13C - HSQC - editing (二維異核相關光譜)

Heteronuclear Single Quantum Coherence - editing Spectroscopy

1H - ACQU: (Data Collection)

1. Mount the sample, a 4.0 cm high solution, on a spinner, wipe the lower part of the glass sample tube with a tissue, gauge the depth of the sample (BBFO = 2.0 cm).

2. Use Lift to send air to the sample inlet above the Magnet.

3. Press Lift to slowly insert the sample with the mounted spinner into the probe inside the Magnet.

4. Enter filename, expno for edc.

5. Use rpar bbfo_1h.

6.  getprosol.

7. Lock (lock according to the solvent used).

8. Perform tuning and matching (atmm).

9. Perform topshim.

10. Shim x, y.

11. Run rga.

 (Note: Measure the 90-degree pulse width before collecting 1H data.)

12. Set ns to 40 (if the field is okay, collect 1H data).

13. Acquire zg (get a 1H spectrum, used as a side plot for 2D).

13C - ACQU: (Data Collection)

14. Enter filename, expno for edc.

15. Use rpar bbfo_13c.

16. getprosol.

17. Perform tuning and matching for 13C (atmm is necessary for this experiment).

18. Set ns to 1k (or 3k) (prepare parameter file, acquire 13C data with zg when time permits).

HSQC - ACQU: (Data Collection)

19. Enter filename, expno for edc.

20. Use rpar bbfo-13c-hsqc-editing.

21. getprosol

22. Run rga.

23. getprosol for 1H (input 90-degree pulse width value) (power of pulse width value in dbw).

24. Set ns to 8 or ns to 16 / otherwise.

25. Acquire zg.

26. Run the experiment (set experiment time).

HSQC - PROC: (Data Processing)

27. Execute Fourier transform in 2D (xfb).

28. Phasing (phase adjustment):

• Enter phase mode in the 2D spectrum.

• Adjust peaks after 120 ppm to positive.

• Adjust peaks in other regions that may be CH2 to negative and peaks that may be CH3 to positive.

29. Calibration (F1 domain = 13C and F2 domain = 1H):

• Identify specific cross peaks' chemical shifts, such as solvent or other distinguishable peaks.

30. Perform abs2D (abs2; abs1).

31. Set contour levels (clev) to 20 - 40.

32. Enter edc2:

• Fill in the left column with 1H (such as expno and procno).

• Fill in the right column with 13C (such as expno and procno).

33. Repeat processing once (rep 1) to update the screen.

HSQC - PLOT: (Data Plotting)

34. Choose Plot.

35. Select the Edit icon and input Xmin, Xmax, Ymin, Ymax for both sides:

• Xmin, Xmax: chemical shifts of 1H's left and right boundaries.

• Ymin, Ymax: chemical shifts of 13C's left and right boundaries.

36. Use the 1D/2D-Edit icon to adjust contour levels.

37. Distinguish peaks with positive and negative phases using different colors.

38. Export the graph as a PDF file using the export option in File or copy it to a Word file.

Note:

• Blue text indicates commands.

• Red text indicates buttons.

• Purple text indicates icons.

• Measure 1D 1H spectrum -> Can be used as a side plot during plotting for F2 projection.

• Measure 1D 13C spectrum -> Can be used as a side plot during plotting for F1 projection.

• The results of HSQC-editing experiments can be used not only to determine the connectivity between CH but also to distinguish the levels of 13C similar to DEPT.

13C - HSQC - editing (二維異核相關光譜)

Heteronuclear Single Quantum Coherence - editing Spectroscopy

1H - ACQU: (數據收集)  

1.      將 4.0 cm 高溶液的樣品套上轉子 (spinner), 用紙巾擦拭玻璃樣品管下半部, 用 gauge 量好樣品

          應置放之深度 (BBFO = 2.0 cm)

2.      按 Lift 將空氣送達 Magnet 上方樣品入口處

3.      按 Lift 將套好轉子的樣品緩緩送入 Magnet 內的探頭中  

4.       edc 鍵入filename, expno

5.       rpar bbfo_1h

6.       getprosol

7.       lock (依據使用的溶劑鎖場)

8.       atmm (tuning and matching - 自動調諧)

9.       topshim

10.    shim x, y

11.    rga

(p.s. 收 1H 數據之前請量測 1H 的 90 度脈寬)

12.   ns 40 (若勻場 OK，收 1H 數據)

13.   zg    (get a 1H spectrum - 當二維之側圖用)

13C - ACQU: (數據收集)  

14.   edc (鍵入filename, expno)

15.   rpar bbfo_13c

16.   getprosol

17.   atmm (13C 的 atmm 在此實驗中一定要做)

18.   ns 1k (or 3k) (建好參數檔，有時間時再 zg 收 13C 數據)

HSQC - ACQU: (數據收集)  

19.   edc (鍵入filename, expno)

20.   rpar bbfo-13c-hsqc-editing

21.   getprosol

22.   rga

23.   getprosol 1H (輸入 90 度脈寬值) (脈寬值的功率 in dbw)

24.   ns 8 or ns 16 / else

25.   zg

26.   expt (實驗時間)

HSQC - PROC: (數據處理)  

27.   xfb (執行二維的傅立葉轉換)

28.   Phasing (相位調整)

(在 2D 圖中，進入 phase 模式，將 120 ppm 後面之峰調成正向，

   將其他區域可能是 CH2 的峰調成負向，可能是 CH3 的峰調成正向)

29.  Calibration (F1 domain = 13C 及 F2 domain = 1H)

(找出特定交叉峰的化學位移，例如溶劑或其他可分辨的峰加以標定)

30.  abs2D (abs2; abs1)

31.  clev 20 ~ 40 (contour level)

32.  edc2

(表格左欄填入 1H ; 如 expno 與 procno 等)

(表格右欄填入 13C; 如 expno 與 procno 等)

33.  rep 1 (re procno 1，可將螢幕更新)

HSQC - PLOT: (數據繪圖) 

34.   挑選 Plot

35.   選取Edit icon，並將左右邊界填入 Xmin, Xmax 及 Ymin, Ymax

         Xmin, Xmax 1H 之左右邊界化學位移

         Ymin, Ymax 13C 之左右邊界化學位移

36.   選取 1D/2D-Edit icon 可以調整 contour 圖的高低層次

37.   將正負向相位之峰以不同顏色來區別

38.   選取 File 中的 export 可將圖形存成 pdf 檔；或使用 copy 鍵複製到 word 檔

  註 :  藍色字表指令  紅色 字表按鍵  紫色字表 icon

  註 :  量測一維之 1H 譜 -> 可在製圖時當側圖使用 - for F2 projection 

          量測一維之 13C 譜 -> 可在製圖時當側圖使用 - for F1 projection 

 註：HSQC-editing 之實驗結果，除可知道 CH 之間的連接關係外，尚可與 DEPT 一樣用來分辨 13C 之級數。