This manual provides systematic instructions on how to perform basic one- and two-dimensional NMR experiments on Bruker Avance DPX400 spectrometer Some features of Bruker Avance DPX400 spectrometer include: 9.40 Tesla standard bore UltraShield magnet B-VT 3200 variable temperature unit GRASP II Z-gradient accessory

QNP probehead (5 mm) with Z-GRASP coil; automatically switchable between

nuclei (F, P, C/H)

Bruker Orthogonal Shim System (BOSS I) with 20 shim gradients Two Frequency Control Units (FCU )

16/18 bit SADC for an effective spectral width up to 150 kHz NMR Case automatic sample changer for 24 samples

Windows 2000 workstation with the NMR Suite software package (including XWIN-NMR 3.5)

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3.1 Sample preparation and positioning

The sample quality can have a significant impact on quality of the NMR spectrum. The following is a brief list of suggestions to ensure high sample quality. Sample preparation:

Use deuterated solvents whenever possible

For H NMR, 1 – 5 mg of the sample is sufficient. For high molecular weight samples, more concentrated solutions are sometimes recommended. However, too concentrated solution leads to lower resolution due to saturation and/or increased viscosity. For C NMR ca. five times the concentration is recommended

For 2D measurements, the sample should be concentrated enough to achieve an acceptable S/N ratio. As a rule of thumb, 25 mg of the sample is sufficient for nearly everything, including H,C HMBC experiment. With samples of only 1 – 5 mg, the homonuclear H,H experiments (e.g. COSY) are still feasible, but those involving C may take overnight to complete

Always use the same sample volume or solution height. This minimizes the

shimming that needs to be done between sample changes. The recommended value is (at least) 0.6 ml or 4 cm solution for 5 mm sample tubes

Always use clean and dry sample tubes to avoid contamination of the sample. See

Appendix A for proper cleaning procedures for NMR sample tubes. Brand new NMR tubes straight from the packet are not usually very clean!

Always use high quality sample tubes to avoid difficulties with shimming (e.g.

Wilmad 527-PP or 528-PP)

Filter the solution. A small plug of fresh medical cotton wool at the neck of a

Pasteur pipette will do the trick. However, it is necessary to pre-rinse it with a little amount of the solvent to be used to flush out any loose fibers Sample positioning:

Use the sample depth gauge to position the sample tube in the spinner. Be careful

with the depth gauge. Do not change the depth! The slider should be in position 5 – 15 mm

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