Introduction to Instrumental

Analysis
Dr. Shoei-Sheng Lee
September 11, 2012

How to obtain Molecular formula?

1. Elemental Analysis: Oxidation (Burning)
CO2, H2O, NO2, etc
Average mass:
H 1.008; C 12.011; N 14.007; O 15.999
e.g. Compound A
Analysis: 66.7% C; 7.9% H
O: (100-66.7-7.9)%= 25.4%
Formula: 66.7/12.011: 7.9/1.008: 25.4/15.999
= 5.553: 7.837: 1.5876= 3.5: 4.94: 1~ 7: 10: 2 => C7H10O2
MS: 126.067

How to obtain Molecular formula?

High Resolution Mass Spectrum
Electron Impact (HR-EI-MS)
Fast Atom Bombardment (HR-FAB-MS
Electron Spray Ionization (HR-ESI-MS)
Acurate Mass:
H 1.0078; C 12.000; N 14.003; O 15.9949
How to distinguish CO and C2H4?
CO: 27.9949; C2H4: 28.0312

How to obtain Molecular formula?

NMR Spectra for estimating the composition of C and H.
BB Decoupled CMR for all types of carbon signals
(1o , 2o , 3o , 4o )
DEPT-90 for CH (methine);
DEPT-135:
CH/CH3 (methyl) (positive or upward);
CH2 (methylene) (negative or downward)

How to verify Functional Groups?

IR, Raman:
carbonyl, amide, ester, substituted phenyl,
hydroxy, amine, alkyl, ether, etc

Optical property
Specific optical rotation: similar skeleton
and conformation
Circular dichroic spectroscopy (CD):
absolute stereochemistry

Effect of Conformation on optical rotation

3

MeO

5
6a NMe

MeO 1
HO

(+)-Norisocorydine
6a-S

MeO

1 NMe
8

MeO

(+)-Isocorydine
6a-S

MeO

NH

MeO
HO

MeO

HO

MeO

HO

HO

(-)-N-methylisococlaurine
1-R

MeO
NH
H

HO

(-)-coclaurine (2)
1-S

HO
HO

NMe
H

MeO

(+)-reticuline (3)
1-S

CD: positive Cotton effects at

290, 235 and 210 nm for 2 and 3

Effect of Conformation on optical

rotation
OMe
MeO

MeO

MeO
NH

HO
H

HO

6.38 H

HO

6.67
OH

(-)-coclaurine (2)
1S

NMe

HO
H

N
14

6.55
OMe

MeO

(+)-reticuline (3)
1S

OH

(-)-14S

How to identify Chromophores?

Chromophore: UV-VIS spectrum

n- and -* electron transition
Enone: -* 215 nm
Diene: homoannular 253 nm
heteroannular 214 nm
Aromatic componds: -*
Carbonyl: n- (low
Substitution effect

7.0

6.0

5.0

ppm

4.0

3.0

2.220

ppm

2.139

ppm

1.089

ppm

5.3804
5.3688
5.3557
5.3456
5.3313
5.3201

4.1473
4.1406

1.015

1.097

2.220

2.139

2.1787
2.1433
2.0563
2.0237
1.9985
1.9830

3.7127
3.7052
3.6898
3.6823

6.2906
6.2509

1.000

1.089

6.7770
6.7566

1.073

1.036

7.0411
7.0364

1.042

6.9500
6.9453
6.9296
6.9250

7.5744
7.5347

0.988

2.1787
2.1433
2.0563
2.0237
1.9985
1.9830

3.3080
3.3039
3.3000
3.2957
3.2918

3.7127
3.7052
3.6898
3.6823

4.1473
4.1406

4.8799

5.3804
5.3688
5.3557
5.3456
5.3313
5.3201

6.2906
6.2509

7.0411
7.0364
6.9500
6.9453
6.9296
6.9250
6.7770
6.7566

7.5744
7.5347

1H

1.097

ppm

1.015

7.0

1.000

1.073

ppm

1.042
1.036

0.988

How to obtain detailed structure

information

NMR spectroscopic analysis

1D NMR: 1H & 13C NMR
2D NMR: COSY, NOESY, TOCSY,
HMQC, HMBC, etc
Conformational analysis

NMR

CCW (08027)-p.3379767-fr. 2/CD3OD-3.30 ppm/06-08-2012, Bruker AV400

ppm

2.0

ppm

7.0

6.0

5.0

4.0
2.3924
2.3849
2.3810
2.3462
2.3388
2.3347

1.028

1.027

40

ppm

3.0
2.3924
2.3849
2.3810
2.3462
2.3388
2.3347

2.8626
2.8564
2.8505
2.8163
2.8104
2.8042

60

1.027

2.8626
2.8564
2.8505
2.8163
2.8104
2.8042

3.3080
3.3038
3.2999
3.2958
3.2918

3.9802
3.9699
3.9634
3.9532

80

1.028

ppm
3.9802
3.9699
3.9634
3.9532

100

1.038

1H

4.4248

4.8168

5.3358
5.3241
5.3191
5.3120
5.3071
5.2952

6.8612
6.8557

120

1.038

5.3241
5.3191
5.3120
5.3071
5.2952

6.8612
6.8557

140

1.067

1.050

1.000

7.0264

160

1.050

1.000

1.893

38.6187
40.0962

49.4246
49.2118
48.9989
48.7859
48.5728

74.3213
72.3080

116.4793
115.4119
115.1338

122.9456

127.8042

149.5244
146.9457
146.7806

168.9399

CCW (08027)-p.3379767-fr. 2/CD3OD-49ppm/06-08-2012, Bruker AV400

DEPT-90

DEPT-135

BBD CMR

ppm

CCW (08027)-p.3379781-peak 2/CD3OD-3.30 ppm/08-01-2012, Bruker AV400

NMR

ppm

ppm

ppm

ppm

CO2H

1
O

3'

1'

5
7'

CCW (08027)-p.3379781-peak 2/CD3OD-49 ppm/08-01-2012, Bruker AV400

OH

DEPT-90

OH

5'
5-O-galloyl-shikimic acid

OH

DEPT-135

200

180

160

140

120

100

80

49.6380
49.4251
49.2122
48.9991
48.7861
48.5730
48.3599

71.6560
69.5010
67.4243

110.1124

121.4080

130.0937

139.9347
139.2232

146.5202

BBD CMR
169.7250
167.6376

HO

60

40

28.6134

HO

20

Structural Fragments: Mass

fragmentation (MS)
Cyclopeptide Alkaloids

+.

+
NMe2

NH
HN O HN
O
O
O
N
NMe2

N
H

-cleavage
N
H

m/z 187 (100%)

Tryptophan moiety

Hemsine C isolated from Paliurus hemsleyanus (Rhamnaceae)

H-Y. Lin, C.-H. Chen, K.C.S.C. Liu, and S.-S. Lee* Helvetica Chimica Acta 2003, 86, 127-138

ppm

Hemsine C
Rf 0.52 [Me2CO-toluene
(3:7), saturated with 25%
NH4OH]
[] 26D -107.0 (c = 1.0,
MeOH).
IR max cm-1: 3398, 3300
(amide NH), 2980, 2930,
2890, 1680, 1650, 1620
(CONHR), 1501, 1480,
1225, 1185, 1110, 1068,
870, 740, 700
(monosubstituted phenyl).

20

19

22

20'

11

HN

13
14
13'
4

O
O

2
NH

5 O

O HN

Intermediate a.a. 1"

3"

15
18

Ring bond a.a.

17

1'
NMe2

3'

6'

N-Terminal amino acid

12'

8'

N
H

11'

Hemsine C

Hemsine C
UV (MeOH) max nm (log ):
214 (4.28), 280 (4.31).
CD (MeOH) nm (): 311 (4.02), 296 (-2.76), 281 (-2.19),
231 (-36.79).
FAB MS (positive) m/z (rel.
int.): [M+H]+ 705 (72), 574 (47),
187 (100), 135 (8).
HRFAB MS (positive) m/z
[M+H]+: 705.3762. Calc. for
C41H49N6O5: 705.3764.

NH
HN

O HN

O
O
N
NMe2
N+Me2
N
H

Hemsine C

N
H

m/z 187

1H

NMR (/ppm, J in Hz ) and 13C NMR (/ppm, m) Data

Hemsine C (CDCl3 )

Position

Ha

13 b

Key HMBCb

Key NOESYb

HC
1

6.39 d (7.6)

114.9 d 2, 14

2, 13, 13'

6.77 dd (10.4, 7.6)

125.8 d 14

6.61 d (10.4)

4
4.25 ddd (11.3, 6.4, 3.0)

6.36 d (6.4)

53.0 d 4
7

20'

5 (vw)

8a

5.23 d (8.6)

1",

5.74 d (6.4)

81.3 d 7, 8, 19, 20

11

56.0 d 9, 7, 1"

12, 20(20'), 8a

155.0 s

12

7.40 dd (8.4, 2.4)

123.7 d 11, 12', 14

12'

7.28 brd (8.1)

124.0 d 11, 12, 14

13

7.15 dd (8.4, 1.5)

130.6 d 1, 11, 13'

13'

7.20 dd (8.1, 1.5)

132.1 d 1, 11, 13

14

15

2
NH

5 O

Intermediate a.a. 1"

3"

13'
4

7
O HN

HN

12', 20(20')
20(20')(w), 9(w)

14 1

11

6 (vw),15

171.1 s
4.52 dd (8.6, 6.4)

19

22

18

Ring bond a.a.

17

1'
NMe2

6'

3'

N-Terminal amino acid

133.1 s

15

1.86 m , 1.35 m

39.4 t

16

1.51 m

24.5 d

17

0.94 d (6.5)

21.1 q 15, 16, 18

18

0.92 d (6.6)

23.5 q 15, 16, 17

19

136.9 s

20 (20)

6.38 brd (7.3)

21 (21)

6.79 dd (7.7, 7.3)

128.7 d 19, 20, 22

7.08 t (7.7)

128.5 d 21, 20,

22

20

1
167.5 s

13

12'
8'

N
H

11'

126.8 d 9, 22

Ceanothic acid from

Rhamnaceous plants

Hemsine C

Symmetric compounds
A glucoside
OMe
OMe
O
HO
HO
HO
HO

OMe
O

Tris-thiophene
3" 4'
2" 5'

4"
5"

3'
2'

3
2

-Glc

Constituents (Vinca Alkaloids)

Alkaloids (>70): dimeric (20)
1. Vinblastine
(Vincaleukoblastine,
N
Velban*): dimer of
H
MeO
catharanthine and
vindoline
2.Vincristine
RN
(Leurocristine;Oncovin*): MeO C
2
2 ppm(2g/1000Kg)
HO
{Vinblastine (N-methyl, AcO
NCH3) Vincristine (Nformyl, -NCHO)}
* Trade name

N
CO2Me
OH

Vinblastine R= Me
Vincristine R= CHO

Hyphenated Techniques
GC-MS: gas chromatography-Mass spectroscopy
LC-MS: liquid chromatography-Mass
spectroscopy
LC-NMR: LC-nuclear magnetic resonance
LC-SPE-NMR: LC-solid phase extract- NMR
LC-MS-NMR
CE-NMR: capillary electrophoresis- NMR

Textbooks:
1. R.M. Silverstein and F.X. Webster "Spectrometric
Identification of Organic Compounds", 7th ed., John
Wiley& Sons: New York, 2005.
2. A.E. Derome, "Modern NMR Techniques for
Chemistry Research", Pergamon Press: New York,
1989.