C13 NMR and 2D NMR

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c-13 n 2D NMR


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13C NMR:

13 C NMR The C-12 is the most abundant and naturally occuring isotope of carbon which is magnetically inactive because of its spin quantum number(I) is zero. The 13 C nucleus is magnetically active as that of H-nucleus and has a spin quantum number ½ The natural abundance of 13 C is only 1.1% that of C-12, as a result it gives extremely weak NMR signals and also its sensitivity is only 1.6% that of H 1 .


IMPORTANCE OF 13 C NMR CMR is a noninvasive and nondestructive method ,i.e. especially used in repetitive In-vivo analysis of the sample without harming the tissues . CMR of biological materials allows for the assessment of the metabolism of carbon, which is so elementary to life on earth. CMR, chemical shift range(0-240 ppm ) is wider which permits easy separation and identification of chemically closely related metabolites. The low natural abundance of 13C nuclei (1.1%) can be made use of tagging a specific carbon position by selective C-13 enrichment, which the signal intensities and helps in tracing the cellular metabolism.

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Labeling is more convenient means of following the metabolism specific carbons throughout the metabolism. C13 nuclei are a stable isotope and hence it is not subjected to dangers related to radiotracers. Labeling of 13C nucleus at multiple carbon sites in the same molecule was possible, as result homonuclear 13C-13C coupling provides novel biochemical information.


CHARACTERISTIC FEATURES OF 13 C NMR The chemical shift of the CMR is wider( δ is 0-240ppm relative to TMS) in comparison to PMR( δ is 0-14ppm relative to TMS). C13-C13 coupling is negligible because of low natural abundance of C13 in the compound. Thus in one type of CMR spectrum(proton de coupled) each magnetically non equivalent carbon gives a single sharp peak that does undergo further splitting. The area under the peak in CMR spectrum is not necessary to be proportional to the number of carbon responsible for the signal. Therefore not necessary to consider the area ratio.


CORRELATION CHART The correlation chart is here divided into sections 1) The saturated carbon atom which appear at highest field nearest to TMS (8-60ppm) 2) Effect of electronegative atom(40-80ppm) 3) Alkanes and aromatic carbon atom(100-170) 4) Carbonyl carbon bond which appear at lowest field value (155-200ppm). ANY USE?


TYPES OF C13 SPECTRA 1) Proton coupled 13 c spectra 2) Proton decoupled c 13 spectra 1) Proton coupled c 13 spectra a) Homonuclear coupling The probability of finding 13 c adjacent carbon is very less therefore homonuclear [carbon- carbon ]splitting is rarely seen. b) Heteronuclear coupling. It involving two different atoms [carbon- hydrogen] Here splitting arises due proton attached directly to 13 c carbon

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2 ) Proton decoupled 13 c spectra Here the decoupling technique obliterates all the interaction between proton and c 13 nuclei thus singlet are observed in proton decoupled c-13 spectra

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NOE SIGNAL ENHANCEMENT NOE may lead to signal enhancement in c13- as the decoupling may interfere with the relaxation times. Major relaxation route –dipolar transfer of its excitation energy to proton attached to it (T 2 ). NOE operates on CH 3 ,CH 2 , CH. ADVANTAGE –Easy identification of carbons without protons. HOW TO ELIMINATE?


ELIMINATION TECHNIQUES Gated decoupling permits the recording of non decoupled spectra while simultaneously harnessing the increase in the line intensities which the NOE generates. Inverse gated decoupling permits the more accurate measurement of line intensities in decoupled c-13 resonances , while at the same time avoiding the distortions of the NOE which invariably accompany decoupling.


QUANTITATIVE MEASUREMENT OF LINE INTENSITIES Two main reasons Nuclear Overhauser Effect Short delay times in pulsed FTR mode- Saturation effect and Solvent peaks METHODS TO ELIMINATE NOE Saturation effect- longer delays between pulses Solvent peaks –paramagnetic ions E.g. chromium acetylacetonate ,shift reagents

Off resonance proton decoupling AND DEUTERIUM COUPLING :

Off resonance proton decoupling AND DEUTERIUM COUPLING Advantage? e.g. p- hydroxy acetophenone CONSEQUENCE?

Structural applications :

Structural applications Differentiation of isomers e.g. C 4 H 10 O DIETHYL ETHER METHYL PROPYL ETHER METHYL ISOPROPYL ETHER


STEPS IN DEDUCING THE STRUCTURE OF ORGANIC COMPOUND Number of signals = nonequivalent carbon environments Assign the chemical shift signals using the correlation chart Note the intensities of the peaks -Low intensity And high intensity peaks Take account of multiplicity of peaks - q ,t ,d , s

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Interpreting NMR Spectra Alkanes 1 H-NMR signals appear in the range of  0.8-1.7. 13 C-NMR signals appear in the considerably wider range of  10-60. Alkenes 1 H-NMR signals appear in the range  4.6-5.7. 1 H-NMR coupling constants are generally larger for trans - vinylic hydrogens (J= 11-18 Hz) compared with cis - vinylic hydrogens (J= 5-10 Hz). 13 C-NMR signals for sp 2 hybridized carbons appear in the range  100-160, which is to higher frequency from the signals of sp 3 hybridized carbons.

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Proton-decoupled 13 C-NMR spectrum of 1-bromobutane

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Aldehydes and ketones 1 H-NMR: aldehyde hydrogens appear at  9.5-10.1. 1 H-NMR: a- hydrogens of aldehydes and ketones appear at  2.2-2.6. 13 C-NMR: carbonyl carbons appear at  180-215. Amines 1 H-NMR: amine hydrogens appear at  0.5-5.0 depending on conditions. Interpreting NMR Spectra

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1 H-NMR spectrum of vinyl acetate.

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Carboxylic acids 1 H-NMR: carboxyl hydrogens appear at - 10-13, higher than most other types of hydrogens . 13 C-NMR: carboxyl carbons in acids and esters appear at - 160-180.


FT NMR These involves irradiation of sample with all the frequency simultaneously ,by supplying a powerful pulse of rf current for few milliseconds. The proton in each environment absorb there appropriate frequency from pulse and these frequency couple to give beats. At the end of excitation pulse the nuclei undergoes relaxation process and reemit the absorbed and coupled energies To give interferogram in the time domain The Fourier transform converts these same into frequency domain as spectrum. Forced induction Free induction

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Free Induction Decay The signals decay away due to interactions with the surroundings. A free induction decay, FID, is the result. Fourier transformation, FT, of this time domain signal produces a frequency domain signal. FT Time Frequency


ADVANTAGES OF FT-NMR TECHNIQUE The scanning takes place rapidly compared to continuous wave NMR. The sensitivity problems are eliminated in NMR, therefore which helps in a) Analysis the sample at very low concentration. b) NMR studies on nuclei with low natural abundance(c13) c) NMR studies on nuclei with low natural abundance and low magnetic moment (C13,N16).

FROM 1D -2D:

FROM 1D -2D A 2D NMR experiment consists of large number of small 1D experiments with a time delay of T 1 between the each pulse. Something (prepare) T 1 Something else (transfer) T 2




1967 Fourier transforms 1971 Jean Jenner - Two dimensional NMR - COSY 1976 Richard Ernst - First two dimensional NMR experiment HISTORY 1980s - application of NMR to protein structures In 1991- Ernst won a Nobel Prize in Chemistry for his contributions to Fourier Transform NMR

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Why 2D NMR ? 1D protein spectra are far too complex for interpretation as most of the signals overlap heavily 1D NMR spectrum of a protein

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B o = 0 B o > 0 Randomly oriented Highly oriented B o Ensemble of Nuclear Spins N S Each nucleus behaves like a bar magnet.

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The net magnetization vector z x y w w z x y M o - net magnetization vector allows us to look at system as a whole z x w one nucleus many nuclei

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Nuclear Spin Dynamics z x y M o z x y M o z x y M o RF off RF on RF off Effect of a 90 o x pulse

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BASIC LAYOUT OF A 2D NMR EXPERIMENT Preparation EVOLUTION T 1 Mixing t Detection t 2 Do something with the nulcei (preparation), let them precess freely (evolution), Do something else (mixing), and detect the result (detection, of course).


MIXING-MAGNETIZATION TRANSFER Scalar J Coupling Electrons have a magnetic moment and are spin 1/2 particles. J coupling is facilitated by the electrons in the bonds separating the two nuclei. This through-bond interaction results in splitting of the nuclei into 2I + 1states. Thus, for a spin 1/2 nucleus the NMR lines are split into 2(1/2) + 1 = 2 states. 1 H 12 C 12 C 1 H Multiplet = 2nI + 1 n - number of identical adjacent nuclei I - spin quantum number

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Scalar J Coupling The magnitude of the J coupling is dictated by the torsion angle between the two coupling nuclei according to the Karplus equation. C C H H H H q J = A + Bcos ( q ) + C cos 2 ( q) A = 1.9, B = -1.4, C = 6.4 q 3 J Karplus Relation A, B and C on the substituent electronegativity .

Resulting 2D spectra:

Resulting 2D spectra 3 types of peaks Diagonal peaks are related to only one transition and occur at the main diagonal of the 2D spectrum Cross peaks correlate different transitions, are off diagonal peaks Axial peaks give information about spin-lattice relaxation processes

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DIAGONAL AND CROSS PEAKS Give information about: Connectivity of transitions in the energy level diagram- The rotation super operator r couples transitions Transverse relaxation processes: Line shapes depend on transverse relaxation times T2 The initial state of the spin system , ρ(0): Conventional NMR only measures allowed transitions, while all matrix elements of ρ(0) can be measured with 2D NMR

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PROCESSING 2D DATA n is the number of increments


GENERAL PROCEDURE FOR RUNNING 2D SPECTRA Insert sample, tune 1 H and 13 C channels Lock and shim (determine 90 o pulse width) Acquire 1 H NMR spectrum Change spectral window to ± 1 ppm of spectrum Re-acquire 1 H spectrum Phase spectrum, apply baseline correction Acquire 13 C spectrum in optimum spectral window Call up macro for 2D experiment. Use 1 H and 13 C parameters for 2D experiments Alter number of transients, number of increments to fit the time available Repeat steps 8 & 9 for other 2D experiments required Set experiments running


SAMPLE REQUIREMENTS  0.25 ml0.5mM protein (=2.5mg for 20kDa protein) 15N,13C,(2H) labelled ( E.coli ) MWT < 60kDa for 3D structure MWT <100(800) kDa for secondary structure, functional tests,etc .

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TYPES OF 2D NMR EXPERIMENTS AUTOCORRELATED Homonuclear J resolved 1 H- 1 H COSY TOCSY NOESY ROESY INADEQUATE CROSS-CORRELATED Heteronuclear J resolved 1 H- 13 C COSY HMQC HSQC HMBC HSQC-TOCSY Three 2D spectra which are widely used for the structure determination of proteins with a mass of up to 10 kD 2D COSY 2D TOCSY 2D NOESY


2D TOCSY In the TOCSY experiment, magnetization is dispersed over a complete spin system of an amino acid by successive scalar coupling. The TOCSY experiment correlates all protons of a spin system. Therefore, not only the red signals are visible (which also appear in a COSY spectrum) but also additional signals (green) which originate from the interaction of all protons of a spin system that are not directly connected via three chemical bonds.


2D NOESY The NOESY experiment is crucial for the determination of protein structure. It uses the dipolar interaction of spins for correlation of protons. The intensity of the NOE is in first approximation propotional to 1/r 6. The NOESY experiment correlates all protons which are close enough. It also correlates protons which are distant in the amino acid sequence but close in space due to tertiary structure. This is the most important information for the determination of protein structures.



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NOE is a consequence of cross-relaxation between 2 spins close to each other in space. NOE is a consequence of modulation of the Dipole-Dipole coupling by motion of the molecule in solution. The NOE intensity is related to the inter nuclear distance r and is a function of the correlation time t c NOE  1 r 6 F ( t c ) NOE and distance r < 5 A Both NOESY and ROESY need long relaxation delays (2 s) The NOE effect ~ 0 at 1000 Da. It works well for small molecules ( t mix ~ 800 ms) and macromolecules ( t mix ~ 100 ms).

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COSY Two-nucleus (2D) NMR experiment called as COSY that evaluates proton-to proton interactions. COSY OVER PMR? Identification of coupling partners in complex structures . COSY spectra can often allow for assignment of coupling relationships in non-first order spectra. In a COSY experiment, the 1D PMR spectrum is plotted along both the x and y axis. Diagonal line in a COSY 1D spectrum. Off-diagonal peaks coupling relationships

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1 H- 1 H COSY ( COrrelated SpectroscopY ) (90 o ) x (90 o ) x t 1 Preparation Evolution Detection 1 H t 2

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2D COSY SPECTRUM OF ETHYLBENZENE The most apparent cross-peak in the spectrum is between H1' and H2' at 2.65 and 1.24 ppm A much weaker four-bond correlation (see the figure below) appears between H1' and H2 at 2.65 and 7.20 ppm

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REQUIREMENTS FOR 1 H- 1 H COSY Number of transients required is half that needed to give decent 1D 1 H NMR spectrum Most of the time we use a ‘double quantum filtered COSY’ (DQF-COSY): Same information as COSY but removes single quantum transitions (large singlet peaks from Me groups), meaning we can see things closer to the diagonal. Solves problems in case where there is a dynamic range problem (very large and very small peaks in same spectrum) It is phase sensitive, we acquire 2 x number of increments (real and imaginary). Get coupling information from phases of correlation peaks.

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COSY spectra recorded by D. Fox, Dept of Chemistry, University of Calgary on a Bruker Advance DRX-400 spectrometer A in the top left corner indicates a coupling interaction between the H at 6.9 ppm and the H at 1.8 ppm .  This corresponds to the coupling of the CH 3 group and the adjacent H on the alkene . B indicates a coupling interaction between the H at 4.15 ppm and the H at 1.25 ppm .  This corresponds to the coupling of the CH 2 and the CH 3 in the ethyl group.

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C 3 H 8 O

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CH 3 – CH 2 – CH 2 – OH CH 3 CH 2 CH 2 OH C 3 H 8 O: COSY

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COSY : 11 B 6/9 2/4 5,7,8,10 1/3

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C 5 H 8 O 2 I= 5 – 8/2 +1 = 2 CH 2 - O CH 2 - O O -C= O

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C 5 H 8 O 2 CH 2 - O d CH 2 O C= O a b c d a CH 2 c CH 2 b CH 2 2 3 4 5

2D NMR applications:

2D NMR applications


2D NMR APPLICATIONS Structural identification in organic and biological chemistry. Since its creation, 2D NMR has been useful for elucidating the structure of small molecules Advanced computing power now allows the structure of large , biological molecules to be solved. Used COSY and NOESY to obtain individual assignments for each proton in the protein backbone in the β-sheet secondary structure of pancreatic trypsin inhibitor COSY spectra taken in 2H2O and H2O were combined to obtain sequential resonance assignments

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Additional experiments were carried out with t1 at different phases to cancel out axial peaks2D COSY 1H NMR for pancreatic trypsin inhibitor at 360 MHz. COSY NMR was used to determine the J connectivities on the protein backbone 2D NMR uses a sequence of two pulses with a series of different evolution times to determine which nuclear spins are coupled to one another. COSY spectra indicate through-bond coupling, and can be used to gain structural information about molecules of a wide range of sizes.

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2D COSY 1H NMR for pancreatic trypsin inhibitor at 360 MHz.

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NOESY was used to determine distance dependent coupling, since NOE can only occur at distances of <5-6 Å A “β-snail” structure was observed for the protein.


REFERENCES Instrumental Methods of Chemical Analysis by GR. Chatwal & Sham K. Anand pg.no-2.18-2.25. Elementary Organic Spetroscopy by Y.R. Sharma pg.no-5.11-5.26. Text book of quantative Chemical Analysis by Vogels . "Principles of NMR". Process NMR Associates. Retrieved 2009-02-23. Organospectroscopy by william kemp pg.no-114-294 Online book http://www.cis.rit.edu/htbooks/nmr/

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