c13 nmr.pptx

advance structure eluicidation of natural products

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2. C13 NMR Spectroscopy Presenter : Farah Naz Advance structure elucidation techniques of Natural products

3. Contents; Introductions of NMR spectroscopy Principle and theory History C13 –NMR Spectroscopy NMR –active Nuclei Information from C13- NMR Chemical shift of C13-NMR Short coming of C13-NMR Spin- Spin splitting of C13-NMR Factor effecting C13-NMR Advantages and dis- advantages 3

4. NMR SPECTROSCOPY Nuclear magnetic resonance spectroscopy ,most commonly known as NMR spectroscopy or magnetic resonance spectroscopy ,is a spectroscopic technique to observe local magnetic fields around atomic nuclei . It results to given a spectrum with frequency on x- axis and intensity of absorption on y-axis. 4

5. Principle and theory The nuclear magnetic resonance occur when nuclei aligned with an applied field are induced to absorb energy and change their spin orientation with respect to the applied field. The energy absorption is quantized process, and energy absorbed must equal to energy difference between the two states involved . E absobed = (E-1/2state –E+1/2state)= hv The stronger the applied magnetic field , greater the energy difference between the possible spin states. 5

6. Principle of nmr when energy in the form of radiofrequency is applied When applied frequency is equal to processional frequency absorption of energy occures . Nucleus in resonance NMR signal is recorded 6

7. HISTORY of c13 nmr The first NMR signal for 1H was observed in 1945 but the first 13C NMR signal was detected in 1957 by Lauterbur . The first 13C NMR of some organic compound was recorded in early 1960. This discovery made structural determination very simple and interesting. This is more helpful to those molecules which have very few C-H bonds. This is because, this type of molecules are not identified completely by 1H NMR. Also, the complex molecules mainly biomolecules found this method of identification more efficient. The study of biosynthetic pathways of natural products and brain metabolism can easily be monitored with the help of 13C NMR spectroscopy . 7

8. C13 – NMR SPECTROSCOPY Carbon-13 nuclear magnetic resonance is the application of nuclear magnetic resonance spectroscopy to carbon. It is analogous to proton NMR and allows the identification of carbon atoms in an organic molecule just as proton NMR identifies hydrogen atoms. ¹³C NMR detects only the ¹³ C isotope.  NUCLEI AND RELATIVE ABUNDANCE OF CARBON ISOTOPES 8

9. NMR Active Nuclei: nuclear spin quantum number (I) atomic mass and atomic number Even mass nuclei that have even number of neutron have I = 0 (NMR inactive) Even mass nuclei that have odd number of neutrons have an integer spin quantum number (I = 1, 2, 3, ) Odd mass nuclei have half-integer spin quantum number (I = 1/2, 3/2, 5/2, ) I = 1/2: 1H, 13C, 19F, 31P , I= 1: 2H, 14N I = 3/2: 15N , I = 0: 12C, 16O 9 The most abundant isotope of carbon is C-12 (nuclear spin, I = 0) and this is NMR inactive . , the natural abundance of C-13 or 13C is 1.1% and has I = 1/2 like a proton. C-13 nuclei are NMR active.

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11. 11 The Spin Quantum Number ( ms ) describes the angular momentum of an electron. An electron spins around an axis and has both angular momentum and orbital angular momentum. Because angular momentum is a vector, the Spin Quantum Number (s) has both a magnitude (1/2) and direction (+ or -) The gyromagnetic ratio , often denoted by the symbol γ (gamma) is the ratio of the magnetic momentum in a particle to its angular momentum. 

12. 12 Information from c13nmr spectrum Analysis of an NMR spectrum may involve analyzing: a) The number of signals a molecule emits b) The frequencies at which signals occur c) The intensity of signals d) The splitting of signals

13. 13 Information from 13C NMR 1.Number of signals: Tells about different types of carbon atoms present (non-equivalent carbons ) 2. Position of Signals: Gives information about either carbons are aliphatic, alkene, aromatic, aldehydic , carbene or carbonyl etc . 3.Splitting of signals: In proton-coupled 13C NMR, splitting of signals gives information of number of hydrogen atoms attached to a particular carbon.

14. 14 Ethyl acetate

15. CHEMICAL SHIFT OF C13 NMR; The frequency at which a nucleus will resonate is dependent on the magnetic field strength . Because this can vary from instrument to instrument, frequency is expressed relative to magnetic field strength, “chemical shift” Chemical Shift = frequency of resonance (Hz) /frequency of instrument(MHz) units = parts per million = ppm The Chemical shift range for 1H is usually in δ 0 – 12 ppm. The Chemical shift range for 13C is quite large and usually in δ – 220 ppm. 15

16. 16 This results is that almost all non-equivalent 13C peaks appear at different Chemical shift values and rarely overlap.

17. Solvents for NMR spectroscopy NMR spectra are usually measured using solutions of the substance being investigated. A commonly used solvent is CDCl 3 . This is a trichloromethane (chloroform) molecule in which the hydrogen has been replaced by its isotope, deuterium. CDCl 3  is also commonly used as the solvent in proton-NMR because it doesn't have any ordinary hydrogen nuclei (protons) which would give a line in a proton-NMR spectrum. It does, of course, have a carbon atom - so why doesn't it give a potentially confusing line in a C-13 NMR spectrum? In fact it  does  give a line, but the line has an easily re- cognisable chemical shift and so can be removed from the final spectrum. 17

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19. Short comings of C13-NMR Spectra Only 1%of the carbon in the molecules is carbon -13. Sensitivity is consequently low. Recording the NMR – spectra is a tedious and time consuming process . Magnetogyric ratio of carbon (g=68)is less than that of proton (g=268 ). 13C nuclei resonance 6000 times weaker than 1H-nuclei. 13C-NMR resonance (25 MHz) frequency ¼less than1H resonance (100 MHz)frequency. The overall sensitivity c13 compound with 1H stands at 1/5700. 19 H 3C –CO- CH3 1.1 % 1.1 % ACETONE

20. 20 T he advent of recent developments in NMR-spectroscopy it is quite possible to eliminate some of these short comings adequately. They are : Development of powerful magnets (‘ super con ’ magnets) has ultimately resulted in relatively stronger NMR-signals from the same number of atoms Improved hardware in NMR-spectroscopy has gainfully accomplished higher sensitivity Development of more sensitive strategies has made it possible to record these C—H correlation spectra in a much easier manner. Eliminate short comings

21. Spin-Spin Splitting in 13C NMR; 1 -Homonuclear(13C-13C)Coupling; low natural abundance of 13C nuclei, the probability of finding two 13C atoms adjacent to each other in the same molecule is very low. Hence, no spin-spin splitting between 13C–13C nuclei.

22. 22 2- proton coupled Spectra: Spin-spin splitting occurs between hydrogen attached to the particular 13C atom. Such spectrum called proton coupled 13C NMR spectrum. The splitting of 13C signal occurs according to n+1 rule. The proton coupled 13C NMR spectrum are very difficult to interpret due to large coupling constant (J = 100-320 Hz). 1H–13C 1J = ~100-320 Hz; 1H–C–13C 2J = 0-60 Hz; 1H–C–C–13C 3J = 0-60 Hz

23. 3- proton decoupled spectra; When there is no coupling between 1H and 13C nuclei, then such 13C spectrum called as proton decoupled 13C NMR spectrum i.e. 13C{1H} NMR spectrum. This gives only singlets and thus simplifies the spectrum. However, this lost the information about attached hydrogens . Majority of 13C NMR spectra are obtained as proton decoupled spectra. 13C{1H} NMR spectrum of [Au(CH3)( carbene )] in CDCl3. The solvent signals are marked with ‘*’. 23

24. 24 Factor effecting c13- nmr

25. Factor effecting C-13NMR (a) α,b-and γ-Substituent Effects The α-substituents results from the replacement of a directly bonded H by an X group : C-H → C-X The b-substituents results from the replacement of a H on an adjacent atom by an X group : C-C-H → C-C-X The gamma -substituents results from the replacement of a H on an adjacent to adjacent atom by an X group : C-C-C-H → C-C-C-X The chemical shift increases on going from primary to secondary to tertiary to quaternary carbon atoms . The average increase per carbon atom lies in the range of 7-10 ppm. The substituents at alpha and beta -position increases the chemical shift value . In other words, the 13C absorption position is shifted downfield . The case with gamma substitution is opposite. In this case, the 13C absorption position is shifted up field i.e. the chemical shift value is decreased. 25

26. 26 The other important factor influencing the substituent effects is the electronegativity of the attached atom. The attachment of electronegative atom shifts the δ value towards higher side. For example, in the 13C NMR spectrum of ethanol the signal for CH3 is observed at higher δ value in comparison to the CH2. This is due to the attachment of CH2 group with electron withdrawing group –OH. (b) Electronegative effect ;

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28. 28 (c) Hybridization Effect; The hybrid state of carbon also affects the chemical shift of carbon. 1-sp3 hybridised carbons The sp3hybridised carbons are either un-substituted or substituted with electron withdrawing groups (EWG). Overall range of resonance observed in between 0 to 50 ppm for un-substituted and between 60-90 ppm for EWG substituted carbons. The influence of strong electronic and steric effect cannot be ruled out. For an extreme case like tetraiodomethane (CI4) where, carbon has been observed at 300 ppm lower than the TMS ( δ = -300 ).

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31. 31 2- sp2 hybridized carbons; The sp2hybridised carbons include alkene and aromatic carbons. These carbons give signals in overlapped regions. The range alkene and aromatic carbon signals are between 90 to 150 and 100 to 170 respectively. For example , >C=O, >C=N etc. The shift in ppm differs for these cases depending upon the electronic and steric factors. The >C=N present in the Schiffs bases are generally observed in the range 130-150 ppm. The 13C NMR signal ranges of important >C=O groups are: ester C=O ;170-175 ppm acid C=O ; 177-185 ppm aldehyde C=O ; 190-200 ppm ketone C=O ; 200-220 ppm 3-sp hybridized carbons The sp hybridized carbons include alkynes, nitriles and isonitriles . The signals for these groups are generally present in very narrow range. The unsubstituted alkynes have been observed in the range of 40-80 ppm.

32. 32 (d)Three-Membered Rings: The three-membered ring has significant effect on the chemical shifts in 13C NMR spectra. The compounds such as cyclopropanes , cyclopropenes , epoxides, aziridines and other 3-membered rings tend to show pronounced upfield shifts i.e. shift towards lower δ values . For example , The C-2 carbon of propane is observed at 16 ppm in 13C NMR spectra whereas the carbon in cyclopropane is observed at -3 ppm . Similar trend has also been observed for propene and dimethyl ether with respect to their cyclic counterpart carbon

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34. 34 (e) Conjugation to carbonyl group; The conjugation to carbonyl carbon also affects the chemical shift. The conjugation may be due to a double bond or aromatic ring. This causes upfield shifts i.e. towards lower δ value about 6-10 ppm for all types of carbonyl compounds. The effect is smaller for nitriles also. For example , The carbonyl carbon of 2-butanone appears at 206 ppm whereas, corresponding conjugated carbonyl carbon for but-3-ene-2-one appears at 197 ppm and acetophenone at 195 ppm

35. 35 2- butanone acetaphenone

36. 36 (f)Hydrogen Bonding Effects; The intra-molecular hydrogen bonding causes substantial downfield shifts in 13C NMR spectra i.e. towards higher δ value. It has been observed that most carbon signals are resistant to solvent effects but the carbonyl groups are an exception. The chemical shifts for carbonyl carbon move to downfield in protic solvents. This may be attributed to hydrogen bonding.

37. 37 ADVANTAGES OF C13 NMR

38. 38 C-13 NMR offers considerably better resolution, largely because the C-13 absorptions for most ordinary organic molecules are spread over 200 instead of 10 ppm . Carbons bearing no protons are directly visible. A count of the number of protons attached to each carbon results from comparison of the broad-band decoupled C-13 NMR spectrum with the off-resonance C-13 spectrum. Thus the number of methyl, methylene, methinyl , and quaternary carbons in a fairly complex molecule is far more readily determined by C-13 than by H-I NMR .

39. 39 Dis-advantages of c13 nmr

40. 40 l . Larger sample size (up to 100 mg) or longer sampling time (up to several days) is sometimes necessary; however, if 100 mg of a sample is available and that sample has high solubility, the time requirement may be only a few minutes; a good spectrum may even be obtained on as little as a I-mg sample when several days are available for scanning the sample . 2. Owing to variations in relaxation times and nuclear Overhauser effects (NOE), the areas of absorption for individual carbons vary considerably (up to a factor of about 10). Thus it is not as easy to tell relative numbers of carbons from C-13 as it is protons from H-l NMR; for this reason only the H-l spectra are integrated in the problems below. 3. Protons attached to heteroatoms are not directly visible.

41. Reference ; MCH 401;Application of Spectroscopy (Organic) UNIT- 4th: Carbon-13 NMR Spectroscopy Introduction to spectroscopy book Pharmaceutical drug analysis book CARBON-13 NMR SPECTRAL PROBLEMS Robert B. Bates and William A. Beavers Tems Eastman Company , 41

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