Steps%20for%20NMR%20Experiments%20small%20molecule

1
Steps for NMR Experimentssmall molecule

• ???
• Casper Wu

2
OUTLINE

• 1. ????
• 2. ????
• 3. NMR????
• 4. ??
• 5. 1D 1H NMR
• 6. 1D 13C NMR
• 7. 2D homonuclear NMR
• 8. 2D heteronuclear NMR

3
1.1 ????

• ????????(10 mg)
• ?????D-solvent
• ?????NMR sample tube
• ??????(5mm)
• ?????? ?? ? 0.5 mL, 3 cm
• ?SHIGEMI??? ?? ? 0.3 mL

4
1.2 ????

• SHIGEMI???

5
2.1 ????
??? ???
????(ppm) ?? ??
????(J, Hz) ?? ??
???(sensitively) ? ?
peak??? ? ?
6
2.2 ????(???)

• 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

7
2.3 ????(???)

• 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

8
2.4 ????(???)
600MHz
500MHz
n-butyl acetate
400MHz
300MHz
ppm
Hz
9
2.5 ????(???)
500MHz
400MHz
uridine
300MHz
10
3.1 NMR????

• Inverse and Observe

1H sensitivity ? 50
11
3.2 NMR????

• 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

12
3.3 NMR????

• 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)

13
3.4 NMR????

• CryoProbe
• ??????????????
• cryoprobe1H, 1H, X
• ?????????
• ??????? (4 K)
• ????????????
• ??signal-to-noise ratio (S/N)

14
3.5 NMR????
3mg sample
ns 100 exp. time 11 min
NS 20000 exp. time 14 hrs
15
3.6 NMR????

• Gradient (????)
• Z-GRAD z gradient
• XYZ-G x, y, z gradient
• ?? 5 ????????
• ??????????

gradient ??
tuning
tuning?matching??
matching
BBO z-grad
16
3.7 NMR????

• ATM Probe
• Automatic Tuning and Matching
• ??????????
• ??????????
• ?(tune)???(match)
• tune ?????????
• match ???????? 50?

17
4.1 (Shim)
18
4.2 ??? (Deuterium Lock)??(Shim)
19
4.3 ??(Shim)
20
4.4 ??(Shim)
21
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.

22
5.2 Solvent Suppression??????????????????
25mg uridine in D2O
23
5.4.1 1D Selective Excitation????????????????
9
1D selective TOCSY
7
8
12
?-ionone
24
5.4.2 1D Selective ExcitationCOSY, TOCSY, NOESY,
ROESY
Cyclosporine
25
5.4.3 1D Selective Excitation
1D selective TOCSY
26
5.4.4 1D Selective Excitation
1D selective NOESY
27
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.

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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.

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6.2.2 Spin-Spin Splitting
2
4
5
1
6
3
?
30
6.3.1 DEPTDistortionless Enhancement by
Polarization Transfer
CH3
CH
CH2
45? 90 ? 135 ?
CH
CH2 ? -
CH3 ?
CH
31
6.3.2 DEPT??????
32
7.1.1 ??J-resolved (??????)
A
M
X
M
X
A
2,3-dibromo- propionic acid
33
7.1.2 ??J-resolved (??????)
11
7
12
3
4
9
8
2
6
5
1
10
?-ionone
34
7.2.2 COSY (??3J????)
1
6
3
5
4
3
5
2
5
1
4
6
4
n-butyl acetate
3
35
7.2.2 COSY90 (??3J????)
A
M
X
M
X
A
2,3-dibromo- propionic acid
36
7.2.3 COSY45 (??3J?2J????)
A
M
X
M
X
A
2,3-dibromo- propionic acid
(a)vicinal spin-spin coupling (3J)
(b)geminal spin-spin coupling (2J)
37
7.3 TOCSY (????J-????)
1
6
3
5
4
3
5
2
1
4
6
n-butyl acetate
3
38
7.4.1 NOESY (???????????)(lt5Å)
11
12
1
9
8
7
11
4
2
9
10
1
5
3
6
8
12
7
?-ionone
9
8
39
7.4.2 NOESY (???????????)(lt5Å)

• C(11)??H --- C(9)??H
• ? 2.5Å
• C(11)??H --- C(8)??H
• ? 3.8Å
• C(11)??H --- C(7)??H
• ? 4.7Å

40
7.5 INADEQUATE (??1JC-C???)
5
4
3
2
5
6
6
4
3
2
1
1
41
8.1.1 HMQC (??1JH-X???)(X 13C, ? gt 0)
1
6
5
4
3
6
5
1
4
3
3
5
2
1
4
6
n-butyl acetate
2
42
8.1.2 HMQC (??1JH-X???)
5
6
9
9
2
3
7
8
9
1
2
6
7
3
9
4
8
5
8
5
6
7
2
uridine
3
43
8.2 HSQC (??1JH-X???)(X 15N, ? lt 0)
1
6
5
4
3
6
5
1
4
3
3
5
2
1
4
6
n-butyl acetate
2
44
8.3.1 HMBC (??nJH-X, n ? 2???)
1
6
5
4
3
6
5
1
4
3
3
5
2
1
4
6
n-butyl acetate
2
45
8.3.2 HMBC satellite
1
6
3
5
4
46
Thank You

• casper_at_rezwave.com.tw