2D NMR experiments at 60 MHz:COSY-90 and COSY-45

NMRApplication Note 10Introduction
In December 2016 it was widely reported† that students from Sydney Grammar School
reproduced the drug Daraprim®, also known as pyrimethamine. The students followed areaction pathway to produce it, and the final product was analysed at the University ofSydney, School of Chemistry (200 MHz 1H NMR spectrum shown in Figure 1 below)‡.
This substance is used as an example to illustrate 60 MHz NMR spectrometer performance(also Figure 1), and the difference between the two-dimensional (2D) experiments,
COSY-90 and COSY-45.

MethodThis application note shows
a comparison of these two
experiments at 60 MHz (Figures
3 and 4), and detailed data
treatment with Mestrelab’s Mnova
software; a full suite of advanced
routines for processing andanalysing data.
1D 1H NMR spectra (as in Figure1) are commonly performed asthey provide rich qualitative andquantitative information about
the environment of atoms inmolecules, which can be used
to identify and characterise
compounds and reactions. 2D
NMR experiments such as COSY
provide further information about
connectivity between atoms,
enabling structural elucidation.

Figure 1. 1H NMR spectra of pyrimethamine at 200 MHz (top) and 60 MHz (bottom). Sample details
at 60 MHz = 16 scans. Approximately four minutes acquisition, 13.5 mg in 0.5 mL DMSO-d6 =approximately 100 mmol dm-3 concentration.

† The Guardian / Melissa Davey. 2016. Australian students recreate Martin Shkreli price-hikedrug in school lab. [ONLINE] Available at: https://www.theguardian.com/science/2016/
dec/01/australian-students-recreate-martin-shkreli-price-hike-drug-in-school-lab.
[Accessed 9 October 2017].
‡ 	Open Notebook Science Network / Alice Williamson. 2016. Open Source Malaria
Daraprim Synthesis. [ONLINE] Available at: http://malaria.ourexperiment.org daraprim_synthesis/15346/post.html. [Accessed 9 October 2017].

2D NMR experiments at 60 MHz: COSY-90 and COSY-45

Basic 1 and 2D NMR experiments
Benchtop NMR spectrometers
have lower sensitivity andresolution than high field
superconducting instruments;
signals are generally broader andoverlap can make interpretationdifficult where spectra are crowded.

Figure 2. The onepulse, COSY-90 and COSY-45 NMR pulse sequences.

Figure 2 shows three basic pulse sequences which illustrate what happens in a spectrometer during experimental acquisition. Each onebegins with an equilibration / relaxation delay (RD) in which spins align with the external magnetic field. In the onepulse experiment, aperpendicular radiofrequency (RF) pulse is applied for a few microseconds, which tilts the bulk magnetisation into the transverse plane.
This then relaxes back to equilibrium (the Free Induction Decay, or FID), and can be detected as it induces current in the receiver electronics.
Mathematical treatment of the FID with a Fourier Transform (FT) turns the time domain signal into a frequency domain signal, the NMRspectrum, which shows information about the different chemical environments of atoms (usually protons) in the sample.
The standard COSY-90 experiment uses two
90° pulses separated by a delay (t1). The
pulse sequence is repeated with an array ofincremented delays. Fourier Transform inboth dimensions allows the J-coupling
between neighbouring protons to be
displayed as off-diagonal crosspeaks.
Results
The COSY-45 sequence is analogous tothe COSY-90, with the exception that thesecond RF pulse has half the duration.
After 2D Fourier Transform, the resulting
spectrum has a reduced diagonal signal,
at a penalty to the signal to noise ratio.
However, the reduced diagonal signals
enable close resonances to be more easily
distinguished as shown in Figure 4.
The aromatic doublet of doublets
crosspeaks at around 7 ppm can moreclearly be seen in the COSY-45 spectrum
after diagonal suppression, notably withthe wavelets and convolution approach.

Figure 3. 1H COSY-90 spectrum of pyrimethamine. Four scans,128 increments, approximately
17 minutes’ acquisition.

NMR
Application Note 10

Figure 4. 1H COSY-45 NMR spectrum ofpyrimethamine. Four scans, approximately 17minutes’ acquisition.

Figure 5. COSY-90 (top left), with convolution
diagonal suppression (top right), shifted convolution(bottom left) and wavelets (bottom right).

Figure 6. COSY-45 (top left), withconvolution diagonal suppression (topright), shifted convolution (bottom left)
and wavelets (bottom right).

Conclusion
Various NMR pulse sequences produce
different results which can enormously help
with interpretation / structural assignment.
COSY-90 and COSY-45 are among the moststraightforward 2D NMR experiments, andyield rich information which can be exploitedfurther with Mnova’s advanced dataprocessing features.

visit nmr.oxinst.com/x–pulse for more information or email magres@oxinst.com

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