EXAMINATION QUESTION PAPER Exam in: KJE-8303 Nuclear Magnetic Resonance Date: 30.05.2017 Time: 4 hours Place: Approved aids: Ruler, pen Type of sheets (sqares/lines): Number of pages incl. cover page: Contact person during the exam: Any 16 Johan Isaksson Phone: 41354726 NB! It is not allowed to submit rough paper along with the answer sheets. If you do submit rough paper it will not be evaluated.
Question 1. Theory (30 p) E B 0 β µ ω -½ µ = γ I h/2π ω 0 = -γ B o v 0 = -γ B o /2π α +½ ω µ ω 0 = larmor frequency (rad -1 ) γ = gyromagnetic ratio µ = magnetic moment γ Η = +2.67 10 8 rad s 1 T 1 1a. Above is a schematic representation of what happens when a spin ½ nuclei is put in a strong magnetic field (B 0). Briefly describe equilibrium, and what characterizes the energy levels. Define polarization. (5 p) 1b. Briefly explain the vector model (vector representation) and use it to describe the magnetization in a sample after a 1 H 90 degree rf pulse. (5 p) B 0 z M ω 0 =γb 0 z ω 1 =γb 1 x y B 1 x M y Equilibrium B 1 Excited z ω M o - net magnetization vector allows us to look at system as a whole z z ω M o - net magnetization vector allows us to look at system as a whole z many nuclei x y x y many nuclei x y x y UiT / Postboks 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmottak@uit.no / uit.no 2
O OH 1 2 3 7 6 HO 4 5 OH OH 600003900 Hz 600001200 Hz H 2 O 1300 1200 1100 1000 900 800 3 700 4 6 5 7 7 DMSO 600 500 400 HO4 HO6 HO5 300 200 100 0-100 5.5 5.0 4.5 f1 (ppm) 1c. What is chemical shift of protons? Briefly explain why different protons in a molecule have different chemical shifts and three factors that greatly affect the observed chemical shift. (6 p) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 1d. Draw in the figure above, what is upfield and downfield, which part of the spectra is shielded, which is deshielded and which part resonates at higher larmor frequency and which resonates at lower larmor frequency. (6 p) f1 f2 90 x 90 x d1 t1 t2 Preparation 2.0 2.5 3.0 Evolution 3.5 4.0 4.5 5.0 5.5 f1 (ppm) Mixing 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 Detection 1e. Explain briefly how the 2D dimensions in the spectra above is constructed using the functional elements to the right. Explain very briefly the origin of crosspeaks (in a general form) (8 p) UiT / Postboks 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmottak@uit.no / uit.no 3
Question 2. 1D NMR (30 p) 2a. i. Assign the peaks based on their multiplicity and their chemical shifts. (5p) 2a. ii. The doublets at 7.1-7.2 PPM are leaning towards each other. Briefly explain the phenomena. (5p) 2b. Above you can see two phosphorous spectra of triphenylphosphate, with and without proton decoupling. Explain what i) proton decoupling is and ii) why is the peak so much sharper and iii) why the peak has (probably) increased in intensity in the proton decoupled spectra? (7 p) UiT / Postboks 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmottak@uit.no / uit.no 4
2c. Zooming in on the phosphorous peak in the decoupled spectra you can see several satellites (magenta, cyan and yellow boxes). Can you assign which couplings they arise from? (3p) 2d. i. In the 1D carbon spectra to the right there are two peaks: a) an aromatic CH and b) an aromatic tertiary carbon. Denote the peaks in the figure. (1p) ii. Explain why it is like this. (2p) iii. What could you do to make a carbon spectra quantitative? (1p) UiT / Postboks 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmottak@uit.no / uit.no 5
2e. Analyze the coupling patterns in the following multiplets and report them using the Hz scale below the multiplets (6p) 525 520 515 510 505 500 f1 (Hz) 495 490 485 480 475 520 515 510 505 500 f1 (Hz) 495 490 485 530 520 510 500 f1 (Hz) 490 480 UiT / Postboks 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmottak@uit.no / uit.no 6
Question 3 KJE-3303. 2D/3D NMR (20 p) E B 0 N+ /2 W 2 W 1 W 0 S I N+ /2 W 0 W 2 S I W 1 N- /2 N- /2 3a. Use the figure above to very briefly explain why Protein size is important for the quality of the NMR data you can acquire. You can also state other reasons. (3p) 3b. Name at least two techniques/tricks used to improve the spectral quality. (2p) 3c. 3D triple-resonance spectra are the foundation of protein NMR. Very briefly explain the principle of triple-resonance experiments and how they are used to assign the protein backbone. (5 p) 2 3 H 3 C CH 3 N H 3 C 1 O UiT / Postboks 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmottak@uit.no / uit.no 7
3d. This is a ROESY of N,N-dimethylacetamide. i) What are the peaks marked by arrows (same sign as the diagonal peaks), ii) how do they arise and iii) what does it tell us about the behavior of the molecule? (5 p) 15 23 24 2 3 21 7 20 11 / 10 21 MeOH 0.5 1.0 O 21 22 Br H 1 18 19 N 20 Br 2 11 17 12 N 14 13 3 16 4 10 Br 15 8 N 9 HN 5 7 6 23 Cl 24 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 f1 (ppm) O 5.5 5.5 5.0 3e. In the structure above, the stereochemistry around the H20 has been removed. Use the assigned ROESY to say if H20 points up or down and provide the argument you used to decide its configuration. (5 p) 4.5 1H 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 UiT / Postboks 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmottak@uit.no / uit.no 8
Question 4. 2D NMR (20 p) Proton 2.00 1.06 1.03 0.92 0.96.5 8.4 8.3 8.2 8.1 7.9 7.8 7.7 7.6 7.4 7.3 7.2 1H (ppm) 7.1 6.9 6.8 6.7 6.6 6.4 6.3 6.2 6.1 6. Carbon 184.94 181.94 156.28 135.31 133.15 132.93 129.41 126.73 122 110.69 200 195 190 185 180 175 170 165 160 155 150 13C (ppm) 145 140 135 130 125 120 115 110 105 100 UiT / Postboks 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmottak@uit.no / uit.no 9
DQF-COSY 1H (ppm) 8.5 8.5 8.4 8.3 8.2 8.1 7.9 7.8 7.7 7.6 7.4 7.3 7.2 1H (ppm) 7.1 6.9 6.8 6.7 6.6 6.4 6.3 6.2 6.1 ROESY 300ms (poor quality) 1H (ppm) 8.5 8.4 8.3 8.2 8.1 7.9 7.8 7.7 7.6 7.4 7.3 7.2 1H (ppm) 7.1 6.9 6.8 6.7 6.6 6.4 6.3 6.2 6.1 HSQC+HMBC UiT / Postboks 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmottak@uit.no / uit.no 10
100 110 120 130 140 150 13C (ppm) 160 170 180 190 8.5 8.4 8.3 8.2 8.1 7.9 7.8 7.7 7.6 7.4 7.3 7.2 1H (ppm) 7.1 6.9 6.8 6.7 6.6 6.4 6.3 6.2 6.1 4a. The spectra above are for an unknown molecule with the molecular formula: C 10H 6O 3. Elucidate the structure and report the 1 H and 13 C assignments in the HSQC+HMBC. All spectra are acquired in CDCl 3 (δ 1H=7.26 PPM, δ 13C=77.16 PPM). (20p) Good luck! Johan UiT / Postboks 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmottak@uit.no / uit.no 11
Compendium Chemical shift tables and coupling constants UiT / Postboks 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmottak@uit.no / uit.no 12
UiT / Postboks 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmottak@uit.no / uit.no 13
UiT / Postboks 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmottak@uit.no / uit.no 14
UiT / Postboks 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmottak@uit.no / uit.no 15