Remember that when you use a D2O quench, the resulting H2O/HOD peak will appear in your spectrum.
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