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核磁共振光譜與影像導論 Introduction to NMR Spectroscopy and Imaging Lecture 02 Chemical Shift and JCoupling (Spring Term, 2011) Department of Chemistry National Sun Yat-sen University Chemical Shift and J-Coupling In the beginning…. All spins were of no difference…same, identical, equal, I/You/He/We/You/They were all the same…or believed to be so….then...the apple of discern came in… (Proctor says: "until it is clearly understood, the accuracy of magnetic moments determined under certain chemical conditions remains somewhat in doubt"). Proctor 1922-2006 Valuable reading: http://www.ebyte.it/library/hist/ProctorWG_Reminiscences.html ? Dickinson? Who can find his photo? Yu 1913-2003 W.G.Proctor, F.C.Yu(虞福春), Phys Rev 1950,77,717. W.C.Dickinson, Phys Rev 1950 77, 736. Norman Ramsey (Phys.Rev. 1950, 78,699): "Furthermore, with heavier nuclei the ratios of the resonance frequencies for the same nucleus in different molecules have been measured with high precision and discrepancies have been found by various observers that are sometimes called chemical shifts". Ramsey Chemical Shift (Shielding) B0 Induced shielding field B loc B0 B induced Binduced B0 chemical shift tensor Chemical Shift: a molecule becomes a dipole B0 m loc 0 B The induced dipole moment shifts the resonance frequency of the nuclear spin. induced Binduced B0 chemical shift tensor Shielding Depends on Chemical Environment Different environments cause different shieldings B (r ) B0 Bs B0 (1 (r )) B B0 B0 0 0 (Representing different local chemical environments, Proctor and Yu, 1951) 0 0 Hence chemical shift (of resonance frequency relative to Zeeman frequency ω0.) Anti-shielding Is Possible B(r) B0 Bs B0 (1 (r)) Anybody cares to find some interesting literature? Some proton chemical shifts The more localized AO/MO, the more shielding Stronger/more bonds mean smaller CS. Less shielded.(downfield) You can tell a lot from this diagram Increasing δ (these trends for σ,δ,B, |ν| are same for γ>0 or γ<0 nuclei. However, for γ<0, ν is negative.) Larger shift/small shielding Small shift/large shielding Downfield (high freq) relative ( ref ) 0 [( 0 ) (ref 0 )] 0 ( ref absolute ) =( ref absolute ) 106 ppm Reference shift Upfield (low freq) These words were from CW NMR. ‘Downfield’ means for a given resonance frequency, the magnetic field used is lower. ‘High frequency’ means at a fixed magnetic field, the spins in this region have higher resonance frequencies. The OH bonding in vaporized water clearly differs from that in liquid water! (hydrogen bonding has significant effect on chemical shift) 1H NMR spectrum of octamer 2 in DMSO–d6. (Nadja Franz a, Laure Menin b and Harm-Anton KlokOrg. Biomol. Chem., 2009, 7, 5207-5218) Why CHn have smaller CS than H atom? B0 C H H atom Some people said: An H in CHn seems to be less shielded because the C has larger electronegativity so it ‘draws’ electrons to its side. But why an H in CHn has smaller CS than H atom? Answer: The electron density at the C-H bonding area is larger than that of an H atom albeit the electron density at other places is smaller. Overall, the H in CHn is more shielded than in H atom. This can also explain why H > H2, H>OH>H2 More bonds, more shielded. Why this CS order: CH>CH2>CH3? More bonds, more shielded. The bonding regions correspond to large shielding (small CS). The CS is smallest when the magnetic field is along the bonding direction. Why this CS order: HF>HCl>HBr>HI? The fewer number of electrons of the bonding partner, the less the shielding the bonded H. (The more clothes you dress, the more you’re shielded.) The shielding of s orbitals is smaller than that of p orbitals which is even smaller than that of d orbitals etc. (The more localized the orbitals, the more shielding) Isotopic Effect • Because CS is generated by electrons, nuclei of isotopic elements (e.g. H1/D2, N14/N15, Cl35/Cl37) have very similar chemical shift but isotope shift does exist: e.g., 1H CS of HOD is 0.035 ppm upfield (more shielded) of that of HOH (the electrons in HOD is a little ‘heavier’ than in HOH lower vib freq/amp more shielding (you are more shielded by your clothes if you shake yourself less violently.) • There is a general rule which says that when one substitutes a nuclide in a chemical group with a heavier isotope then all other nuclides in the group become a bit more shielded (this has to do with an overall reduction in vibrational amplitudes). • Consequently, the chemical shift of protons in standard bulk water should be 4.795 + 0.035 = 4.830 ppm, give or take 0.02 ppm. Of course, heavy water and normal water do not even have the same bulk properties (such as density and magnetic susceptibility) and this introduces a further uncertainty when trying to deduce the chemical shift of normal water from the data on HDO traces in D2O. You may be able to memorize this table or you may explain it based on your good scientific intuition Most internal NMR referencing standards are pH and temperature sensitive. Proton and Carbon Standards for Organic Solents Chemical Shift Chemical Formula Chemical Structure Boiling Point H1 C13 Tetramethylsilane (TMS) C4H12Si 27 0.000 0.000 Dioxane C4H8O -- 3.75 -- 2 Proton Standards for Aqueous Solents 3-(Trimethylsilyl)- Propionic acid-D4, sodium salt (TSP) C6H9D4NaO2SI 302 0.000 0.000 2,2-Dimethyl-2-silapentane5-sulfonate sodium salt (DSS) C6H15NaO3SSi 120 0.000 (labelled as DSS) -- More info http://www.bmrb.wisc.edu/home/iupac.pdf. P31 Standards B. P. (oC) Chemical Shift H3PO4 -- 0.00 (CH3O)3PO -- 0.00 Chemical Name Chemical Formula 85% Phosphoric Acid (external) 10% trimethylphosphate (internal) Chemical Structure OTHER NMR RESOURCES N15 Standards Chemical Name Chemical Formula liquid NH3> (external) NH3 Chemical Structure B. P. (oC) Chemical Shift -- 0.00 NMR Internal Referencing Standard Samples Chemical Name (oC) Solvent 1H Chemical Shift (multiplicity) JHD (Hz) HOD in solvent (approx.) 13C Chemical Shift (multiplicity) JCD (Hz) B.P. (oC) M.P. (oC) 11.65 2.04 1 5 -2.2 11.5 178.99 20.0 1 7 -20 118 17 Acetone-d6 2.05 5 2.2 2.8 206.68 29.92 13 7 0.9 19.4 57 -94 Acetonitrile-d3 1.94 5 2.5 2.1 118.69 1.39 1 7 -21 82 -45 Benzene-d6 7.16 1 -- 0.4 128.39 3 24.3 80 5 Chloroform-d 7.27 1 -- 1.5 77.23 3 32.0 62 -64 Cyclohexane-d12 1.38 1 -- -- 26.43 5 19 81 6 Deuterium Oxide 4.80 (DSS) 1 -- 4.8 -- -- -- 101.4 3.8 N,N-Dimethylformamide 8.03 2.92 2.75 1 5 5 -1.9 1.9 3.5 163.15 34.89 29.76 3 7 7 29.4 21.0 21.1 153 -61 Dimethyl Sulfoxide-d6 2.50 5 1.9 3.3 39.51 7 21.0 189 18 p-Dioxane-d6 3.53 m -- 2.4 66.66 5 21.9 101 12 Ethanol-d6 5.29 3.56 1.11 1 1 m -- 5.3 -56.96 17.31 -5 7 -22 19 79 <-130 Methanol-d4 4.87 3.31 1 5 -1.7 4.9 -49.15 -5 -21.4 65 -98 Methylene Chloride-d2 5.32 3 1.1 1.5 54.00 5 24.2 40 -95 Pyridine-d5 8.74 7.58 7.22 1 1 1 -- 5.0 150.35 135.91 123.87 3 3 5 27.5 24.5 25 116 -42 Tetrahydrofurand8 3.58 1.73 1 1 -- 2.4 - 2.5 67.57 25.37 5 5 22.2 20.2 66 -109 Toluene-d8 -7.09 7.00 6.98 2.09 -m 1 m 5 ----2.3 0.4 137.86 129.24 128.33 125.49 20.4 1 3 3 3 7 -23 24 24 19 111 -95 Trifluoroacetic Acid-d 11.50 1 -- 11.5 164.2 116.6 4 4 72 -15 Trifluoroethanold3 5.02 3.88 1 4x3 -2 (9) 5 126.3 61.5 4 4x5 75 -44 1 and C 13 Chemical Shifts of NMR Solvents -22 H Acetic Acid-d4 NOTES: o1H chemical shifts are in PPM, relative to 0.05% TMS (v/v), at 295 K. o13C chemical shifts are in PPM, relative to 1.0% TMS (v/v), at 295 K. o'm' denotes broad peak with some fine structures (at 200 MHz). oHOD peak positions may vary depending upon concentration in solvent, pH and temperature. oM.P. and B.P. values are for the corresponding non-deuterated solvent (except for D2O). o(DSS) denotes chemical shifts relative to 2,2-Dimethyl-2-silapentane- 5-sulfonate sodium salt. o See NMR Referencing for more information CHARACTERISTIC PROTON CHEMICAL SHIFTS Type of Proton Structure Chemical Shift, ppm Cyclopropane C3H6 0.2 Primary R-CH3 0.9 Secondary R2-CH2 1.3 Tertiary R3-C-H 1.5 Vinylic C=C-H 4.6-5.9 Acetylenic triple bond,CC-H 2-3 Aromatic Ar-H 6-8.5 Benzylic Ar-C-H 2.2-3 Allylic C=C-CH3 1.7 Fluorides H-C-F 4-4.5 Chlorides H-C-Cl 3-4 Bromides H-C-Br 2.5-4 Iodides H-C-I 2-4 Alcohols H-C-OH 3.4-4 Ethers H-C-OR 3.3-4 Esters RCOO-C-H 3.7-4.1 Esters H-C-COOR 2-2.2 Acids H-C-COOH 2-2.6 Carbonyl Compounds H-C-C=O 2-2.7 Aldehydic R-(H-)C=O 9-10 Hydroxylic R-C-OH 1-5.5 Phenolic Ar-OH 4-12 Enolic C=C-OH 15-17 Carboxylic RCOOH 10.5-12 Amino RNH2 1-5 Carbon-13 Chemical Shifts Carbon-13* Environment Chemical Shift Range (ppm) (CH3)2C*O -12 CS2 0 CH3C*OOH 16 C6H6 65 CHCl=CHCl (cis) 71 CH3C*N 73 CCl4 97 dioxane 126 C*H3CN 196 CHI3 332 You may be able to memorize this table or you may explain it based on your good scientific intuition Be aware of “abnormal” chemical shifts …... 1H 1H 13C Temperature dependence of the 1H NMR spectrum of Ni Ni dissolved in toluene-d8. The temperature runs from 183 (lowest trace) to 385 K. S = solvent. Harald Hilbig and Frank H. Koehler, New J Chem, 2001. • • For most organic compounds, the 1H chemical shift is in the range of 12 ppm, but the chemical shift range for hydrides (organometallic compounds) is approximately +25 to 60 ppm, the largest range could possibly reach 200 ppm!. The downfield shifts are most common in d0, d10 and early transition metal cases whereas those with other dn counts and late transition metals tend to be upfield of zero. Similar phenomenon occurs for other nuclei such as 13C, 31P etc. Phosphorous-31 Chemical Shifts Phosphorous-31 Environment Chemical Shift Range (ppm) PBr3 -228 (C2H5O)3 P -137 PF3 -97 85% phosphoric acid 0 PCl5 80 PH3 238 P4 450 Compoun d Chemical Shift (ppm) Relative to 85% H3PO4 PMe3 -62 PEt3 -20 PPr(n)3 -33 PPr(i)3 +19.4 PBu(n)3 -32.5 PBu(i)3 -45.3 PBu(s)3 +7.9 PBu(t)3 +63 PMeF2 245 PMeH2 -163.5 PMeCl2 +192 PMeBr2 +184 PMe2F +186 PMe2H -99 PMe2Cl -96.5 PMe2Br -90.5 Phosphorous (III) Chemical Shift Table (from Bruker Almanac 1991) Compound Chemical Shift (ppm) Relative to 85% H3PO4 Me3PO +36.2 Et3PO +48.3 [Me4P]+1 +24.4 [PO4]-3 +6.0 PF5 -80.3 PCl5 -80 MePF4 -29.9 Me3PF2 -158 Me3PS +59.1 Et3PS +54.5 [Et4p]+1 +40.1 [PS4]-3 +87 [PF6]-1 -145 [PCl4]+1 +86 [PCl6]-1 -295 Me2PF3 +8.0 Phosphorous (V) Chemical Shift Table (from Bruker Almanac 1991) Fluorine-19 Chemical Shifts Fluorine-19 Environment Chemical Shift Range (ppm) UF6 -540 FNO -269 F2 -210 bare nucleus 0 C(CF3)4 284 CF3(COOH) 297 fluorobenzene 333 F- 338 BF3 345 HF 415 Nitrogen-14 Chemical Shifts Nitrogen-14* Environment Chemical Shift Range (ppm) NO2Na -355 NO3- (aqueous) -115 N2 (liquid) -101 pyridine -93 bare nucleus 0 CH3CN 25 CH3CONH2 (aqueous) 152 NH4+ (aqueous) 245 NH3 (liquid) 266 B-11 Chemical Shift Almost all quadrupolar nuclei have rather small CS range. Factors Affecting Chemical Shift • Temperature • Solvents (pH, concentration) • Pressure • Sample shape (susceptibility) • …… NMR can be used as a thermometer, a pH meter or a barometer. (Only very smart guys would like to buy an NMR spectrometer for those purposes though) Solvent H2O D2O DMSO acetone CD Cl3 C6D6 * Relative to TMS. Shift *(H2O) 4.83 4.79 3.3 2.5 1.4 0.3 Amide proton chemical shifts of NHA in CDCl2CDCl2 as a function of temperature and concentration. Derr et al. J. Chem. Soc., Perkin Trans. 1, 2000. Chemical Shift The surrounding electrons cause a shielding magnetic field at the nucleus B B0 Bs B0 (1 ) Shielding Anisotropy (CSA) Electron clouds are seldom spherically symmetrical. They are anisotropic in almost all molecules. B0 B0 B B 0 (1 ) Chemical shift anisotropy (CSA) tensor In liquids, CSA is averaged out by rapid molecular tumbling; in solids, CSA is kept. Oriented Molecules B0 Oriented Single Crystals B0 Powder (Polycrystalline Solid) B0 Chemical Shift Tensor E B0 (r ) Applications of Chemical Shift Applications of Chemical Shift Applications of Chemical Shift Applications of Chemical Shift Relaxation, dynamics Solid state NMR CS Imaging …… Story Goes On Indirect Dipolar Interaction (J-Coupling) Interaction between spins mediated by electrons around them. J-coupling is usually much smaller than direct dipolar coupling. J-Coupling NMR/I Homonuclear system A Heteronuclear System AX System X X J AX J AX A X General Cases of Two-Site Homonuclear Systems 1=“up” 0=“down” Spin A: 1 : Cn1 : Cn2 : Cn3 Cnn 1 : 1 Spin B: m 1 m 1 : C : C : : C 1 m 2 m 00000…000 000…00 10000…000 100…00 01000…000 010…00 00100…000 001…00 … … 11000…000 110…00 01100…000 011…00 00110…000 0011..00 … … :1 11111…101 111…01 11111…110 111…10 11111…111 111…11 Spin A Spin B Exercise: Who are They? ABC System ABCD system 200 MHz 1H-NMR spectrum of dibromo benzonorbornene derivative in CDCl3 and expansions of the signals. Equivalent Spins Coupled with Quadrupolar Spins Strong Coupling and Quantum Mechanical Treatment Example E is broad becaue of exchange. Ha Hb Hc Ha(Hoye) Analysis Analysis Hc Hd Hd Result Result Karplus Equation Φ Karplus Equation showing the relationship between the observed coupling constant and the φ(=θ-135o) angle. Note that unique solutions are obtained only for J > 8 Hz and J <5 Hz . Karplus Equations Karplus Equations 3J 2 0 H-C-C-H = 10 cos q for 0 £<q <90 , and 3J 2 0 H-C-C-H = 12 cos q for 90 £<q £< 180 Typical J-coupling constants • • • • • • • • • • • • • • 3JCOCH Mulloy et al. Carbohydr. Res. 184 (1988) 39-46 Tvaroska et al. Carbohydr. Res. 189 (1989) 359-362 Anderson et al. J. Chem. Soc., Perkin 2 (1994) 1965-1967 3JCOCC B. Bose et al. J. Am. Chem. Soc. 120 (1998) 11158-11173 Q. Xu and A. Bush Carbohydr. Res. 306 (1998) 335-339 M.J. Milton et al. Glycobiology 8 (1998) 147-153 3JCCCH R. Aydin & H. Günther Mag. Reson. Chem. 28 (1990) 448457 A. de Marco et al. Biochemistry 18 (1979) 38473JPOCH Lankhorst et al. J. Biomol. Struct. Dyn. 1 (1984) 1387-1405 3JCCOP Lankhorst et al. J. Biomol. Struct. Dyn. 1 (1984) 1387-1405 3JHNCH S. Ludvigsen et al. J. Mol. Biol. 217 (1991) 731- A. Pardi et al. J. Mol. Biol. 180 (1985) 741V.F. Bystrov,Prog. NMR Spectrosc. 10 (1976) 413JCNCH L.-F. Kao et al. J. Am. Chem. Soc. 107 (1985) 23233JCNCC L.-F. Kao et al. J. Am. Chem. Soc. 107 (1985) 23233JHCOH R.R. Fraser et al. Can. J. Chem. 47 (1969) 403-409 Applying the Karplus Equation Applying the Karplus Equation Long Range Coupling The doublet splitting arises from the coupling with the geminal proton Ha. The fact that the Hb, proton does not couple with the bridgehead protons Hc is attributed to the dihedral angle, which is nearly 90°. At the same time, proton Ha couples with the geminal proton Hb and bridgehead protons Hc. Furthermore, proton Ha has long-range coupling to the Hj protons. This can be clearly seen by the further triplet splitting of the signals. This long-range coupling arises from the zigzag orientation of protons Ha and Hd. The zigzag orientation of protons Hb and Hd is impossible because of the rigid geometry. Consequently, there is no long-range coupling between these protons. The fact that proton Ha has long-range coupling to Hd protons clearly indicates the exo configuration of the bromine atoms. In the case of the endo configuration we should not observe any long-range coupling. Amino Acids Amino Acid, Name, Abbr. R= Alanine, ala,A CH3- Arginine, arg,R H2N-C(=NH2+)-, NH-(CH2)3- Asparagines,asn,N H2NC(O)CH2- Aspartic acid, asp,D HOOC-CH2- Cysteine, cys,C HS-CH2- Glutamic acid, glu,E HOOC-(CH2)2- Glutamine, gln,Q H2NC(O)CH2-, CH2- Glycine, gly,G H- Histidine, his,H Isoleucine, ile,I CH3CH2- Leucine, leu,L (CH3)2CHCH2- Lysine, lys,K +H3N(CH2)4- Methionine, met,M CH3SCH2CH2- Phenylalanine,phe,F Ph-CH2- CH(CH3)- Praline, pro,P Serine, ser,S HOCH2- Threonine,thr,T CH3CH(OH)- Tryptophan,trp,W Tyrosine,tyr,Y HO-Ph-CH2- Valine,val,V (CH3)2CH- Summary of one-bond heteronuclear couplings along the polypeptide chain utilized in 3D and 4D NMR experiments Structure of an A-U (A) and a C-G (B) Watson-Crick base pair. Notice that in each case, there is a single N-H ... N hydrogen bond. Scalar coupling across this bond was determined to be approximately 6.3 Hz for the GC bp and 6.7 Hz for the AU bp. Non-Watson Crick bp schemes (such as Hoogsteen) contain different hydrogen bonds that can be distinguished from traditional Watson-Crick. (CH3)2CH (CH3)2CH Coupled Decoupled Varian parameters: dn, dm, dmm, dpwr C-H Coupling and 13C Broadband Decoupling 13C-1H Coupling and 13C Broadband Decoupling Selective Decoupling of 1H-1H Selective Decoupling of 1H-1H