2. NMR Automation Overview
• 1. NMR
a) history
b) Automation
• 2. ChenoMX
a) Processor
b) Profiler
c) Compound Builder
• 3. XML
• 4. NMRlib
3. 1. NMR History
• 1945 - First
experimentally
observed by Felix
Bloch (Stanford) and
Edward Purcell
(Harvard)
• 1946 - Spectra first
published in January
edition of Physical
Review
• 1952 - Nobel prize
for physics shared
by Bloch and Purcell
Felix Bloch
4. NMR (Nuclear Magnetic
Resonance)
• Nuclei of atomic isotopes (1H, 13C, 31P, 15N,
29Si etc.) interact with a static magnetic field
• Magnetic field makes spin states of nuclei
differ in energy, and transitions between
energy states can be observed
• Chemically distinct nuclei differ in resonance
frequency in the same magnetic field –
5. NMR
• Nuclei are aligned by a magnetic field
• An oscillating, perpendicular magnetic field is
applied
6. NMR
• Precession is a change in the
orientation of the rotational
axis of a rotating body.
• Dependent on rate of spin,
size and shape of gyro,
strength of gravity.
• Find out the magnetic field
strength to get the precession
frequency (frequency it takes
to get the nucleus precessing)
• 2nd magnetic field varies in
time at the same rate as the
precession - i.e. resonant with
the nuclear motion
(continuous wave NMR)
• Nuclei which are not aligned
7. • This non-equilibrium magnetisation can be induced,
generally by applying a pulse of resonant radio-
frequency close to the Larmor frequency of the nuclear
spins.
• Fourier Transform NMR, free induction decay (FID) is the
observable NMR signal generated by non-equilibrium
nuclear spin magnetisation precessing about the
magnetic field.
• The resultant oscillating magnetization vector of the
nuclei induces a current in a pickup coil, creating an
electrical signal oscillating at the NMR frequency (aka
FID).
• The FID contains the vector sum of the NMR responses
from all the excited spins
• To obtain the frequency-domain NMR spectrum (NMR
absorption intensity vs. NMR frequency) this time-
NMR - FID
8. NMR Spectra
• The chemical shift of a nucleus is the
difference between the resonance frequency of
the nucleus and a standard, relative to the
standard. (We use DSS: 4,4-dimethyl-4-
silapentane-1-sulfonic acid)
9. NMR Automation
• Why Automation?
-time: countless hours
spent processing/
profiling spectra
-inconsistencies between
different users (user
bias)
-misidentification in
profiling
-user fatigue results in
profiling errors
10. NMR Automation
Automation
-time: Faster (minutes vs.
hours)
-consistent profiling
-misidentification also
consistent, can correct
and re-run spectra
(saves time – hopefully!)
-fatigue is negligible
17. Phasing
• Phasing corrects shifts that may have occurred
in data acquisition
• Asymmetric peaks/clusters may be inverted
• Larger phase shifts may have an oscillation to
the baseline
• 3 phase angle controls: normal (1°), fine (0.1°)
and very fine (0.01°)
Sorry,
William
Shatner, not
this kind of
32. If you see wild
distortions, do not
panic. Move points to
centre of noise and it
will smooth out – hold
control and click on
the line to add points,
hold shift and click to
33. Line Broadening (Optional)
• Multiplies fid by an exponential
function before fid is Fourier
transformed
• Increases linewidth in spectrum and
smoothes out instrument noise
• Broader peaks, less noisy
• Without broadening – sharper peaks
but baseline may be noisy
43. Black: Spectrum
Red: Sum of profiled
compounds
Green: Subtraction line
Blue: Current compound
selected
Shortcuts: Space – automatic
fit
(bottom left will say it can’t fit
if
autofit doesn’t work)
44. These numbers will go
green if there’s a good
fit (NB: will not go
green all the time even
if fit well)
Click these numbers to
view/move different
clusters
NB: Name here is the
compound selected
45. Bottom blue arrow will
shift cluster selected on
x axis
Right side arrow will
shift all clusters on y
axis
Clicking and dragging
on the blue peak will
move all clusters x +
y
61. 3. XML
• Extensible Markup
Language (.xml files)
• A (document) markup
language is a modern
system for annotating
a document in a way
that is syntactically
distinguishable from
the text. [Wikipedia]
• Advantage: Machine
and Human readable
65. More XML
• <clusters>
• <cluster>
• <center>1.17166</center>
• <lowerBound>1.17166</
lowerBound>
• <upperBound>1.17166</
upperBound>
• <peaks>
• <peak>
• <center>1.15747</center>
Bounds limit
how far the
cluster centres
can shift
Peaks are
described by
their centres,
66. BAYESIL
• “Siamak’s Program”
• Processes NMR spectra similarly to
ChenoMX – phases, baseline corrects
etc.
• Global fit – uses area under the curve
to fit spectra
• Future improvements: UI (User
interface), flagging false positives/
negatives, concentration corrections,
69. 4. NMRlib
• “Roman’s Program”
• GUI (Graphical User Interface), web
based application
(nmrlib.wishartlab.com) that uses
BAYESIL to process spectra and create
XML files
• Faster than ChenoMX approach,
eliminates typographical errors
• Doesn’t allow for overlapping peaks
NMRlib
72. NMRlib
• Fill in fields, Name,
HMDBID, NMR Freq.
Metabolite
concentration (as
measured by
ChenoMX or
experimental), and
ratio of [metabolite]/
[DSS]
• -Spectrum files must
be .zip files and the
fid must be in a
folder named
HMDBxxxxx.fid
eg.
