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1、40-042 Analytische Chemie IVStructure Determination byNMRProf. Bernhard JaunOffice: ETH Hnggerberg HCI E317Phone: 01 632 31 44e-mail: jaunorg.chem.ethz.chhome page: http:/www.jaun.chem.ethz.chProf. B. Jaun: Structure determination by NMR / Analytische Chemie IV2Contents1.Practical aspects of pulse F

2、ourier transform NMR spectroscopy1.1The basic NMR experiment: physical description1.2Excitation by RF pulses1.3Digitization, window functions and fourier transform1.4Quadrature detection1.5Phase cycles and Z-gradients1.6Dynamic range and solvent suppression1.7The principal components of an NMR spect

3、rometer2.The principle of 2D-NMR2.1The basic idea2.2A meaningless experiment2.3Quadrature detection in 13.Homonuclear shift correlation through scalar couplings3.1COSY.3.2Determination of coupling constants from COSY cross peaks.3.3TOCSY3.4INADEQUATE: homonuclear 13C-13C double quantum spectroscopy.

4、4.Spectral editing4.1Spin echo building blocks4.2Heteronuclear J-modulation4.3Polarisation transfer: INEPT and DEPT5.Heteronuclear shift correlation through one bond and long range couplings5.1Proton detected methods5.2Proton detected H,X-COSY5.3HSQC and HMQC5.4HMBCProf. B. Jaun: Structure determina

5、tion by NMR / Analytische Chemie IV36.Relaxation, dipole-dipole couplings and NOE6.1Longitudinal and transversal relaxation6.2NOE in a two spin system without scalar coupling6.3The mechanism of dipolar relaxation7.Homonuclear 1D-NOE difference spectroscopy7.1The steady state NOE in homonuclear multi

6、-spin systems7.2Practice of 1D-NOE difference spectroscopy8.Kinetic NOE-spectroscopy8.1NOESY8.2NOE in the rotating frame: ROESY9.On the influence of dynamic processes on NOE spectra9.1Overall molecular tumbling, internal rotation (conformational changes) und chemical exchange9.2How to proceed with m

7、olecules with 0c near 110.Combined application of several methods10.1 Typical structure problems with organic molecules and suitable strategies11.References and additional reading11.1 Textbooks11.2 Homonuclear correlation through scalar coupling11.3 Heteronucleare correlation through scalar coupling

8、11.4 NOE11.5 Coupling constants und Karplus relationships11.6 Abbreviations and Acronyms11.7 On the presentation of 2D-NMR data in the experimental part of a thesis or publication.Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV4What we would like to achieve with this course:-Th

9、e student knows which method yields which type of structural information.-The student is able to choose the most promising combination of methods for the solution of a given structural problem. Since spectrometer capacity is expensive, the selection of an optimal strategy depends on technical (feasi

10、bility) as well as on economical (fast and simple) factors.-The student knows how to interpret each type of spectrum and how to extract the pertinent data.-The student is aware of the possible artifacts and errors of interpretation for each method.-The student knows the NMR vocabulary and is able to

11、 correctly describe NMR data in publications.Subjects that are not part of this course:-Comprehensive treatment of the underlying theory (quantum mechanics of NMR)(- courses by Prof. Schweiger and Meier).- Solid state NMR-Practical nuts and bolts of carrying out the measurements on the spectrometer.

12、(- Praktikum Analytische Chemie)-Application to large biopolymers such as proteins and nucleic acids (- course by Prof. Wthrich) and calculation methods (simulated annealing, distance geometry etc.) usedto derive solution structures of biopolymers from NMR data. Shorter oligopeptides, oligonucleotid

13、es und oligosaccharides, however, will be discussed as examples or problems.Outline of the course:-Lectures on the individual methods with discussion of example spectra.-Problems (real life spectra) for each of the discussed methods.-Problems on structure elucidation of medium to complex organic mol

14、ecules with combined methods (in the last ca. 1/3) of the course.Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV1.11.Practical aspects of pulse Fourier transform NMR spectroscopy1.1 The basic NMR experiment: physical descriptionSpinComponent of the angular momentum of nuclei, e

15、lectrons (and other elementary particles) that cannotbe described as orbital momentum. Its origin is only understandable in terms of relativistic quantum mechanics.The magnetic momentThe magnetic moment () associated with the orbital angular momentum (L) of a charged particle is given by: L = r x pL

16、pe-r = LThe spin angular momentum Jis associated with a magnetic moment as well:J= , the gyromagnetic ratio is a fundamental property of each nuclear isotope with non-zero spinInteraction between the magnetic moment und an external magnetic fieldClassical physics:zB0The torque T acts on . In respons

17、e, precesses around the direction of B 0 (analogousto a spinning top under the force of gravity) with the circular frequency 0 rad/s, which is called the Larmor frequency. T = x B00Ty0 = B 0x Epot = - . B0Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV1.2Quantum mechanics: Quan

18、tum mechanical description of the spin angular momentum J : J = h II : nuclear spin operatorI : spin quantum number of the nucleus, a property of each isotope (I = 1/2.n; n=0,1,2).a)The z-component (parallel to the external field) of the spin angular momentum can only assume certain values governed

19、by the magnetic quantum number mI :J = h mm = - I, - I + 1,.,0, I - 1, Im magnetic quantum numberzIIIThis leads to 2I+1 allowed states. For nuclei with I = 1/2, which are of predominant interest in organicchemistry, only the two states with mI = -1/2 and mI = +1/2 are possible.The interaction energy

20、 for each state with a static external magnetic field along the z-axis isE = - z . B0 = - J z B0E = - h mI B0The energy difference between the two states is:E = - (1/2 - (- 1/2) B0= - B0In order to achieve resonance, the energy of the irradiated radio frequency has to match the energydifference betw

21、een the two states:E = h = = - B0The Larmor (circular) frequency is: i = - (1-i)B0zWith I = resonance frequency (rad/s) of spin i with shielding constant iThe resonance frequency for a given isotope is proportional to the gyromagnetic ratio and to the external magnetic field.a) The spin quantum numb

22、ers of nuclei follow the rules:A = Z + NA = mass numberZ = number of protons(nuclear charge)N = number of neutronsA even - I = integerZ even, N even - I=0Z odd, N odd - I=1,2,3. A odd - I= 1/2, 3/2, 5/2 .Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV1.3Macroscopic magnetizatio

23、n MExperimentally, only the total magnetization M of the sample inside the RF coil can be detected.Mcorresponds to the vector sum of the magnetic moments of all spins.M = iisum over total sample volume inside the coilFor I = 1/2, 0 (e.g. 1H):B0B0mI = + 1/2zzyMyxxmI = - 1/2Because the magnetic moment

24、s are distributed statistically in the xy plane, there is no net transversemagnetization. In the Boltzmann equilibrium and for 0, the population of the (mI = +1/2) state isslightly larger than that of the (mI = 1/2) state. This leads to a small residual magnetization in thedirection of the external

25、field B 0 .The energy difference between the two states (: mI = +1/2; : mI = 1/2) is very small.For 1H at 14.1 Tesla (600 MHz) the ratio of the two populations is only: N+1/2 / N-1/2 = 1.0002.Because 0 is proportional to and B 0 , nuclei with high are more sensitive than those with low,and higher ma

26、gnetic fields increase the sensitivity dramatically. In practice, the sensitivity of NMRspectrometers increases approximately according to B3/2.1.2 Excitation by radio frequency pulsesRotating coordinate frame: The Larmor frequencies in modern NMR spectrometers are in the orderof 30 - 900 MHz. On th

27、e other hand, the differences between the individual spins of the observed nucleus (chemical shifts and scalar couplings) are typically in the 1 Hz to 20 kHz range. In order to make the description of the dynamics of the magnetization both mathematically and visually easier, itis usual to use a coor

28、dinate frame which rotates around the B0 = z-axis with the circular frequency 0.Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV1.4The resulting stroboscope effect allows to describe the precession in terms of frequency differences = - 0. In the following, we will use the rotati

29、ng frame for all vector diagrams.In modern NMR spectrometers with superconducting magnet coils, the magnetic field is parallel to the axis of the sample tube. The radiofrequency coil, which transmits the excitation pulses and the induced signal to and from the sample to transmitter and detector, res

30、pectively, is a saddle coil that generates and detects RF fields having their magnetic component B1(t) orthogonal to the constant external field B0. The relative orientation of B1 vectors in the xy plane can be controlled by changing the relative phase of the irradiating RF.Irradiation of radiofrequ

31、ency corresponding to the Larmor frequency of a given nucleus for a short time(an RF pulse of frequency = 0/2 and duration ) induces a complicated spiral movement of themacroscopic magnetization M away from the z-axis towards the xy plane. In the rotating coordinateframe this process, which is calle

32、d nutation, is a simple rotation of M around the axis of the field B 1.The nutation angle () is a function of both, the RF field strength B1 and of the pulse duration (it is proportional to the integral of the RF pulse): = B1 rad. In practical work, the amplitude of the RFfield is usually given as B

33、1/2 Hz. It can be calculated if the duration necessary for a nutation of1 =90 is known: B /2 = 1/(4. (90).Note: The spectrometer software uses parameters in the unit decibel (dB) attenuation from the maximal output in order to control the amplitude of RF pulses. Since these values are different for

34、each instrument/amplifier/probe head combination, one should always use the absolute RF amplitude B1/2 Hz in publications.zzMz0B1yyMy0xx2 -xProf. B. Jaun: Structure determination by NMR / Analytische Chemie IV1.5Dependence of the excitation band width on the duration of the pulseBecause the nutation

35、al angle is proportional to the integral of the RF pulse, the same nutation can be achieved either with a long weak pulse or with a short intense one. However, this holds only for spins, which resonate exactly at the frequency of the transmitter ( = 0). The bandwidth of excitation ( the frequency re

36、gion in which spins are more or less equally excited) is directly dependent on the intensityof the pulse (peak to peak voltage, B1 amplitude). The first zero crossing of the excitation function occurs at 0/2 B1/2 Hz. Short intense pulses (so called hard pulses) are unselective and excite abroad regi

37、on of the spectrum, long weak pulses (so called soft pulses) are selective and excite only a narrow region around the transmitter frequency. Continuous wave irradiation with very weak amplitude during 0.5-5s allows to irradiate a single line and is used for homodecoupling and presaturation of solven

38、t signals.Phase and amplitude of excitationB10Phase and amplitude of excitationB10Offset EffectsSpins resonating at frequencies different from the transmitter frequency experience an effective RFfield B1 eff that is the vector sum of B1 and of a component along B0:tan = 2(-o)/B1Nutation around B1 ef

39、f with (90) no longer follows a grand circle. For 90 pulses, the longer path and slightly stronger field B1eff compensate each other. Therefore, 90pulses are much less sensitive to offset effects than180 pulses. Pulse sequences are usually based on the assumption that offset effects are negligible.

40、In reality,offset effects lead to artifacts and loss of signal in pulse sequences such as DEPT and heteronuclear shiftProf. B. Jaun: Structure determination by NMR / Analytische Chemie IV1.6correlation, which depend on accurate 180 pulses. In order to minimize offset effects, high amplitude pulses f

41、orunselective excitation are standard in modern instruments. In practice, probe heads and amplifiers (typically1300W for X-nuclei) on a modern high resolution spectrometer can deliver 90 pulses as short as ca. 7 s (B /2= 35 kHz). Higher power would lead to arcing in the probe and could destroy the p

42、robe head or amplifier. Practical example: 11.7 T (125 MHz for 13 C / 500 MHz for 1H): chemical shift range 13 C: -10 to 240 ppm = 15.6 kHz. Offsetof a carbonyl signal: 13.5 kHz.- With B1/2 = 35 kHz and transmitter frequency at 110 ppm- = 21.Offset effectzzMBeffyxM0B effyB1tan = 2 ( - ) / B01xEvolut

43、ion of magnetization in the xy plane after excitation by an RF pulseIf equilibrium magnetization Mz is excited with an RF pulse, transverse magnetization (with components along x and y) is created. This corresponds to promotion of spins to the state and therefore to the generation of single quantum

44、coherence (excitation of a mI = 1 transition). After the pulse, the magnetization in the xy plane evolves due to the chemical shift and the scalar coupling between spins as shown below:Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV1.7Evolution of transverse magnetization under chemical shifts and scalar couplingsMx = My0sin(-Jt)M = Mxy0sin(t)