Friday, 5 June 2009

Fighting against peak overlap – Introducing Global Spectral Deconvolution (GSD)

1H NMR is for sure the most powerful technique for structure elucidation, especially for small organic molecules. Typically, an organic chemist uses the chemical shift, coupling constants and integration information contained in an 1H-NMR spectrum to either verify or elucidate an unknown compound. Of course, it’s quite common that a simple 1H-NMR spectrum is not enough to unambiguously confirm a structure and thus other NMR experiments (e.g. 13C-NMR, HSQC, COSY, etc) are used to get more structural information.
Nevertheless, I have often found that many organic chemists do not always try to get the most out of 1H-NMR spectra (which is the cheapest experiment), in particular when some multiplets are complex to interpret (strong coupling) or when peaks overlap prevents valuable information to be detected in some multiplets. Overlapping peaks and new ways to get around it will be the subject of this post.
As it is well known, there are two principal factors limiting the resolution power in a spectrum. First, we have the natural line width limitation imposed by the T2 (spin-spin relaxation). For example, if T2 is about 1 second, the peak linewidth at half height cannot be less than 0.32 Hz (remember, line width at half height = 1 / (pi * T2) = 1 / 3.1415 = 0.32) no matter how powerful is our NMR instrument or the field homogeneity. On the other hand, there are instrumental shortcomings (e.g. spatial uniformity of the applied magnetic field, etc).
Nonetheless, there is an additional limiting factor, and whose importance is generally underestimated which has to do with the generally large number of transitions in 1H-NMR spectra. In short, the peaks we can observe in a 1H-NMR are just a small fraction of the actual transition resonances which are not observable because of the limited digital resolution. In fact, every peak in an 1H-NMR spectrum is basically an envelope of a large number of transitions and its shape is dominated by the coupling pattern of the spin system. Even in molecules of modest size the number of distinct peaks is tens to thousands times smaller than that of quantum transitions. As a very simple example, consider an A3B2 spin system. Depending on the second order interaction and on the available digital resolution, we might observe the expected triplet / quadruplet multiplet patterns. This is illustrated in the figure below.

However, if we use Mnova capabilities to display all main transitions of any coupled spin system by simply hovering with the mouse over the particle of interest, we can appreciate the additional number of resonances (see below):

Furthermore, I can easily increase the digital resolution of the A3B2 spectrum above by just reducing the line width used in the spin simulation module of Mnova. As a result, it’s now possible to observe more resonances in this particular A3B2 spin system (although not all of them, of course):

Of course, this way of increasing the digital resolution is only possible with synthetic spectra and cannot be applied to experimental data. Obviously there are many resolution enhancement techniques being Resolution Booster one of the most powerful ones. As a nice example of the application of this technique, let me tell you this story:
A couple of weeks ago, a very good friend of mine, a professor of organic chemist, came to me with an interesting structural problem. His research group had carried out a reaction which resulted in one single product whose 1H-NMR spectrum was, in principle, compatible with two potential structures. In order to ambiguously find the right structure, they acquired more NMR spectra (DEPT, HSQC, HMBC, COSY) which allowed them to find the correct molecule However, while discussing the problem having a few beers at a bar in Santiago, we found that just the 1H spectrum was more than enough in order to discard one of the two structures and completely assign the correct one without the necessity to acquire any other NMR experiment.
The key was the ability to resolve a long range coupling (homo-allylic) with the assistance of Resolution Booster. Basically, the 1H-NMR showed a clean double doublet which was compatible with both structures (I’m sorry, but I cannot reveal those structures). This multiplet is shown below:

After appling Resolution Booster, we could clearly appreciate a further splitting which we could assign to the expected homo-allylic coupling with a value of 1.76 Hz. This coupling was also found in its corresponding multiplet partner confirming the structure:

At this point, it’s worth mentioning that Resolution Booster is a very powerful method to resolve overlapped peaks, but it cannot be used for integration as the area of the peaks get distorted by this process. The good news is that we have developed a new method which in addition to taking advantage of the power of resolution booster, it yields accurate integrals.

This method has been named as Global Spectral Deconvolution (GSD) and as its name says, it automatically deconvolves all the peaks in a spectrum. In short, this method first recognizes all significant peaks in a spectrum, then assigns a realistic a-priori bounds to all peak parameters (chemical shift, heights, line widths, etc) and finally fits all these parameters in a very reasonable time.
Following with the example above, if we apply GSD, we get a multiplet with all the individual peaks clearly resolved and this time, with accurate integrals.

It’s important to mention that we haven’t just fitted the multiplet above, but we have actually fitted the whole spectrum!

We are confident that GSD will open new avenues in NMR data interpretation and quantitative analysis (qNMR). I will blog about these points in future posts.