Sample Preparation

As undergraduate students, it is very rare that we are afforded the opportunity to work with equipment such as the Nuclear Magnetic Resonance (NMR). Dr. Timothy Bates, our supervisor for this project, kindly organised for our group to undertake some valuable training with Dr. Stephen Prior, who is an Instrumental Scientist, at the University of Lincoln.

Before we could start preparing our samples, Dr. Prior described NMR spectroscopy and its capabilities. We learned that NMR spectroscopy can be used to determine the chemical and physical properties of atoms or the molecules that they are contained in, and in addition can be used to confirm the identity of a substance within a mixture of compounds.

NMR machine

Bruker 500 MHz NMR with auto sampler

Once we understood the basic theory of NMR spectroscopy and what information we could obtain from using it with biological samples and the various constraints on sample preparation, we moved on to preparing the tissue extracts. The first task was to clean the NMR tubes that we would be using to hold our samples, and it was important this was done thoroughly or the results would be affected by any water residue or other chemicals left from previous samples. We used highly purified distilled water (18.2 Mega Ohm grade) and the tubes were then dried out with an air tap supply and a small plastic tube.

Keita and Lee-Anne cleaning out the NMR tubes.

Keita and Lee-Anne cleaning out the NMR tubes.

The next step was to prepare the samples: (1) glycine, (2) l-lysine, (3) bovine serum albumin, (4) deoxyribonucleic acid [DNA] and (5) bile salts. All of the samples had been freeze-dried by Dr. Bates the day before. In order to prepare them we first had to clean a syringe using ethanol, then we had to fill the syringe half way with the solvent used to dissolve the dry tissue extracts in, Deuterium Oxide (D2O). D2O allows the protons from the tissue extracts to produce an NMR signal without showing up on the spectrum itself, making identification of signals from the tissue extract metabolites much easier. Once the sample and D2O were mixed together the syringe was filled to around the half mark again. This tissue sample solution was transferred to the NMR tube and then placed in a holder, ready for proton (1H) NMR spectroscopy.

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Sam, Corinne and Luke working on preparing the samples.

Dr. Prior instructed the group on how to use the software, which is complex and powerful. When the NMR spectra were produced we could then use the software to interpret the results.

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Keita analysing the spectra.

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