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Sana 13 nmr

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  • 1. CARBON-13 NMr school of pharmaceutical science (sops, utd-rgpv, Bhopal (m.p.) Prepared BySana iram M.PHARM (Q.A) 1st sem.
  • 2. INTRODUCTION:NUCLEAR MAGNETIC RESONANCE (NMR Spectroscopy) • A spectroscopic technique that gives us information about the number and types of atoms in a molecule. • Nuclear magnetic resonance spectroscopy is a powerful analytical technique used to characterize organic molecules by identifying carbon-hydrogen frameworks within molecules. Nuclear In the Nucleus Magnetic Resonance Involves Magnets In the Nucleus 2
  • 3.  It is concerned with the magnetic properties of certain atomic nuclei.  Involves change in the spin state at the nuclear level. SPINNIG NUCLEUS:  proton acts as a tiny spinning bar magnet and possesses both electrical charge and mechanical spin.  NMR is the most powerful tool available for organic structure determination.  It is used to study a wide variety of nuclei: 1. 1H 2. 13C 3. 15N 4. 19F 3 5. 31P
  • 4. INTRODUCTION TO 13C-NMR • Proton NMR used often for the complete elucidation of the unknown compound. • Carbon NMR can used to determine the number of non-equivalent carbons and to identify the types of carbon atoms(methyl, methylene, aromatic,carbonyl….) which may present in compound. 4
  • 5. INTRODUCTION ( CONT : ) • 12C has no magnetic spin. . 13C has a magnetic spin, but is only 1% of the carbon in a sample. •The gyromagnetic ratio of 13C is one-fourth of that of 1H. •Signals are weak, getting lost in noise. Hundreds of spectra are taken, averaged 5
  • 6. IMPORTANCE OF 13C NMR • CMR is a noninvasive and nondestrutive 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 compared to HNMR(0-14 ppm), which permits easy seperation 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. • Labelling is more convenient means of followimg the metabolism specific carbons throughout the metabolism. • C13 nuclei are a stable isotope and hence it is not subjected to dangers related to radiotracers. • Labelling of 13C nucleus at multiple carbon sites in the same molecule was possible, as result homonuclear 13C-13C coupling provides novel biochemical information. 6
  • 7. CHARACTERISTIC FEATURES OF 13C 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. • In proton-coupled spectra, the signal for each carbon or a groupof magnetically equivalent carbon is split by proton bonded directly to that carbon and the n+1 rule is followed. 7
  • 8. Types of 13c spectra 1) proton coupled 13c spectra 2) proton decoupled 13c spectra 1) PROTON COUPLED 13c SPECTRA a) Homoannular coupling the probablity of finding 13c adjacent carbon is very less therefore homonuclear [carbon- carbon ]splitting is rearaly seen b) Hetronuclear coupling. it involving two different atoms [carbon- hydrogen] Here splitting arises due proton attached directly to 13c carbon 8
  • 9. COUPLING TO ATTACHED PROTONS 3 protons 2 protons H 13 0 protons H C H 13 H n+1 = 4 Methyl carbon 1 proton C H n+1 = 3 Methylene carbon 13 C H n+1 = 2 Methine carbon The effect of attached protons on 13C resonances ( n+1 rule applies ) (J‟s are large ~ 100 - 200 Hz) 13 C n+1 = 1 Quaternary carbon
  • 10. 2) proton decoupled 13c spectra Here the decouplig technique obliterates all the interaction between proton and c13nuclei thus singlet are observed in proton decoupled c-13 spectra 10
  • 11. ETHYL PHENYLACETATE in some cases the peaks of the multiplets will overlap 13C coupled to the hydrogens 13C decoupled from the hydrogens this is an easier spectrum to interpret
  • 12. Problems of 13C-NMR 13 C-NMR signal is 6000 times weaker than NMR why??? NATURAL ABUNDANCE GYRO MAGNETIC RATIO COUPLING PHENOMENON 12 1 H-
  • 13. NATURAL ABUNDANCE: 13 C natural abundance is very low (1.08%). GYRO MAGNETIC RATIO: 13 C nucleus gyro magnetic ratio is much lesser than proton nucleus. C-1.404; H-5.585. This shows that CMR is more sensitive than PMR which is overcome by using FT-NMR technique 13
  • 14. Coupling phenomenon: 13 C & 1H have I=1/2 so that coupling between them probability occur. 13 C-13C coupling no! 13 1 C- H coupling YES! Not probable Very commo Extremely complex spectra and overlap of multiplets difficult to interprete. 14
  • 15. Problems of 13C-NMR can overcome by FOURIER TRANSFORM TECHNIQUE. DECOUPLING TECHNIQUE. NUCLEAR OVERHAUSER PHENOMENON. 15
  • 16. FTnmr • 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 interferogrm in the time domin The Fourier transform converts these same into frequency domin as spectrum.
  • 17. FREE INDUCTION DECAY ( relaxation ) 1 O CH2 C CH3 2 3 1, 2, 3 have different half lifes
  • 18. COMPOSITE FID “time domain“ spectrum 1 time + 2 + 3 + ......
  • 19. FOURIER TRANSFORM A mathematical technique that resolves a complex FID signal into the individual frequencies that add ( Details not given here. ) together to make it. TIME DOMAIN converted to FID FREQUENCY DOMAIN NMR SPECTRUM DOMAINS ARE MATHEMATICAL TERMS FT-NMR computer COMPLEX SIGNAL Fourier Transform a mixture of frequencies decaying (with time) 1 + 2 + 3 + ...... individual frequencies converted to a spectrum
  • 20. 20 December 2013 M.M.C.P. 21
  • 21. FT-NMR FID 22
  • 22. • 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).
  • 23. Nuclear overhausner enhancement
  • 24. 13C-NMR and the Nuclear Overhauser Effect NOE is an interaction between a target nucleus and its radiatively-saturated neighbors. Spin is transferred from the target nucleus to its saturated neighbors, increasing the population of low-spin target nuclei. This increases the signal of the target nucleus. As a result, during a standard Hdecoupled experiment, 13C atoms attached to
  • 25. PROTON OR NOISE DECOUPLING COHERENT OR BROAD BAND DECOUPLING OFF-RESONANCE DECOUPLING 26
  • 26. DECOUPLED SPECTRA
  • 27. DECOUPLING THE PROTON SPINS PROTON-DECOUPLED SPECTRA A common method used in determining a carbon-13 NMR spectrum is to irradiate all of the hydrogen nuclei in the molecule at the same time the carbon resonances are being measured. This requires a second radiofrequency (RF) source (the decoupler) tuned to the frequency of the hydrogen nuclei, while the primary RF source is tuned to the 13C frequency. RF source 2 “the decoupler” 1H-13C continuously saturates hydrogens 13C RF source 1 pulse tuned to carbon-13 signal (FID) measured
  • 28. ETHYL PHENYLACETATE in some cases the peaks of the multiplets will overlap 13C coupled to the hydrogens 13C decoupled from the hydrogens this is an easier spectrum to interpret
  • 29. SOME INSTRUMENTS SHOW THE MULTIPLICITIES OF THE PEAKS ON THE DECOUPLED SPECTRA s = singlet d = doublet CODE : d t = triplet q = quartet d q s s t t d This method gives the best of both worlds.
  • 30. Broadband decoupling  Also called Noise decoupling or 1H decoupling  Double irradiation at the resonance frequency is carried out  Hetronuclear decoupling in which spin-spin splitting of 13C lines by 1H nuclei is avoided  Broadband Radiofrequency signal encompassing entire proton spectral region is irradiated
  • 31. Off- resonnance decoupling  Decoupling frequency is set at 1000-2000 Hz      above the proton spectral region Partially decoupled spectrum are obtained Primary carbon nuclei- quartet Seconadry carbon nuclei- triplet Tertiary carbon nuclei- doublet Qurternary carbon nuclei- single line
  • 32. CHEMICAL SHIFT “Chemical shift is the difference between the absorption position of the sample proton and the absorption position of reference standard” Variations of the positions of NMR absorptions due to the electronic shielding and deshielding. 33 M.M.C.P. 20 December 2013
  • 33. Chemical Shifts…. • Measured in parts per million (ppm). • It is the ratio of shift downfield from TMS (Hz) to total spectrometer frequency (MHz). • The chemical shift is independent of the operating frequency of the spectrometer. • Same value for 60, 100, or 300 MHz machine. • Common scale used is the delta (δ) scale. 34 M.M.C.P. 20 December 2013
  • 34. Measurement of Chemical shift. chemical shift is measured in frequency unit „Hertz‟. Most of routine instruments operate at 60, 90, 100 MHz. More sophisticated instruments operate as high as 600MHz. The chemical shift recorded in Hz may vary with the spectrometer. To avoid this complication the chemical shift values are expressed in terms of delta or tau scale. Which are independent of field strength. Chemical shift in delta scale are expressed in parts per million (ppm).
  • 35. 13C NMRcc cchekjjjhhffdmical shift ranges in chemical shifts
  • 36. Interpreting 13C NMR • The number of different signals indicates the number of different kinds of carbon. • The location (chemical shift) indicates the type of functional group. • The peak area indicates the numbers of carbons (if integrated). • The splitting pattern of off-resonance decoupled spectrum indicates the number of protons attached to the carbon. 39
  • 37. 1-PROPANOL HO-CH2-CH2-CH3 c b a PROTON DECOUPLED 200 150 100 50 Proton-decoupled 13C spectrum of 1-propanol (22.5 MHz) 0
  • 38. Applications of c-13 nmr  Cmr is noninvasive and nondestructive method.i.e ,     especiallly used in repetitive in vivo analysis of the sample without harming the tissues. Cmr of biological materials allows for the assesssment of the metabolism of carbon, which is so elementary to life on earth. 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 Labelling is more conveinent means of following the metabolism specific carbons throughout the metabolism Labelling of C-13 nucleus at multiple carbon sites in the same molecule was possible, as result homonuclear 13C-13C coupling provides novel biochemical information
  • 39. REFERENCES  William Kemp: Organic spectroscopy, 3rd edition.  Robert M. Silverstein: Spectroscopic identification of Organic compounds, 6th edition.  Pavia: Introduction to spectroscopy, 3rd edition.  Y.R. Sharma: Elementary organic spectroscopy, principle and chemical applications.  Instrumental Methods of Chemical Analysis , Gurdeep R,Chatwal , Sham K. Anand 43
  • 40. THANK YOU 44

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