In this video, I'm going to discuss Nuclear Magnetic
Resonance spectroscopy.
NMR spectroscopy is often described
in organic chemistry textbooks in a very
dry and difficult to understand way.
In this video, I'd like to bring NMR to life
and make it more approachable.
NMR spectroscopy is a versatile analytical technique
that is used to elucidate the structure of chemical
compounds.
Sample sizes of 50 milligrams or less
can be analyzed in a non destructive way.
Before the widespread use of NMR,
chemical compounds underwent chemical tests
to help determine their structure.
Chemical tests such as the Tollen's test, the 2,4 D and P
test, or the idoform test were used
to detect functional groups inside chemical compounds.
Unfortunately, during the process of this analysis,
the sample was destroyed because it undergoes a chemical
reaction and cannot be recovered.
By contrast, in NMR spectroscopy,
after analysis is complete, the sample
can be recovered and used for other purposes.
This is particularly important in cases where there's
very little sample available or the sample
itself is prohibitively expensive to purchase.
In the biological sciences, elucidation
of protein structures often begins with NMR analysis.
Even after spending weeks, months,
or longer as an NMR sample, the pure protein
can be recovered and used for other purposes.
More routinely, organic chemists working
in research laboratories often use
NMR as a quick check of reaction products
or the progress of their reactions.
In the remainder of this video, I'd
like to show you a spectrometer that
has been taken apart so that you can see inside.
And lastly, I will use a live, working spectrometer
to obtain the spectrum of a prepared sample
and show you how it's analyzed.
Before we actually look at the spectrometer,
there are few key points of theory we need to keep in mind.
First, the sample is placed into a magnetic field.
The magnetic field aligns the spins of the nuclei.
It can align them in two different ways.
It can go with magnetic field, and that's the lower spin
state of alpha, or they can align
against the magnetic field, and that's the higher energy
spin state of beta.
The strength of the magnetic field
determines how far apart these energy levels are split.
The stronger the magnetic field, the further that they're split.
For high resolution NMR, we require
very strong magnetic fields.
And so we use superconducting magnets.
Because we are using superconducting magnets,
they must be kept very cold.
Once the sample is in the magnetic field,
it can be irradiated with RF radiation.
And this causes the alpha spin state
to excite to the beta spin state.
After the pulse is over, the nuclei,
which have been excited into the beta state,
relax back to the lower energy state of the alpha.
This is detected by the NMR instrument
as the time it takes the nuclei to relax back.
This is then transformed through a mathematical formula
into our readable spectrum.
Now let's take a look at the actual instrument.
Now let's have a look at how the instrument actually works.
Here's our prepared NMR sample.
This is usually three to 50 milligrams of sample,
dissolved in one milliliter of a deuterated solvent.
The sample is placed inside the spinner like this.
We'll have a look at this again in just a moment.
At the heart of the NMR spectrometer
is the superconducting magnet.
The superconducting magnet is kept
in the liquid helium chamber, which is kept at 4.2 Kelvin.
Superconducting magnets only superconduct
at very low temperatures, so we must maintain liquid
helium inside this chamber and covering the magnet.
Because liquid helium is so very cold,
it must be kept isolated from the outside environment
or it would evaporate very quickly.
To help do this, there are two layers of vacuum at the bottom.
Between these two layers of vacuum,
there's a protective layer of liquid nitrogen,
which is at 77 Kelvin.
This liquid nitrogen helps slow the rate
of evaporation of the liquid helium
by helping to prevent heat from transferring in from outside.
After the liquid nitrogen chamber,
there's two more layers of vacuum.
The liquid nitrogen must be refilled periodically.
Now we'll take a look at how the sample
goes into the spectrometer and sits in the probe.
Now we have our NMR sample prepared
and it's positioned inside the spinner.
Now we need to put it into the spectrometer for analysis.
The sample enters and exits the spectrometer here on top
through this hole.
The sample floats into and out of the spectrometer
on a cushion of air.
As the sample travels down the tube,
it goes into the magnetic field and sits
on top of a probe, which is where the data is collected.
Now let's have a look at the probe.
This is our probe.
And it sits down in the magnet, right about here.
The sample then travels down the tube
and sits into the probe just like this.
We need to homogenize the magnetic field
across the sample.
So we spin the sample much like this,
except it's done more smoothly in real life.
And so it spins.
The pulse of radio frequency radiation is transmitted here.
And it's also received here.
Different nucleus have different resonant frequencies.
And so the probe must be tuned to the different resonant
frequencies.
It's exactly analogous to turning in a radio station
on in your car on a radio.
And so the probe detects the resonance
and this data is then collected by the computer
and is transformed into the spectrum.
Now let's take a look at our live spectrometer
and run the spectrum collected on our sample.
So now we have our sample that's in the spinner.
Let's put it in the spectrometer so it can collect some data.
Now remember I said that the sample floats in
and floats out on a cushion of air.
So we'll get to see that here.
Now let's start our experiment.
Once the experiment is started, inside the spectrometer,
the probe will tune to the specific frequency
of the nucleus of interest and ensure
the magnetic field is held constant and homogeneous
through locking and shimming.
This is what the lock symbol looks like.
The pull sequence consists of three segments--
a delay period for all nuclei to align to the magnetic field,
a short pulse of irradiation to excite the spin states,
and a few seconds to measure and record the relaxation of spins.
The sequence is repeated several times
to improve the signal to noise ratio.
For example, in this spectrum, this spectrum
was collected with one scan.
Compared to this spectrum, which was collected with 64 scans,
we can see that the baseline is considerably smooth.
The more number of scans that are recorded,
the better the signal to noise ratio will be
and the smoother the baseline.
This is the actual data the instrument acquires.
This plot shows the Free Induction Decay, or FID,
of the excited spin state with time.
This FID contains all the frequencies
that will be a part of our spectrum
after mathematical treatment.
Let us now convert the FID into a readable spectrum,
using a FA transform function.
After a few cosmetic adjustments,
we are ready to reference the frequency
scale to the internal standard, of trimethyl silane
at zero PPM.
The size of each signal is proportional to the number
of magnetically equivalent nuclei producing the signal.
With the integration software, we're
able to calculate the area under each signal
and correlate that to the number of protons
that is creating that signal.
This is a sample of ethanol.
The spectrum shows one quartet for the three hydrogens
on the methyl group, a triplet for methylene unit,
and a singlet for the hydroxy group.