Chem343 (Fall 2009) NMR Presentation

Transcription

1 Chem343 (Fall 2009) NMR Presentation Y Ishii Oct 16, NMR Experiment Cautions Before you start, Read the handouts for background information. Read NMR procedure handouts for the procedures of the experiments. Don t be late The schedule of the experiment is tight. You have only 5 min to answer the pre-lab questions. As soon as you arrive at the lab, go to TA and answer the questions. You will meet your instructor at 5 min past the lab starting time at Pchem Lab. Meet your NMR instructor. He/She will take you to the NMR lab. If you are late more than 20 mins, we will start without you. If both of you are late more than 30 mins, the lab will be cancelled,, and you will automatically have Grade E for the NMR experiment. 2

2 What shall we do? Signal assignment for an unknown sample ( 1 H NMR, 13 C NMR, DEPT, 2D 13 C/ 1 H Correlation NMR, 2D 1 H/ 1 H Correlation NMR) Analysis of 13 C T 1 inversion recovery experiment for the same sample (we may give you the data for analysis, if time is not enough) 3 NMR Samples for Rot Jaglone (C 10 H 6 O 3 ) 2. Menthol (C 10 H 20 O) 3. Camphor (C 10 H 16 O) 4. 2-bromonapthalene 5. Ibuprofen 6. Methyl Nicotonate (C 10 H 7 Br) (C 13 H 18 O 2 ) (C 7 H 7 NO 2 ) 7. Ethyl sorbate 8. 2-heptanone 9. 3-heptanone (C 8 H 12 O 2 ) (C 7 H 14 O) (C 7 H 14 O) 9. 4-heptanone 10. Diacetone-D-glucose (C 7 H 14 O) (C 12 H 20 O 6 ) 4

3 NMR Experiments Score Sheet [ /100] Abstract [ /7] (1) Writing ( /3) (2) Contents ( /4) Introduction [ /22] (3) Principles of 1D FT NMR (Spin magnetic moment/angular momentum, FID, How NMR signals are generated?, chemical shift FT/FT NMR) ( /8) (4) Brief summary of 13C & 1H NMR, 13 C DEPT, 2D NMR (usefulness, how it works) ( /6) (5) Brief summary of inversion recovery (T1, relaxation, inversion recovery) ( /4) (6) Overall Writing ( /4) Spectra [ /26] (7) 1H and 13C spectra of your unknown sample with structures and 13C, 1H chemical shift assignments made ( /8) (8) 13 C DEPT spectra ( /5) (9) 2D 13 C/ 1 H HETCOR spectra & 2D 1 H/ 1 H COSY spectra with assignments ( /8) (10) T1 Inversion Recovery stackplot ( /5) Analysis [ /16] (7) Table of τ vs I(τ) ( /3) (8) Table of τ vs y(τ) ( /3) (9) Correct calculation for the above table (+ explanation) ( /3) (9) Plot for y(τ) vs Fitting for the above plots ( /3) (10) Plots of I(τ) vsτ τ with fitting curves (T1 value needed ) ( /4) Discussion [ /29] (11) Reasoning of 13 C NMR assignments with 1D 13 C NMR and DEPT (Intensity, chemical shift, which is protonaed?) ( /7) (12) Assignment based on 1D 1H NMR.(2D 13C/1H & 1H/1H correlation, 1H multiplet, intensity/integral, shift) ( /7) (13) What is the sample? Discuss reasoning for identification of the sample. ( /5) (14) Discuss how the 2D NMR enhances resolution and change the way of your assignments. ( /5) (15) Comment on the difference in 13C T1 values in terms of the dipolar relaxation mechanism. Does the relaxation behavior of the various 13C nuclei assist in making chemical shift assignments? Discuss why the fitting of the inversion recovery data is poor for some of the peaks ( /5) 5 Schedule DAY1 (RRC East) Explanation of 1D 1 H and 13 C NMR experiments Hands-on 1D experiments of 1 H and 13 C NMR DEPT 45, 90, 135 & 2D 13 C/ 1 H (HETCOR) & 2D 1 H/ 1 H (COSY) experiments for your unknown sample(s). 13 C T 1 inversion recovery exp. (Data should be handed to you) Make sure you have all the spectra and printout. Please ask your instructor r values for the inversion n recovery r exp. DAY2 (Pchem Lab) Plot 13 C T 1 inversion recovery Obtain T 1 values Analysis of data; signal assignments and interpretation of DEPT & 2D data. Helpful Hint: Obtain info on principle and applications of NMR from Physical and Organic Chemistry text books before writing the report. 6

4 13 C and 1 H Fourier Transform NMR: Determination of 13 C T 1 and Signal Assignments 2D 15 N/ 1 H Correlation Spectrum of GroEL-GroES Wuthrich et al. Nature 418 p207 (2002) 7 What is NMR? Nuclear Magnetic Resonance No More Resonances MRI Imaging Chemistry/Drugs Protein Structures 8

5 Spin & Quantum Angular Momentum Like an electron spin, a nuclear spin also has a quantized angular momentum I of I = [I(I+1)] 1/2 ħ [1] The z-component of I, I Z is quantized as I Z = m ħ, [2] where m = -I, -(I-1),, (I-1), I. 9 Why does a spin function as a magnet? I + + μ S N μ=γι Rotation of a charged particle (Remember a nucleus has a positive charge) A magnetic field! 10

6 Spin and Magnetic Moment When I 0, a spin has nuclear magnetic moment μ that can be related to I as μ = γi, where γ is a constant characteristic of nucleus. The z-component of μ is given by μ Z = γi = Z γmħ. Nucleus I γ (Ts) -1 Natural Abundance (%) 1 H 1/2 2.7* % 13 C 1/2 6.7* % 2 H 1 4.1* % Zeeman Interaction Spin in a Magnetic Field When a spin is placed in a magnetic field, the energy is given by E m = - μ Z B 0 = -γmħb 0 1 Figure 1. Zeeman energy levels for spins of I =1/2 and 1. Lower energy level attracts more spin population. It induces spin polarization (bulk magnetic moment) 12

7 NMR Frequency The transition energy between the two states is E = -γ[m (m 1)]ħB 0 = γħb 0. NMR frequency is easily obtained as 0 = E/h = B 0 γ/2π. 1 The frequency is typically in a range of MHz RF (Radio Frequency) 13 Bulk Magnetic Moment (Spin Polarization) P m = exp(-mγħb 0 /kt)/ exp(-mγħb 0 /kt) ~ (1 -mγħb 0 /kt)/(2i+1) (Note: mγħb 0 << kt) M 0 = NP m μ Zm = N 2 ħb 0 /4kT (For I =1/2) M 0 N, B 0, 2, 1/T = 14

8 800 MHz NMR at UIC Center for Structural Biology 15 Vector Model NMR A. FT NMR Equipment B. Bloch Model C. Effects of RF pulses D. /2-pulse and -pulse E. Free Induction Decay 16

9 A. FT NMR Equipment Observe Receiver Lock Receiver Field Controller Computer Probe Observe Transmitter Lock Transmitter Decoupler Transmitter 17 B. Bloch Model The time dependence of the bulk magnetic moment M(t) in a magnetic field is given by Bloch equation: dm(t)/dt = M(t) B(t), [3.1] When the magnetic field is a static magnetic field applied along the z-axis, it is described as dm(t)/dt = M(t) B 0. [3.2] In the equilibrium state, M(0) is parallel to B 0. In this case, dm(t)/dt = M(0) B 0 = 0. [Q1] Thus, no thing will happen 18

10 Precession & NMR signal Vector product Bloch Equation dm(t)/dt = M(t) B 0. [3.2] However, when M(t) is not parallel to B 0, M X (t) = M X (0)cos B 0 t M Y (0)sin B 0 t, [3.3] M Y (t) = M Y (0)cos B 0 t + M X (0)sin B 0 t, [3.4] M Z (t) = M Z (0), [3.5] a b absin a b 19 How NMR pulse works! t B 1 at frequency: NMR = B 0 /2 B 1 t = Flip Angle 20

11 1D Pulse NMR The RF pulse is turned off when B 1 t = /2, M (t) is flipped into the x-y plane. The RF pulse is called a /2-pulse or 90 pulse. After the RF pulse is off, dm (t)/dt = M (t) [- ]. [3.10] 21 Summary of 1D The motion of the magnetic moment is simply summarized as I Z [π/2 I Y ] I X [3.11] Z [ Y] X [ ] and I X -[ t I Z ] I X cos( t) + I Y sin( t). [3.12] 22

12 E. Free Induction Decay (FID) A typical time domain signal is given in a real form, s(t) = cos( t)exp(-t/t 2 ) T 2 : Transverse (or Spin-spin) relaxation time 23 Examples of FT FT 24

13 Inversion Recovery for Measurements of 13 C T 1 Relaxation T 1 : Longitudinal (or spin-lattice) relaxation M 0 M 0 M 0 M 0 M( ) = M 0 {1-2exp(-t/T 1 )} /2 25 How is the relaxation introduced? Dipolar Relaxation Mechanism 1 H r 13 C 13 C T 1 r 6 26

14 Analysis of Inversion Recovery Data Step 1 (Input data) Exp I(tau) at the peak position tau (s) 165 (ppm) 153 (ppm) 150 (ppm) Step 2 (Plot I( ) vs for each peak) 8 dependence of I( ) at 165 ppm 6 I(tau au) (arbitraryunit) (ppm) -6-8 tau (s) 27 Inversion Recovery Data 20 s 10 s 5 s 2 s 1 s 0.01 s 1. Label each spectrum with tau value 2. Measure peak intensity by a ruler 3. Prepare a Table 28

15 Step 3 (Fitting data) Fitting equation: I( ) = I( ){1-2exp(-t/T 1 )} I 0 = I( min ) or I( max ), whichever larger for the peak. Assume I( ) = I 0 Then, I( ) = I 0 {1-2exp(- /T 1 )} {I 0 - I( )}/I 0 = 2exp(- /T 1 ) Define y( ) ln{[i 0 - I( )]/I 0 }. Then, y( )= - /T 1 + ln2 If you plot vs y( ), the slope A = -1/T 1 and dthe intercept tb = ln2 (0.693). If B is far from ln2, check your data sheet calculations. 0.8 vs Ln{I0 -I( )} Ln{ n{i0 - I( )} y = x R 2 = (ppm) Linear (165 (ppm)) (s) 29 Step 4: Calculate the fitting curve and add it to the plot created in Step 2 I0(1-2exp(At)) tau 165 ppm 153 ppm 150 ppm A (-1/T1) dependence of I( ) at 165 ppm t) I( ) (arbitrary unit (ppm) -4 Fitting -6-8 (s) Repeat this for all the peak positions 30

16 Inversion Recovery 8 dependence of I( ) at 165 ppm I( ) (arb bitrary unit) (ppm) -8 (s) T 1 = 1/0.105 = 9.53 (s) 31 Signal Assignments 13 C NMR (Shifts, Intensity, # of lines) 1 H NMR (Shifts, Multiplet) l t) DEPT (CH/CH 3, CH 2, C) 2D 13 C/ 1 H HMQC and 1 H/ 1 HCOSYNMR (Connectivity of 13 C- 1 H & 1 H- 1 H spins) 32

17 2D 13 C/ 1 H HETCOR of Ethylbenzene Signal from a solvent 13 C Shift Cross peak 1 H Shift - 13 CH 2 -CH 3 33 Analysis of 2D 1 H/ 1 H COSY for Ethylbenzene ring CH 2 CH 3 CH 3 CH 2 -CH 2 -CH 3 J-coupling ring 34

18 Report 1) Cover page: Title & Your Name Name of TA, Your partner 2) Abstract (1p) 3) Introduction (2-4p in double space) 4) Datasheet & Calculation - Tables, Calculations, Graphs 5) Results - Your estimation of the sample - Tables for assignments ( 1 H, 13 C) & T 1 values 6) Discussion (5-8 p) List answers for the assigned questions in separate paragraphs Discuss other issues 7) Spectra (Clearly label the data & include assignments) 8) Reference Format: Double space, 12 point font, print only one side 35