CHEM 335 - NMR Laboratory Tour

Vintage (1970s) NMR system: 60 MHz

(Continuous Wave NMR with Electromagnet)

A spectrometer similar to this Varian EM-360A was in use in our department from 1979 until 1995.


Vintage (1980s) NMR system: 100 MHz

(Pulse / Fourier Transform NMR with Superconducting Magnet)

This Bruker AC-100 NMR instrument and a similar AC-300 are still in service in our department and are suitable for most research samples from our synthesis groups.


Modern (2010) NMR system: 600 MHz

Our most recent acquisition is a Bruker Avance III 600 and it is primarily used for the structure determination of organic and biochemical samples in solution or in solid state.


Schematic and Inside View of Superconducting NMR Magnets.

The innermost container holds the superconducting coil in a bath of liquid Helium.
Surrounded by several vacuum layers with liquid Nitrogen between them, the Helium loss is minimized to 10-20 liters per month.


Superconducting NMR Magnet during Helium Refill

Periodic cryogen refills are necessary to keep the magnet in operation.


The inside of a modern NMR Console with essential components highlighted.

RF pulses from the signal generators are amplified and sent to the probe (see below) to excite specific nuclei, i.e. 1H or 13C in the sample.
Gradients separate wanted from unwanted signals. Shims make the magnetic field uniform to optimize signal resolution and sensitivity.


NMR Probes

The probe, which is normally located in the center of the magnet, detects signals from the excited nuclei and sends them back to the receiver.
Probes usually have more than one coil to be able to manipulate several nuclei (i.e.: 1H, 13C, 15N, 31P) simultaneously.


The NMR Experiment - Sample Preparation

NMR sample tubes are filled with 0.5 ml of a solution of a compound of interest.
The use of deuterated solvents minimizes solvent signals. Tetramethylsilane (TMS) may be added as a reference compound.


The NMR Experiment - Sample Change

The sample tube is placed into a sample holder ("Spinner") and inserted into the magnet.


The NMR Experiment - Deuterium NMR Signal for Field/Frequency Lock

The 2H atom in the deuterated solvent can be observed via a separate NMR experiment.
It allows us to monitor the stability of the magnetic field and to improve its homogeneity ("Shimming").

Unlocked Sweep Signal vs. Shimmed Lock Signal


The NMR Experiment - Data Acquisition

A 90o RF Pulse generates a Free Induction Decay (FID), which is recorded during the aqcuisition time ("The Scan").
Repeated scanning will improve the signal/noise ratio (Signal Averaging) and is essential for the direct observation of less abundent isotopes, i.e.: 13C or 15N.

          


300MHz Proton NMR Spectrum of Ethyl Benzene after Fourier Transformation of the FID.


75 MHz 13C NMR Spectra of Ethyl Benzene

top: 1H coupled: Multiplicities due to 1JC,H couplings allow the identification of CH3, CH2, CH, and Cq signals.
bottom: 1H decoupled: Proton decoupling simplifies the spectrum by collapsing each multiplet into one single peak.


Bigger molecules have more complicated (more interesting) spectra.

1H NMR Spectrum of the disaccharide Cellobiose
Integration of the individual proton signals confirms the presence of 14 H atoms on the rings.
The H-1 signals for each ring are easily identified as doublets, as each is only coupling to one proton.
All other protons are coupling with more than one proton and show more complicated multiplicities.


To fully characterize the structure, two-dimensional NMR techniques have to be employed:

1H,1H COSY Spectrum of Cellobiose
Starting from proton H-1 at 6.26 ppm, all other protons can be found by following the crosspeak patterns.
The connectivities for one of the two monosaccharide units have been drawn into the spectrum.
Try finding the connections in the other ring by starting from the H-1' doublet at 4.52 ppm.


Once all proton signals have been assigned, carbon signals can be found through a C,H correlation experiment:

1H,13C HSQC Spectrum of Cellobiose
The HSQC spectrum allows the immediate identification of direct Carbon-Proton bonds (1J coupling).
Note the negative CH2 signals (red) which are easily distinguishable from CH and CH3 (blue).



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