NMR spectroscopy
From Biocrawler, the free encyclopedia.
Nuclear Magnetic Resonance Spectroscopy is the name given to the technique which exploits the magnetic properties of nuclei. This phenomenon and its origins is detailed in a separate section on Nuclear magnetic resonance (NMR).
Many areas of information can be obtained from this single phenomenon. In its simplest form NMR allows identification of individual atoms in a pure molecule. Much like using infrared_spectroscopy to identify functional groups, analysis of a 1D NMR spectrum tells the scientist what atom environments (like a methyl proton), and in some cases how many atoms of each type, exist within the sample. NMR is based in quantum mechanical properties of nuclei, and as such is very reliable, predictable and reproducible.
NMR Spectroscopy is much more powerful than this everyday usage. It can be used to study mixtures of analytes; to understand dynamic effects such as change in temperature and reaction mechanisms; it can be used in the solution and solid state; and critically it is an invaluable tool in understanding protein and nucleic acid structure and function.
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Basic NMR Techniques
When placed in a magnet, NMR active nuclei (like 1H or 13C) resonate at a specific frequency. Since frequency is dependent on the strength of the magnet. In a 21 Tesla magnet protons resonate at 900MHz. It is common to refer to a 21T magnet as a 900 MHz magnet but it is worth remembering that different nuclei resonate at a different frequency at this field strength.
1D NMR
What does a 1D Spectrum look like?
At 21T, protons resonate at around 900MHz. Different protons in a molecule each resonate at slightly different frequencies dependent on their local environment. Since this frequency is dependent on the strength of the magnetic field, it is converted into a field-independent value known as the chemical shift.
So nuclei in different environments have different chemical shifts. By understanding the different values of chemical shift we can assign each signal to an atom or group of atoms in the molecule under study.
For example, in a proton spectrum for ethanol (CH3CH2OH) we would expect three specific signals at three specific chemical shifts. One for the CH3 group, one for the CH2 group and one for the OH. From experience I know that a typical CH3 group as a shift around 1ppm, the CH2 attached to a OH has a shift of around 4ppm and the OH has a shift around 2-3ppm.
Why do we not get three signals for the CH3 group? It is because during the course of the NMR experiment (which typically takes a few ms) molecular motion makes each of the three methyl protons average out - they become "degenerate" which is a scientific way of implying identical.
The area of peaks
Interestingly the shape and size of peaks are indicators of chemical structure too. In the example above - the proton spectrum of ethanol - the CH3 peak would be three times as large as the OH. Similarly the CH2 peak would be twice the size of the OH peak, but only 2/3 the size of the CH3 peak!
Modern analysis software allows analysis of the size of peaks to understand how many protons give rise to the peak. This is known as integration - a mathematical process which gives the area under a graph (essentially what a spectrum is). It is important to note that the analyst must integrate the peak and not measure its height because the peaks also have width - and thus its size is dependent on its area not its height.
Multiplets - The appearance of peaks
The chemical shift is not the only indicator we can use to assign a molecule. Because nuclei themselves are little magnets they influence each other, changing the energy and hence frequency of nearby nuclei as they resonate - this is known as spin-spin coupling. The most important type in basic NMR is scalar coupling. This interaction between two nuclei occurs through chemical bonds, and can typically be seen up to three bonds away.
To understand the effect of scalar coupling lets look at a proton which has a signal at 1ppm. If we now say that our proton is in a molecule where three bonds away exists another proton (in a CH-CH group for instance), the neighbouring group (a magnetic field) causes the signal at 1ppm to split into two, with one peak being a few Hertz higher than 1ppm and the other peak being the same number of Hertz lower than 1ppm. These peaks have half the area of the former singlet peak. The magnitude of this splitting (difference in frequency between peaks) is known as the coupling constant. A typical coupling constant value would be 7Hz.
The coupling constant is independent of magnetic field strength because it is caused by the magnetic field of another nucleus, not the spectrometer magnet. Therefore it is quoted in Hertz(frequency) and not ppm (chemical shift).
So in our spectrum we have a signal centered at 1ppm, split into two - but we've introduced an extra proton. Lets say that proton resonates at 2.5ppm, what would the spectrum look like? Well, that proton would also be split into two by the proton at 1ppm. Because the magnitude of interaction is the same the splitting would have the same coupling constant 7Hz apart. Our spectrum would have two signals, each being a doublet. The area of the doublets will be the same as another, because they're both produced by one proton each.
Beyond Doublets? Triplets!
Lets take our two doublets at 1ppm and 2.5ppm from our fictional molecule CH-CH. What happens if we change it to CH2-CH? From what we know above we can rationalise the following:
- The total area of the 1ppm CH2 peak will be twice that of the 2.5ppm CH peak.
- The CH2 peak will be split into a doublet by the CH peak - with one peak at 1ppm+3.5Hz and one at 1ppm-3.5Hz (total splitting or coupling constant is 7Hz).
What will happen to the splitting of the CH peak at 2.5ppm? It will be split twice by each proton from the CH2. The first proton will split the peak into two equal intensities and will go from one peak at 2.5ppm two peaks, one at 2.5ppm+3.5Hz and the other at 2.5ppm-3.5Hz - each having equal intensities. However these will be split again by the second proton. Lets see what happens to the frequencys:
- The 2.5ppm+3.5Hz signal will be split into 2.5ppm+7Hz and 2.5ppm
- The 2.5ppm-3.5Hz signal will be split into 2.5ppm and 2.5ppm-7Hz
So do we get four peaks? The careful reader will note that we get three: one signal at 7Hz above 2.5ppm, two signals occur at 2.5ppm, and a final one at 7Hz below 2.5ppm. The ration of height between them is 1:2:1. This is known as a triplet and is an indicator that the proton is three-bonds from a CH2 group.
Multiplets
We can extend this to any CHn group. Lets change our CH2-CH to CH3-CH2 but keep the chemical shift and coupling constants idential.
- The relative areas between the CH3 and CH2 subunits will be 3:2.
- The CH3 is coupled to two protons into a 1:2:1 triplet around 1ppm.
- The CH3 is coupled to three protons. What shape will it have?
Something split by three identical protons takes a shape known as a quartet, each peak having relative intensities of 1:3:3:1.
The multiplicity of a peak coupled to n number of identical protons follows the values set in Pascal's triangle:
n 0 1 1 1 1 2 1 2 1 3 1 3 3 1 4 1 4 6 4 1 etc. 1 5 10 10 5 1
Lets try an extreme example. Take 2-methylpropane: (CH3)3CH. The CH proton is attached to three identical methyl groups. That's 9 protons! Looking at Pascal's triangle for n=9, we see that the peak in the spectrum would be split into ten with relative intensities of: 1:9:36:84:126:126:84:36:9:1!
External Links
- The Science of Spectroscopy (http://www.scienceofspectroscopy.info) - supported by NASA, includes OpenSpectrum, a Wiki-based learning tool for spectroscopy that anyone can edit
