In 2020, a program called AlphaFold won the biennial challenge called CASP. The challenge is all about predicting the structure of different proteins using Artificial Intelligence and algorithms. AlphaFold was made by Google’s AI offshoot, DeepMind, and beat over 100 other teams to win first prize. This is a big deal for science because knowing the structure of proteins accurately helps us create drugs and understand how cells are built. Before AlphaFold, scientists had to use complicated experimental techniques like X-ray crystallography, cryo-electron microscopy, and NMR spectroscopy to figure out the structure of proteins. One type of NMR spectroscopy is called Carbon-13 NMR. It's an analytic technique that helps us identify molecules and work out their structure.

What is carbon-13 NMR?

Let's talk about carbon-13 NMR, a type of NMR spectroscopy that helps identify the structure and identity of a molecule by using carbon atoms. In this article, we will focus solely on carbon-13 NMR. We'll learn how it works, and how to interpret its spectra. We'll go over important terms such as spin, resonance frequency, and chemical shift. You'll get to practice spotting different carbon environments and identifying different molecules based on their spectra. But before we dive deeper, let's refresh our understanding of NMR spectroscopy and why it's important.

Spin

As a quick recap, you may recall from our previous discussion on NMR that nuclei with an odd mass number have something called spin. This property allows them to behave similarly to bar magnets when exposed to external magnetic fields. When placed in an external magnetic field, these nuclei align in a way that their spin is either parallel or antiparallel to the field. If they are spin-aligned, we call them in their parallel state. If they are spin-opposed, we say they are in their antiparallel state.

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Resonance

It's important to note that most nuclei with spin in a magnetic field are typically spin-aligned or in their parallel state. This is because it's more energetically stable than being in the antiparallel state. Think of it as swimming in a stream of water - it's much easier to swim with the current rather than swim against it. However, if we supply enough energy, we can flip a nucleus from its parallel to its antiparallel state, which is known as resonance. The energy required to do this is called the magnetic resonance frequency. If we supply a sample of nuclei with energy in a range of frequencies, some of them will absorb energy equal to their resonance frequency and flip to their antiparallel state.

Magnetic field strength

It's worth noting that different nuclei can feel the strength of the magnetic field differently, and this is due to electrons shielding nuclei from external magnetic fields. For instance, in the case of the C=O bond we discussed in the previous article, oxygen is significantly more electronegative than carbon, which causes the shared pair of electrons to be pulled more towards oxygen, leaving the carbon atom electron-deficient. As a result, the carbon atom feels the magnetic field much more strongly than the oxygen atom and has a higher resonance frequency.

Moreover, shielding also means that the resonance frequency of nuclei of the same element can vary depending on the atoms or groups surrounding them. A less well-shielded nucleus feels the strength of the magnetic field much more strongly and has a higher resonance frequency than a more well-shielded nucleus.

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To summarize, if we have a sample of a substance with nuclei that have spin, we can supply energy to it and then plot the energy absorbed against chemical shift on a spectrum graph. Chemical shift is a value that's related to resonance frequency, and we've learned that different nuclei will have different resonance frequencies based on the groups surrounding them, leading to different chemical shifts.

By comparing the chemical shift values to those in a data table, we can then work out the structure of the substance. This approach is known as nuclear magnetic resonance spectroscopy, or NMR spectroscopy, and it's a powerful technique that's widely used in chemistry, biochemistry, and materials science for the analysis of chemical structure and composition.

How does carbon-13 NMR work?

Any old nucleus can't be analysed using NMR spectroscopy. It has to be a nucleus with an odd mass number. Carbon-13 is one such example. A carbon-13 nucleus contains six protons and seven neutrons, giving it a mass number of 13. This means it has spin. We can therefore analyse organic molecules containing carbon using carbon-13 NMR, as we mentioned earlier.

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Carbon-13 is a less abundant isotope of carbon, making up only about 1% of all carbon atoms. However, when we analyze a sample containing a large number of molecules, it's highly likely that at least some of the carbon atoms in the molecule are carbon-13 atoms, which will produce a peak on the NMR spectrum.

To carry out carbon-13 NMR, we typically dissolve the sample in a particular solvent such as CCl4, and then add a small amount of a reference compound such as TMS (tetramethylsilane). The reference compound serves as a calibration standard, as it produces a single peak at a known chemical shift value on the NMR spectrum.

Once the sample and reference have been prepared and mixed, we can then analyze the spectrum produced, which shows the energy absorbed by the carbon-13 nuclei against their chemical shift values. By studying the pattern of peaks on the spectrum, we can work out the different environments of the carbon-13 atoms in the molecule, and use this information to determine its structure and composition.

TMS

TMS, systematically known as tetramethylsilane, is an organic molecule used as a reference in NMR spectroscopy. It takes the chemical shift value 0. We use it because it is cheap, inert, non-toxic, easy to remove, and gives a clear signal.

Environment

We’ve mentioned this term a couple of times now, but what does it actually mean?

An atom’s environment is simply all the other atoms or groups of atoms surrounding it.

When looking at environments, we don’t just look at the species directly bonded to the atom in question - we look at the molecule as a whole. Atoms are only in the same environment if they have exactly the same atoms, groups and side chains bonded to them. We'll have a go at working out environments in just a minute.

Chemical shift

To summarize, chemical shift is a value that's related to resonance frequency and is measured in parts per million (ppm) compared to a reference molecule such as TMS. In carbon-13 spectra, chemical shift values typically range from 0-200.

Each carbon atom in a molecule produces a specific chemical shift value, which varies depending on its environment, i.e., the other atoms or groups attached to the carbon atom. Carbon atoms in different environments have different chemical shifts, and a less well-shielded atom will have a higher chemical shift value than a more shielded atom. These chemical shift values always fall within certain ranges for carbon atoms in specific environments, and these ranges are used to interpret NMR spectra and determine the structure and composition of molecules.

Spectra

Spectra are graphs produced showing chemical shift plotted against energy absorbed by the molecule. By looking at spectra, we can infer the structure of our molecule.

Working out environments

Here’s an example of an organic molecule, propanal. How many different carbon-13 environments do you think this molecule has?

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The carbon atom on the left, shown below circled in green, is bonded to three hydrogen atoms and a  group. The middle carbon, circled in red, is bonded to a  methyl group and a  group. The carbon on the right, circled in blue, is bonded to an oxygen atom with a double bond, a hydrogen atom and a  group. These three carbon atoms are all bonded to different species. We can therefore say that they are in different environments.

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How about this next molecule, propanone?

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The carbon in the centre, shown below circled in red, is bonded to two methyl groups. The carbon on the left is bonded to three hydrogen atoms and a  group. The carbon on the right is also bonded to three hydrogen atoms and a  group. Because they are both bonded to exactly the same atoms and groups, the two carbon atoms are in the same environment. Both are circled in green.

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In general, if a molecule is symmetrical, it contains multiple carbon atoms in the same environment.

How do we interpret carbon-13 spectra?

Now we know what carbon-13 NMR spectroscopy is, we can have a go at interpreting a spectrum. To do this, we need a data table. This table shows chemical shift values produced by carbon atoms in certain environments.

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Let’s look at a typical carbon-13 NMR spectrum. Take this one, produced using propanal.

Based on the NMR spectrum analysis, we can conclude that the molecule in question, propanal, contains carbon atoms in three different environments. The left-hand peak on the spectrum, with a chemical shift value of about 190 ppm, corresponds to the carbon atom in the aldehyde group of propanal. The next peak, with a value of around 40 ppm, corresponds to the carbon atom bonded to the oxygen atom in the propanal molecule. The peak on the right-hand side of the spectrum, with a value of about 10 ppm, corresponds to the carbon atom bonded to the two hydrogen atoms in the methyl group of propanal.

Overall, the NMR spectrum analysis provides valuable information about the different carbon environments in a molecule, which is crucial for understanding its structure and composition.

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Pulling together what we’ve learnt, we can conclude the following things:

The peak at 5 ppm shows the carbon atom circled in green, a methyl group.The peak at 40 ppm shows the carbon atom circled in red. We know this because we can see it is bonded to a methyl group.The peak at 190 ppm shows the carbon atom circled in blue. Again, we know this because it contains a  bond.

Let’s now look at another example, the carbon-13 NMR spectrum for but-1-en-3-one.

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Based on the information provided, we can conclude that the NMR spectrum being referred to contains peaks corresponding to different carbon environments in a molecule. The peak at 190 ppm indicates the presence of a double bond in the molecule. The two peaks at 130 and 140 ppm correspond to carbon atoms at either end of the double bond, which are in two distinct environments as indicated by the separate peaks. The peak at around 25 ppm corresponds to a methyl group that is bonded to an oxygen atom, falling within the chemical shift range of 20-50 ppm for this type of group. Overall, the NMR spectrum analysis provides valuable information about the different carbon environments in a molecule, which can be used to determine its structure and composition.

But-1-en-3-one.

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That is correct. In carbon-13 NMR, the heights of the peaks do not correlate with the number of carbons in that environment. The peak heights are determined by the relative number of carbon-13 nuclei in each environment, as well as the relaxation times of those nuclei. This means that a peak with a lower height could correspond to a larger number of carbon atoms in that environment, depending on the relaxation time and the other factors affecting peak height.

In summary, carbon-13 NMR is a powerful analytical technique used to identify and determine the structure of different molecules. It works by detecting the chemical shift value of carbon-13 nuclei in a sample and comparing it to a data table to deduce the different environments of those nuclei. The peak heights in carbon-13 NMR do not correlate with the number of carbons in each environment, but instead depend on factors such as the relative number of carbon-13 nuclei and their relaxation times.

Carbon -13 NMR

What is the difference between proton NMR and carbon NMR? 

Proton NMR looks at the environments of hydrogen-1 atoms whilst carbon NMR looks at the environments of carbon-13 atoms.

What is carbon-13 NMR? 

Carbon-13 NMR is an analytical technique used to identify and work out the structure of molecules. It produces graphs called spectra, which contain various peaks that show the different environments of carbon atoms in a molecule.

Why is carbon-13 used in NMR? 

Carbon-13 is used in NMR because it has an odd mass number. This means that it has a property called spin and behaves a bit like a bar magnet when placed in an external magnetic field. Because of this, carbon-13 atoms show up in NMR spectra.

How does carbon NMR work? 

Carbon-13 atoms have an odd mass number. This means that they have a property called spin. When placed in an external magnetic field, they act like bar magnets and line up with the magnetic field. Supplying them with enough energy causes them to flip in the opposite direction, but this energy varies depending on the other atoms and chemical groups bonded to the carbon atom in a molecule. By plotting a graph of energy against a value called chemical shift, we can identify which groups the carbon atom is bonded to and work out the structure of the molecule. 

What does carbon-13 NMR tell you?

Carbon-13 NMR tells you the different environments of carbon atoms and helps you work out the structure of an organic molecule.

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