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Trinity College of Dublin
M.Sc. Pharmaceutical Analysis
TMA Module 3 Spectroscopy Dr. Sasse
MSc. Pharmaceutical Analysis 2012/2013
Name: Mustafa Hamido
Student Number: 11263930
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TMAModule 3 Spectroscopy Dr. Sasse
MSc. Pharmaceutical Analysis 2012/2013
Mustafa Hamido [email protected]
1. What are HETCOR, INADEQUATE, TOCSY in NMR? How are they related? What is theirimportance in structure elucidation?
NMR
Nuclear magnetic resonance (NMR) is a physical phenomenon in which magnetic nuclei in a
magnetic field absorb and re-emit electromagnetic radiation. This energy depends on the strength of
the magnetic field and the magnetic properties of the isotope of the atoms. The resonance
frequency of a particular substance is directly proportional to the strength of the applied magnetic
field.
Spectroscopy is the study of the interaction of electromagnetic radiation with matter. Nuclearmagnetic resonance spectroscopy is the use of the NMR phenomenon to study physical, chemical,
and biological properties of matter. NMR spectroscopy finds applications in several areas of science.
NMR spectroscopy is routinely used by chemists to study chemical structure using simple one-
dimensional techniques. Two-dimensional techniques are used to determine the structure of more
complicated molecules. These techniques are replacing x-ray crystallography for the determination
of protein structure. Time domain NMR spectroscopic techniques are used to probe molecular
dynamics in solutions. Solid state NMR spectroscopy is used to determine the molecular structure of
solids. Other scientists have developed NMR methods of measuring diffusion coefficients.
Two Dimensional NMR
Conventional NMR spectra (one-dimensional spectra) are plots of intensity vs. frequency; in two-
dimensional spectroscopy intensity is plotted as a
function of two frequencies, usually called F1 and
F2. There are various ways of representing such a
spectrum on paper, but the one most usually used
is to make a contour plot in which the intensity of
the peaks is represented by contour lines drawn at
suitable intervals, in the same way as a
topographical map. The position of each peak isspecified by two frequency co-ordinates
corresponding to F1 and F2. Two dimensional NMR spectra are
always arranged so that the F2 co-ordinates of the peaks
correspond to those found in the normal one dimensional
spectrum, and this relation is often emphasized by plotting the
one dimensional spectrum alongside the F2 axis.
The figure shows a schematic COSY spectrum of a hypothetical
molecule containing just two protons, A and X, which are coupled
Figure 1 Classical H 1 Dimesnional NMR
Figure 2 Example of COSY
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together. The one dimensional spectrum is plotted alongside the F2 axis, and consists of the familiar
pair of doublets centered on the chemical shifts of A and X, A and X respectively.
In the COSY spectrum, the F1 co-ordinates of the peaks in the two-dimensional spectrum also
correspond to those found in the normal one dimensional spectrum and to emphasize this point the
one dimensional spectrum has been plotted alongside the F1 axis. It is immediately clear that thisCOSY spectrum has some symmetry about the diagonal F1 = F2 which has been indicated with a
dashed line.
In one-dimensional pulsed Fourier transform NMR the signal is recorded as a function of one time
variable and then Fourier transformed to give a spectrum which is a function of one frequency
variable. In two-dimensional NMR the signal is recorded as a function of two time variables, t1 and
t2, and the resulting data Fourier transformed twice to yield a spectrum which is a function of two
frequency variables.
The two dimensional NMR spectroscopy is used to get more information not obtainable from one-dimension spectra. The most common used techniques for the two dimensional NMR are: HETCOR,
TOCSY and INADEQUATE. A lot other two dimensional techniques are available such as COSY,
HMBC...etc. (1)
HETCOR (Heteronuclear Correlation Spectroscopy)
HETCOR is a 2D Proton-Carbon NMR spectroscopy where two different nucleuses are correlated
through single bond spin-spin couplings. It uses Use the JHC interaction to correlate proton and
carbon shifts. It Uses JHC interaction to correlate protons with neighboring carbons.
The principles of HETCOR are
precisely analogous to COSY. A
different experimental regime
however is required since two
observing nuclei with different
Larmor frequencies are involved.
That is why this technique is refer to
H, X-COSY, where X could be 13C,
15N, 31P, 29Si etc.
The experiment is used to correlate
the chemical shifts of X-nuclei with
the chemical shifts of protons
coupled with the X-nuclei. The
assignment of one member of a spin-
coupled pair leads immediately to the
assignment of the other. Most NMR
instruments with two channels can
perform the experiment. The 90degree pulses for X nucleus and proton need to be calibrated. HETCOR has a lower sensitivity in
Figure 3 HETCOR pulse sequence. Where d90 specifies the proton 90
degree pulse, p90 is X nucleus
Figure 4(HETCOR spectra recorded by D. Fox, Dept of Chemistry,
University of Calgary on a Bruker Advance DRX-400 spectrometer)
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comparison to other 2D Proton-Carbon NMR spectroscopy techniques. It is useful in case we need
high resolution in C dimension.
INADEQUATE
INADEQUATE is an NMRexperiment of analyzing
adjacent spin pairs from the
correlation of double quantum
transition and chemical shifts. Jc-
c spin coupling constants are
valuable for the structure
elucidation of chemical
compounds. However the Jc-c
is much difficult to determine in
nature abundance, since the
amount of 13C in the nature is about 1% of 12C isotope, the
probability of two 13C nuclei being adjacent is one out of ten
thousand. In normal proton decoupled 13C spectra, almost of all
carbon signals are those of isolated 13C. In this experiment the
13C signals from isolated 13Cs are suppressed and the coupled
13C (doublets, coupled to another 13C) are observed, so that the
connectivity of carbons can be determined.
It has a very low sensitivity as this technique needs pairs of C
which is very rare. A very concentrated sample must be used to
get good results. .It considers a nice way to trace carbon
skeleton of organic compound. This technique is used when the
sensitivity is not a big issue.
TOCSY
TOCSY is a 2D Proton NMR
spectroscopy. It is a correlation
between all protons within
spin system. It shows all
protons in the spin system .It
produces narrow line shapes.
Cross peaks are observed
nuclei which are connected by a chain of couplings. This property makes easy to identify the larger
interconnected networks of spin couplings. It is useful for assigning resonances in side-chains of
proteins.
1H-1H TOCSY (Total Correlated Spectroscopy also known as HOHAHA Homonuclear Hartmann
Hahn) is useful for dividing the proton signals into groups or coupling networks, especially when themultiplets overlap (have very similar chemical shifts) or there is extensive second order coupling. A
Figure 6 The pulse sequence uses 90 and 180 degree pulses. Extensive phase
cycling is carried out to suppress the singlet 13C signals. JC-C is one bond
carbon-carbon coupling constant.
Figure 5 An INADEQUATE spectrum
of menthol in C6D6.
Figure 7 Pulse Sequence of TOCSY
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TOCSY spectrum yields through bond correlations via spin-spin coupling. Correlations are seen
throughout the coupling network and intensity is not related in a simple fashion to the number of
bonds connecting the protons. Therefore a five-bond correlation may or may not be stronger than a
three-bond correlation. TOCSY is
usually used in large molecules
with many separated coupling
networks such as peptides,
proteins, oligosaccharides and
polysaccharides. If an indication
of the number of bonds
connecting the protons is
required, for example in order to
determine the order in which
they are connected, a COSY
spectrum is preferable.
The pulse sequence used in our
laboratory is the gradient
enhanced TOCSY that means that
during the pulse sequence,
magnetic field gradients are
applied. The spin-lock is a
composite pulse and should be applied for between 20 and 200ms with a pulse power sufficient to
cover the spectral width. A short spin-lock makes the TOCSY more COSY-like in that more distantcorrelations will usually be weaker than short-range ones. A long spin-lock holds the magnetic vector
in the x-y plane allowing correlations over large coupling networks. The length of the spin-lock is
roughly related to the distance through the coupling network that correlations are seen. However,
too long a spin-lock will heat the sample causing signal distortion and can damage the electronics of
the spectrometer. Therefore care should be taken.
Figure 8 Artifacts in the TOCSY spectrum of ethylbenzene
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2. Deduce a structure for C10H6O3 that corresponds to the spectra below. Show your work.
UV
The UV spectrum above doesnt tell a lot about the chemical compound. UV spectrum is not
an effective tool to deduce the structure of any compound. It can tell about the nature of
the compound or the chemical environment of the compound.
The compound is definitely has a chromophore as it absorbs UV light.
We notice a change in the PH environment between the two solvents. NaOH has a high PH
in comparison to Ethanol. Changing the pH of the solvent leads to a bathochromic shift to a
higher wavelength and a hyperchromic effect (increase in intensity). This indicates that the
spectrum above is for an Aromatic compound. The spectrum suggests that the compound
has a benzene ring which has a wavelength of with a carboxyl substituent.
Substituentmax
-C(O)OH 273
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IR (KBr)
The IR spectrum above has many stretches which can identify the chemical compound I have. It is
clear that the main stretching area is between 3000-3500 wavenumber/cm. The stretch in this area
let me think that we have an Aromatic group and OH group.
Stretching Group
3100 Aromatic
3500 Hydroxyl Group
1600C=C in Aromatic
1650 Alkene C=C
1680 C=O group
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1H-NMR (400 MHz, DMSO-d6)
In addition, there is a signal at 13 ppm, which integrates for 1H and is exchangeable in D2O.
It seems from the spectrum has three main regions for chemical shifts. The first is at 6 ppm,
the second between 7.8ppn and 8.0 ppm and the third is exchangeable signal at 13 ppm.Theregion between 7.8 and 8.0 tells that the compound is Aromatic while the shift at 13 ppm tells
that the compound has a (CO2)H group.
Shift Splitting Chemical Environment
7.8ppm-8ppmMultiplet Aromatic Ring
6.109 ppm Singlet Alkene C=C
13ppm singlet (CO2)H
The compound structure could be:
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APT13
C-NMR (DMSO-d6)
APT and DEPT are techniques for 1H-decoupled13C spectra which use the phase (normal or
upside-down) or selective deletion (certain peaks missing) of the13C peaks as a way to encode
information about the number of protons attached to a carbon (C, CH, CH2 or CH3).
APT gives all of the information of a normal carbon spectrum with somewhat reduced
Sensitivity, and it tells you if the number of attached protons is odd (CH3 or CH) or even (CH2
or quaternary). The APT spectrum shows all carbons including the quaternary C=O and solvent
carbons, and sorts the carbons into categories of CH and CH3 (up peaks) and quaternary and
CH2 (down peaks).The CH and CH3 groups appear as positive peaks while those from CH2
and quaternary carbons are negative. In comparison to the DEPT technique, all carbon nuclei
are visible in one spectrum.
110 125 126 130 132 133 134 160 182 185
+ + + - - + - + - -
CH CH CH CH2 CH2 CH CH CH2 C=O C=O
The compound could be:
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EI-MS
The mass spectrum is a plot of ion abundance versus mass-to-charge ratio. The most
abundant ion formed in the ionization chamber gives rise to the tallest peak in the mass
spectrum, called the base peak. In the mass spectrum of C10H6O3, the base peak is indicatedat an m/z value of 174. The relative abundances of all of the other peaks in the spectrum are
reported as percentages of the abundance of the base peak.
- When a beam of high-energy electrons impinges upon a stream of sample molecules,
ionization of electrons from the molecules takes place. The resulting ions, called molecular
ions, are then accelerated, sent through a magnetic field, and detected. If these molecular
ions have lifetimes of at least 105 seconds, they reach the detector without breaking into
fragments. The mass spectrometer can distinguish between masses of particles bearing the
most common isotopes of the elements and particles bearing heavier isotopes.
- Consequently, the masses which are observed for molecular ions are the masses of the
molecules in which every atom is present as its most common isotope.
- Molecules subjected to bombardment by electrons may break apart into fragment ions. As
a result of this fragmentation, mass spectra can be quite complex, with peaks appearing at a
variety of m/e ratios.
- Fragmentation can provide useful evidence for the structure of the compound. A chemist
pieces together the fragments to form a picture of the complete molecule.
- The largest m/z ratio in the mass spectrum is 174 m/z. This is the molecular ion peak which
means that the molecule has a relative molecular mass of 174 m/z .
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- The other peaks with smaller m/z ratios result from fragmentation of molecule. The most
abundant fragment ions appear to have relative masses abundant fragment ions with
masses of 146 m/z, 118 m/z and 105 m/z.
- The compound we are dealing with is unsaturated and contains an Aromatic Ring. The
saturation of the compound is calculated by :
UnSaturation= 2C-(H+2)/2
= 14+2/2=8 as the compound is C10H6O3
Molecular Peak Name
174 Base Peak
Fragments are seen in the spectrum as following:
The compound structure could be:
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Sources:
1. NMR Spectroscopy Explained: Simplified Theory, Applications and Examples for Organic
Chemistry and Structural Biology, Neil E. Jacobsen , John Wiley & Sons
2. Understanding NMR Spectroscopy. James Keeler, John Wiley & Sons
3. 1D and 2D NMR:Experiment Methods , Emory University 2011