TWO-DIMENSIONAL NMR SPECTROSCOPY

Goal: The goal of this experiment is the determination of the covalent structure and if the data permit the conformation of an organic compound. NMR methods will be used but the mass spectrum will also be measured as an aid in the determination of the molecular formula.

References:

R. M. Silverstein, G. C. Bassler, and T. C. Morrill, Spectrometric Identification of Organic Compounds, 5th. ed., Wiley, New York.

H. Friebolin, Basic One- and Two-Dimensional NMR Spectroscopy, Verlag Chemie, Weinheim, 1991.

S. Braun, H.-O. Kalinowski, and S. Berger, 150 and More Basic NMR Experiments, Wiley-VCH, Weinheim, 1998.

Experimental:

1) Mass spectrometry. Prepare a 1% w/w solution of the unknown in methanol and inject it in the Hewlett-Packard gas chromatograph/mass spectrometer system. View the videotape for the instrument before coming to lab. The instructor will brief you in the use of the instrument. Obtain the molecular weight and candidates for the molecular formula from the mass spectrum.

2) NMR spectroscopy.

a) Sample Preparation. First select a solvent using small amounts of the sample and normal solvents. Once a solvent has been selected, the instructor will provide 0.5 ml of deuteriated solvent which is used to prepare a solution of the unknown for the NMR measurements. Tranfer the solution to a 5 mm NMR tube.

b) Initial Adjustments of the NMR Spectrometer. Log on to the student account on the workstation and start the NMR software by entering the command xwinnmr. Insert the sample and turn on the variable-temperature unit. 25C is a good choice for the temperature. Lock the spectrometer on your deuteriated solvent and adjust the magnet homogeneity. Tune the probe for both the proton and carbon channels.

c) Initial NMR spectroscopy. Recommended parameter sets are given in parentheses. Run a proton spectrum and integrate it (PROTONEXP). Also run a carbon-13 spectrum with NS = 32 (C13CPD32) and a carbon-13 APT spectrum (C13APT). In the case of the APT spectrum, make sure that the time delay d20 is set to 6.90 ms. Determine the molecular formula of the compound from a combination of the NMR and mass spectrometric information. Also draw inferences about groups present in your molecule. How many double bonds and rings? How many C, CH, CH2, and CH3 groups? What stereochemistry and conformation are implied by the coupling constants?

d) Run a carbon-13/proton hetero COSY experiment to connect the proton and carbon-13 data (INV4GSTPSW). The routine defined by the parameter set INV4GSTPSW uses gradients and inverse detection and yields excellent spectra in short order. The spectrum may need to be rephased.

e) Run a proton COSY to establish the connectivities between the proton spin systems (COSYGSSW). This routine also uses gradients and is very efficient.

f) Use the information obtained from the COSY spectra to map out the spin systems in the molecule.

g) Measure a long-range proton-carbon COSY spectrum to connect the spin systems that were identified in step f (INV4GSLRNDSW).

h) Additional information might be obtained by running a ROESY spectrum. Use the Shaka modification of this experiment. This spectrum will identify pairs of protons that are within 4-5 Angstrom of one another. The method is very useful in the determination of the three-dimensional structure of proteins but is not as useful in the case of small molecules.

3) Devise and test a model for the structure of compound. You may choose to use other two-dimensional techniques to remove ambiguity and confirm your hypothesis.

Report. Write the report in the style of a journal article. The Experimental section should address sample preparation and the types of techniques used as well as an relevant acquisition parameters such as the mixing time. The Results section should present the spectroscopic evidence. Make effective use of labeled figures prepared from your spectra or portions thereof. Include a table giving the proton and carbon-13 assignments (chemical shifts and coupling constants) for your compound. The Discussion section should present and support a structure or possible structures for your compound. Address any unresolved questions.

twodnmr.doc, WES, 9 June 2000