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

TMA Module 3 Spectroscopy Dr. Sasse MSc. Pharmaceutical Analysis 2012/2013 Mustafa Hamido mustafahamido@gmail.com

1. What are HETCOR, INADEQUATE, TOCSY in NMR? How are they related? What is their importance 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. Nuclear magnetic 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 onedimensional 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 twodimensional 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 is specified by two frequency co-ordinates
Figure 1 Classical H 1 Dimesnional NMR

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 2 Example of COSY Spectrum

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 this COSY 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 onedimension 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
Figure 3 HETCOR pulse sequence. Where d90 specifies the proton 90 degree pulse, p90 is X nucleus

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 spincoupled pair leads immediately to the assignment of the other. Most NMR Figure 4(HETCOR spectra recorded by D. Fox, Dept of Chemistry, instruments with two channels can University of Calgary on a Bruker Advance DRX-400 spectrometer) perform the experiment. The 90 degree pulses for X nucleus and proton need to be calibrated. HETCOR has a lower sensitivity in

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 NMR experiment of analyzing adjacent spin pairs from the correlation of double quantum transition and chemical shifts. Jcc spin coupling constants are valuable for the structure elucidation of chemical Figure 6 The pulse sequence uses 90 and 180 degree pulses. Extensive phase compounds. However the Jc-c cycling is carried out to suppress the singlet 13C signals. JC-C is one bond is much difficult to determine in carbon-carbon coupling constant. 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
Figure 5 An INADEQUATE spectrum of menthol in C6D6.

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.
Figure 7 Pulse Sequence of TOCSY

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 the multiplets overlap (have very similar chemical shifts) or there is extensive second order coupling. A

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,
Figure 8 Artifacts in the TOCSY spectrum of ethylbenzene

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 distant correlations 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.

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.

Substituent -C(O)OH

max 273

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

1600 1650

C=C in Aromatic Alkene C=C

1680

C=O group

H-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.The region 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 7.8ppm-8ppm Splitting Multiplet Singlet singlet Chemical Environment Aromatic Ring Alkene C=C (CO2)H

6.109 ppm

13ppm

The compound structure could be:

APT 13C-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 + CH

125 + CH

126 + CH

130 CH2

132 CH2

133 + CH

134 CH

160 + CH2

182 C=O

185 C=O

The compound could be:

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 indicated at 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 .

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:

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