CHEM 335 - NMR Laboratory Tour

Vintage (1970s) NMR system: 60 MHz (B0 = 1.41 Tesla = 14100 Gauss)
(Continuous Wave NMR with Electromagnet)

A spectrometer similar to this Varian EM-360A was in use in our department from 1979 until 1995.
It observes protons at a frequency of 60 MHz in a magnetic field of 1.41 Tesla.

For comparison: Today's benchtop NMRs are much more compact and user-friendly.
These instruments use Rare Earth Permanent Magnets with fields up to 2.35 Tesla.
Primary applications are undergraduate teaching and industry process control.
Nanalysis 100
Nanalysis 100 MHz
Bruker Fourier 80
Bruker Fourier 80 MHz
Oxford Instruments X-Pulse-60
Oxford Instruments X-Pulse 60 MHz

However, most research samples require much stronger fields of 7 Tesla or higher for improved resolution and sensitivity.
Uniform magnetic fields of such strength can currently only be achieved with superconducting magnets (present limit: 28 Tesla).


BU's Chemistry Department currently operates several NMR spectrometers between 100 and 600 MHz (B0 = 2.35 - 14.1 Tesla).

Vintage (1980s) NMR system: 100 MHz (B0 = 2.35 T)

(Pulse / Fourier Transform NMR with Superconducting Magnet)

This Bruker AC-100 NMR instrument and a similar AC-300
(operating at proton resonance frequencies of 100 and 300 MHz, respectively)
are still in service in our department and are primarily used by our instructional labs.
A 300 MHz NMR is suitable for most research samples from our synthesis groups.


Modern NMR system: 600 MHz (B0 = 14.09 T)

Our most recent acquisition is a Bruker Avance III 600.
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 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.

   
center: Probe for Solution NMR
     right : Probe for Solid State NMR


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.


Click the picture to view the video.


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

The 2H atom in the deuterated solvent (i.e.: C6D6 or D2O) is observed via a separate NMR experiment.
It allows us to monitor the stability of the magnetic field and to improve the field 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 abundant isotopes, i.e.: 13C or 15N.

          


300 MHz Proton NMR Spectrum of Ethyl Benzene after Fourier Transformation of the FID.
Different protons resonate at different frequencies (=> chemical shifts).
Proton signals often are split into multiplets, indicating couplings with other protons.


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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