Spectroscopy Assignment by Mustafa Hamido

7/29/2019 Spectroscopy Assignment by Mustafa Hamido

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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

Spectroscopy Assignment by Mustafa Hamido

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