Steps for NMR Experiments small molecule 吳英彥 Casper Wu

OUTLINE 1. 樣品配製 2. 儀器選擇 3. NMR探頭選擇 4. 勻場 5. 1D 1H NMR 6. 1D 13C NMR 7. 2D homonuclear NMR 8. 2D heteronuclear NMR

1.1 樣品配製 取用適當的樣品量(10 mg) 選擇合適之D-solvent 取用合適的NMR sample tube 配製適當的量(5mm) 一般樣品管: 體積  0.5 mL, 3 cm SHIGEMI樣品管: 體積  0.3 mL

1.2 樣品配製 SHIGEMI樣品管

2.1 儀器選擇 低磁場 高磁場 化學位移(ppm) 相同 偶合常數(J, Hz) 靈敏度(sensitively) 低 高 peak鑑別度

2.2 儀器選擇(靈敏度) For 1H:  = 26753 (s-1Gauss-1) = 2.6753 (s-1Tesla-1) (1Tesla = 104 Gauss) when Bo = 4.69T  E = h = (hBo)/2   = Bo/2 = 2108 (s-1) = 200 MHz

2.3 儀器選擇(靈敏度) Boltzmann equation: N/N = exp(-E/kT) = exp[(hBo)/(2kT)] when Bo = 4.69T (200MHz)  N/N = 0.999967 i.e. N = 106  N = 1000033 when Bo = 9.38T (400MHz) N = 106  N = 1000066  field  2  sensitively  2

2.4 儀器選擇(鑑別度) 600MHz 500MHz n-butyl acetate 400MHz 300MHz ppm Hz

2.5 儀器選擇(鑑別度) 500MHz 400MHz uridine 300MHz

3.1 NMR探頭選擇 Inverse and Observe 1H sensitivity  50% 1H/2D X gradient

3.2 NMR探頭選擇 Fixed-channel probe SEL: 1H selective DUL: 13C and 1H (inner 13C, outer 1H) SEI: selective inverse (inner 1H, outer X) TXO: triple resonance observe TXI: triple resonance inverse QXI: quattro resonance inverce

3.3 NMR探頭選擇 Broad-band probe BBO: broad band observe BBI: broad band inverse TBO: tripe resonance broad band observe TBI: tripe resonance broad band inverse QNP: quattro nucleus probe (1H/13C/19F/31P, 1H/13C/15N/31P)

3.4 NMR探頭選擇 CryoProbe 樣品熱絕緣式冷卻核磁共振探頭 cryoprobe{1H}, {1H, X} 將探頭內之線圈系統 冷卻到液氦溫度 (4 K) 降低電子線路所產生之雜訊 提高signal-to-noise ratio (S/N)

3.5 NMR探頭選擇 3mg sample ns = 100 exp. time = 11 min NS = 20000 exp. time = 14 hrs

3.6 NMR探頭選擇 Gradient (磁場梯度) Z-GRAD: z gradient XYZ-G: x, y, z gradient 節省 5 倍以上的實驗時間 執行自動化的勻場動作 gradient 接頭 tuning tuning與matching數值 matching BBO z-grad

3.7 NMR探頭選擇 ATM Probe Automatic Tuning and Matching 以簡單的方式使機器自 動調整樣品於探頭中頻 率(tune)與阻抗(match) tune: 輸出正確的共振頻率 match: 使系統的阻抗值為 50

4.1 (Shim)

4.2 氘鎖定 (Deuterium Lock) 勻場(Shim) B0 = 0 CDCl3 B0  0 hn(CDCl3) digital & shaped pulse deuterium lock Better shimming

4.3 勻場(Shim) Z -Z2, -Z4 Z3, Z5 Z2, Z4

4.4 勻場(Shim) Z4 Z2 Z2 and Z4

5.1 1H NMR The number of signals shows how many different kinds of protons are present. The location of the signals shows how shielded or deshielded the proton is. The intensity of the signal shows the number of protons of that type. Signal splitting shows the number of protons on adjacent atoms.

5.2 Solvent Suppression 提高濃度較稀或高含水量之樣品的靈敏度 H2O suppression 25mg uridine in D2O H2O

5.4.1 1D Selective Excitation 凸顯目標、簡化問題、減少實驗時間 TOCSY 9 1 2 3 4 5 6 7 8 9 10 11 12 7 8 12 -ionone

5.4.2 1D Selective Excitation COSY, TOCSY, NOESY, ROESY Cyclosporine

5.4.3 1D Selective Excitation 1D selective TOCSY

5.4.4 1D Selective Excitation 1D selective NOESY

6.1 13C NMR 12C has no magnetic spin. 13C has a magnetic spin, but is only 1.1% of the carbon in a sample. The gyromagnetic ratio of 13C is one-fourth of that of 1H. Signals are weak, getting lost in noise.

6.2.1 Spin-Spin Splitting It is unlikely that a 13C would be adjacent to another 13C, so splitting by carbon is negligible. 13C will magnetically couple with attached protons and adjacent protons. These complex splitting patterns are difficult to interpret.

6.2.2 Spin-Spin Splitting 13C{1H} NMR (1H decoupling) ethylbenzene  1 4 5 3 ethylbenzene 1 4 5 2 3 6 5 4 2 1 6 3 

6.3.1 DEPT Distortionless Enhancement by Polarization Transfer CH3 CH CH2 45 90  135  CH + CH2  - CH3 CH

6.3.2 DEPT 區分碳的級數 6 5 4 3 2 1 s ethylbenzene 1 4 5 2 3 6

7.1.1 同核J-resolved (獲得偶合常數) M A X M X A 2,3-dibromo- propionic acid

7.1.2 異核J-resolved (獲得偶合常數) 11 7 12 4 3 9 8 2 5 6 10 1 1 2 3 4 5 6 7 8 9 10 11 12 -ionone

7.2.2 COSY (獲得3J偶合關係) 1 6 3 4 5 3 5 2 5 1 4 6 4 n-butyl acetate 3

7.2.2 COSY90 (獲得3J偶合關係) M A X M X A 2,3-dibromo- propionic acid

7.2.3 COSY45 (獲得3J與2J偶合關係) M A X M X a b A 2,3-dibromo- propionic acid (a)vicinal spin-spin coupling (3J) (b)geminal spin-spin coupling (2J)

7.3 TOCSY (獲得所有J-偶合關係) 1 6 3 4 5 3 5 2 1 4 6 n-butyl acetate 3

7.4.1 NOESY (獲得分子於空間中之關係) (<5Å) 12 11 1 7 8 9 11 4 9 10 2 5 1 3 6 8 12 7 -ionone 9 8

7.4.2 NOESY (獲得分子於空間中之關係) (<5Å) C(11)上的H --- C(9)上的H  2.5Å C(11)上的H --- C(8)上的H  3.8Å C(11)上的H --- C(7)上的H  4.7Å

7.5 INADEQUATE (獲得1JC-C之關係) 3 5 2 4 2 3 4 6 1 6 1 Sucrose 1 4 5 6 3 2

8.1.1 HMQC (獲得1JH-X之關係) (X = 13C,  > 0) 6 4 5 3 6 5 1 4 3 3 5 2 1 4 6 n-butyl acetate 2

8.1.2 HMQC (獲得1JH-X之關係) uridine 5 6 9 9 2 3 7 8 9 1 2 6 7 3 9 4 8 8 5

8.2 HSQC (獲得1JH-X之關係) (X = 15N,  < 0) 6 4 5 3 6 5 1 4 3 3 5 2 1 4 6 n-butyl acetate 2

8.3.1 HMBC (獲得nJH-X, n  2之關係) n-butyl acetate 1 6 4 5 3 6 5 1 4 3 3 5

8.3.2 HMBC satellite 1 n-butyl acetate 1 2 3 4 5 6 6 3 4 5

Thank You casper@rezwave.com.tw