Application Note 7: Observing Spin
Systems using COSY
H-1H COSY experiments produce 2D NMR spectra that identify proton coupling partners, which
in many cases is directly related to the carbon skeleton connectivity. Thus COSY is one of themost informative and widely used 2D NMR experiments.
1In this application note we demonstrate how COSY NMR spectra can be used to determine thecomposition of amino acids present in peptides using the Spinsolve benchtop NMRspectrometer.

9,8,7,6,52O

5673

13a3b214

2

9NH2OH3842567
9,8,7,6,589987654
f2 (ppm)

32f1 (ppm)

3a3b‘COSY’ (Correlated Spectroscopy) is a 2D homonuclear (1H, 1H)-correlated NMR experiment in which proton
coupling partners can be identified. A COSY spectrum is shown as a contour plot in which the 1D proton NMRspectrum is plotted along the diagonal and protons that couple to each other result in a peak off the diagonal in linewith each of the coupling protons (Figure 1).

Figure 1: COSY Spectrum of 1,3-dichloro-6-nitro-benzene. Short range coupling is indicated on the spectrum withdashed lines and on the compound with double-headed arrows.
The analysis of COSY spectra often allows substantial piecing together of fragments in the molecule as coupling
partners and therefore adjacent nuclei can be identified. In the process, the positions of functional groups or atoms
that are not protonated can be identified as they provide a ‘road-block’ in the coupling for most molecules1 (forexample, a carbonyl). Fragments that are separated by these road blocks are known as spin systems. Spin systems
are fragments of molecules that show short-range coupling within themselves, with no short-range coupling to therest of the molecule. Often these form ‘puzzle pieces’ in structural elucidation that can be pieced together using
other 2D NMR experiments.

Longer range coupling (>3 bonds) may be observed in the COSY spectrum for some systems, particularly those withπ-systems, e.g. benzene derivatives and alkenes.
1

EXAMPLE: GLYCYL-L-PHENYLALANINE
One area where COSY spectra and the ability to identify spin systems is particularly useful is in the characterisationof peptides. Peptides are made up of amino acids (Figure 2) which each have a characteristic fingerprint in the COSYspectrum.

Figure 2: Formation of peptides through condensation of amino acids.

There is no coupling between different amino acidmoieties as they are separated by peptide linkages(road-blocks) (Figure 3). Therefore COSY spectra canbe used to identify the amino acids that make up
simple peptide molecules.

Figure 3: Peptide linkage.

Glycyl-L-Phenylalanine is produced in a third year
synthesis laboratory experiment where students
synthesise the dipeptide from the corresponding
amino acids. Analysing the COSY NMR spectra of theamino acids shows that the dipeptide can be easily
characterised.

In the COSY spectrum of phenylalanine, two different spin systems can be observed (Figure 4). The two spin
systems are shown in different colours, and coupling is where the lines intersect on a peak off the diagonal (crosspeak). The protons in line with this cross-peak couple to each other.
Note that the methylene protons occur at different chemical shifts despite being attached to the same carbon
atom, as the neighbouring carbon atom is chiral. This means that the two protons experience a different chemicalenvironment (through space), resulting in a difference in chemical shift.

9,8,7,6,5

2

1

3a3b2
33b4

2D2O

56
7

9,8,7,6,5

8
9
987

Figure 4: COSY spectrum of phenylalanine in D2O (4.89 ppm).

65
4f2 (ppm)

32f1 (ppm)

3aThe COSY spectrum of glycine (Figure 5) has only one peak along the diagonal and therefore no COSY correlations
off the diagonal. This is because there is no chirality in the molecule (the only non-chiral amino acid) therefore thetwo hydrogen nuclei are equivalent.

223D2O

56
78
998Figure 5: COSY spectrum of glycine in D2O (4.89 ppm).

76

5f2 (ppm)

43

2

f1 (ppm)42

7,8,9,10,11

3

1

5a5b2
35b1

4

3
D2O

5f1 (ppm)

5a6
7
7,8,9,10,118
9
98

7

6

5
4
f2 (ppm)

321Figure 6: COSY spectrum of glycyl-L-phenylalanine in D2O (4.89 ppm).
In the COSY spectrum of the dipeptide (Figure 6), the two amino acid moieties can easily be recognised due to theircharacteristic fingerprints that were observed in the COSY spectrum of the un-coupled amino acid. The same spin
systems observed for the amino acids are translated into the COSY spectrum of the dipeptide.

Conclusion
Spin systems are fragments of molecules that have short-range coupling within themselves. Correlated
Spectroscopy (COSY) experiments are used to observe short-range coupling, and therefore spin systems canbe identified. The 2D homonuclear COSY experiment was used to observe the spin systems in the amino acids
L-phenylalanine and glycine, and the corresponding dipeptide glycyl-L-phenylalanine. It was observed that the spinsystems in each amino acid moiety were preserved as peptide linkages prevent coupling between amino acids. This
example was a simple demonstration of how spin systems of amino acids have a characteristic fingerprint in theCOSY spectrum of peptides which can be used to identify the amino acids. This analysis can be applied to larger
peptides, which provides a non-destructive method for determining the amino acid composition.

References:E. Gonzalez, D. Dolino, D. Schwartzenburg, and M. A. Steiger, Dipeptide Structural Analysis Using Two-Dimensional
NMR for the Undergraduate Advanced Laboratory, Journal of Chemical Education, 2014.
H. Friebolin, Basic one- and two-dimensional NMR spectroscopy, Wiley-VCH Verlag GmbH & Co. KGaA, 2011.

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