Applicazioni della spettroscopia di risonanza magnetica nucleare (NMR) nel settore ambientale, vegetale e agroalimentare Dr.

Pellegrino Conte Dipartimento di Scienze del Suolo, della Pianta e dellAmbiente

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

Magnetic moment

Ih = = h I 2

gyromagnetic ratio Planck constant

h h= 2

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

M z (t ) = M (t ) B0 (t ) z R1 (M z (t ) M 0 ) t M x (t ) = M (t ) B0 (t ) x R2 M x (t ) t M y (t ) = M (t ) B0 (t ) y R2 M y (t ) (t )

R1=1/T1 spin lattice relaxation

time R2=1/T2 spin-spin relaxation time

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

149 148 145 126 125 114 112 173 109 57 O 10 OH 8 1 6 4 OH 2 3 O CH 3 7 9 1=126 ppm 2=125 ppm 3=148 ppm 4=149 ppm 5=114 ppm 6=112 ppm 7=56 ppm 8=145 ppm 9=109 ppm 10=173 ppm

150

100 Chemical Shift (ppm)

50

= B0 (1 ) 2

S R = 10 6 R

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

Direct detection
pulse

preparation

acquisition

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

CSA, strong dipolar couplings

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

Elimination of the strong dipolar couplings pulse

13C

preparation

acquisition

1H

decoupling

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

Elimination of the Chemical Shift Anisotropy (CSA)

h I S

2r 3

(3 cos 2 1)

Magic angle spinning (MAS)

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

Sensitivity enhancement by cross polarization Cross polarization

13C

Contact Time (CT)

acquisition

pulse
Contact Time (CT)

1H

decoupling

Short introduction to the theory of nuclear magnetic resonance spectroscopy (NMR)

Static, no decoupling Static, decoupling MAS, no decoupling

MAS, decoupling and/or CP

Application of solid state NMR spectroscopy to the analysis of the soil organic matter
O 9 OH

CH3 H3C H3C CH3 CH3

12
CH3

11

10

9 8

7 6

5 4

8 1 2

SO4-

6 5

Sodium dodecilsulphonate (SDS)

Hexamethylbenzene (HMB)

Ferulic Acid

4 3 O OH CH3 10

OH

* O

6 4 5
HO

O H 2C

2 3
O

O O

Carboxymethylcellulose sodium salt (CMC)

Application of solid state NMR spectroscopy to the analysis of the soil organic matter
Methyl C Quaternary Aromatic C

CH3 H3C H3C CH3 CH3 CH3

OH

SSB

SSB

HMB

* O

6 4 5
HO

O H2C

2 3
O

O O

COO-

C1

C2-C6

CMC
C10 C11 C12 C1

C2-C9

12
SDS

11

10

9 8

7 6

5 4

SO4-

O 9 OH

C3-C4 C9 SSB

C7 C1-C2 C5, C6, C8 C10

7 1 6 5

8 2

Ferulic Acid
100 0 -100 -200

4 3 O OH CH3 10

400

300

200

Chemical Shift (ppm)

Application of solid state NMR spectroscopy to the analysis of the soil organic matter
H A: COOH (CMC), C9 (Ferulic acid) E
Mixture 1 OCT=6ms

C G

B: C3,4,7 (Ferulic acid) C: C1,2 (Ferulic acid), Car(1-6) (HMB)

A B D
400 300

F
-100 -200

D: C5,6, 8 (Ferulic acid), C1 (CMC) E: C2-C5 (CMC), C1 (SDS) F: C10 (Ferulic acid), -CH2O-, and C6 (CMC) G: C2-C11 (SDS) H: C12 (SDS), Cal(7-12) (HMB)

200 100 0 Chemical Shift (ppm)

E G B D C
Mixture 2 OCT=1.2ms

F H

400

300

200 100 0 Chemical Shift (ppm)

-100

-200

Application of solid state NMR spectroscopy to the analysis of the soil organic matter
Effect of spin diffusion mediated by water
H3C H3C H3C H3C H3C H3C CH3 CH3 CH3 CH3 SO4 CH3 CH3 H H O H OH CH3 HO HO HO CH3 CH3 OH CH3 H2C CH3 CH3 H OH HO O H CH3 HO HO CH3 CH3 H2C CH3 O H H3C H
H2 C H2 C H2 C H2 C H2 C SO4 H2 C C H2 H2 C H2 C H2 C H2 C H2 C SO4

H3C

C H2

C H2

C H2

C H2

C H2

H2 C

C H2

H2 C

C H2

H2 C

C H2

H2 C

C H2

CH3

C H2

C H2

C H2

C H2

C H2

O H H OH

O H OH

O O H H H2C

CH3 HO

CH3

HO CH3 HO OH

CH3

OH

CH3 CH3

OH CH3

OH CH3

Application of solid state NMR spectroscopy to the analysis of the soil organic matter
Typical 13C-NMR spectra of natural organic matter in the solid state A wood A humic acid

200 ppm (t1)

150

100

50

0
ppm (f1)

300

200

100

-100

A protein (prion)

Carboxylic groups Aromatic systems Anomeric carbons C-O groups (i.e. Carbohydrates) C-N groups (i.e. peptides)

250 ppm (t1)

200

150

100

50

Aliphatic C

36

131 107 75 60

13C-CPMAS

176

Bulk HA 243 HA4 70 39 28 13 29 HA8 35 28 14 HA9 HA10 HA11 250 200 150 100 50 0

HA1

199

HA5

HA2

HA6

HA3 250 200 150 100 50 0

HA7 250 200 150 100 50 0

Chemical Shift (ppm)

49

69

Analyses of Tea leaves by CPMAS 13C-NMR and 1H-HRMAS

174 156 145 97 73 63

B 129 56

C 105 30

D 300 200 100 0 Chemical Shift (ppm) -100

I 300 200 100 0 Chemical Shift (ppm) -100

N 300 200 100 0 Chemical Shift (ppm) -100

A = Star Green Tea B = Gu Zhan Mao Jian Green Tea C = Sir Winston Green Tea D = ScelgoBio Green Tea

E = Star Black Tea F = PG-Tips Black Tea G = Gepa Black Tea H = Twinings Lemon Scented Black Tea I = Lipton Yellow Label Black Tea

J = Harrods n. 42 Earl Grey Tea K = Darjeeling Earl Grey Tea L = Twinings Earl Grey Tea M = GS Earl Grey Tea N = Infr Decaffeinated Tea

Analyses of Tea leaves by CPMAS 13C-NMR and 1H-HRMAS

A = Star Green Tea B = Gu Zhan Mao Jian Green Tea C = Sir Winston Green Tea D = ScelgoBio Green Tea

E = Star Black Tea F = PG-Tips Black Tea G = Gepa Black Tea H = Twinings Lemon Scented Black Tea I = Lipton Yellow Label Black Tea

J = Harrods n. 42 Earl Grey Tea K = Darjeeling Earl Grey Tea L = Twinings Earl Grey Tea M = GS Earl Grey Tea N = Infr Decaffeinated Tea

Analyses of Radish seeds during germination

24h

48h

72h

96h

Analyses of Radish seeds during germination

Cotyledon

Tegument

24h

48h Roots

72h

96h

Analyses of Radish seeds during germination: CPMAS 13C-NMR

0h

24h

48h

72h
200 ppm (t1) 150 100 50 0

Analyses of Radish seeds during germination: CPMAS 13C-NMR

Cotyledon

Cotyledon

0h

24h 72h
300 ppm (f1) 200 100 0

50 45 40 35 Content (%) 30 25 20 15 10 5 0 189-164 ppm 164-113 ppm 113-46 ppm 46-0 ppm 0h 24h 72h
-100

Analyses of Radish seeds during germination: CPMAS 13C-NMR

Tegument 0h

Tegument

24h

72h
300 ppm (f1) 200 100 0

70 60 50 Content (%) 40 30 20 10 0 189-164 ppm 164-113 ppm 113-46 ppm 46-0 ppm 0h 24h 72h
-100

Analyses of Radish seeds during germination: CPMAS 13C-NMR

Roots 0h

Root

24h

72h
300 ppm (f1) 200 100 0

70 60 50 Content (%) 40 30 20 10 0 189-164 ppm 164-113 ppm 113-46 ppm 46-0 ppm 0h 24h 72h
-100

Analyses of Radish seeds during germination: HRMAS 1H-NMR

Cotyledon

0h

24h

48h

72h

96h
10.0 ppm (t1) 5.0 0.0

Analyses of Radish seeds during germination: HRMAS 1H-NMR

Cotyledon
100 90 80 Content (%) 70 60 50 40 30 20 10 0 8-6 ppm 6-5 ppm 4.5-3.0 ppm 3.0-2.5 ppm 2.5-0 ppm 0h 24h 48h 72h 96h

Analyses of Radish seeds during germination: HRMAS 1H-NMR

Tegument

0h

24h

48h

96h
10.0 ppm (f1) 5.0 0.0

Analyses of Radish seeds during germination: HRMAS 1H-NMR

Tegument

40 35 30 Content (%) 25 20 15 10 5 0 8-6 ppm 6-5 ppm 4.5-3.0 ppm 3.0-2.5 ppm 2.5-0 ppm 0h 24h 48h 96h

Analyses of Radish seeds during germination: HRMAS 1H-NMR

Roots

0h 24h 48h 72h

96h
10.0 ppm (f1) 5.0 0.0

Analyses of Radish seeds during germination: HRMAS 1H-NMR

Roots

90 80 70 Content (%) 60 50 40 30 20 10 0 8-6 ppm 6-5 ppm 4.5-3.0 ppm 3.0-2.5 ppm 2.5-0 ppm 0h 24h 48h 72h 96h

Analyses of Radish seeds during germination: HRMAS 1H-NMR

Teguments 4.5-3.0 ppm 2.5-0 ppm

38 36 34 32 30 28 26 24 22 20 18 16 14 12 10 8 6 4 2

90 80 70 1H-HRMAS Content (%) 60 50 40 30 20 10 0

Cotyledons
4.5-3.0 ppm 2.5-0 ppm

1H-HRMAS Content (%)

24

48

96

20

40

60

80

100

Time (h)
Roots
90 80 4.5-3.0 ppm 2.5-0 ppm
80 70

Time (H)

1H-HRMAS Content (%)

70 1H-HRMAS Content (%) 60 50 40 30 20 10 0 0 24 48 Time (h) 72 96

60 50 40 30 20 10 -10 0 10 20 30 40 50 60

Total Seeds 4.5-3.0 ppm 2.5-0 ppm

70

80

Time (h)

Conclusions 1. NMR spectroscopy is widely used for structural analyses of natural products. Typical NMR spectra are obtained in liquid state by applying monodimensional sequences for measurements of proton and carbons, or bidimensional sequences useful to obtain HC(=X) correlations 2. Unconventional uses of NMR spectroscopy are those involving solid state samples and materials whose physical state is in between the solid and the liquid one. 3. Solid state NMR spectroscopy allows analyses of soil organic matter and all the systems (i.e. soils, minerals, polymer blends, pharmaceutycal products, and so on) hardly dissolving in the common deuterated solvents. 4. High resolution magic angle spinning (HRMAS) NMR spectroscopy is used for the analyses of complex mixtures such as plant residues or food products.

CERMANU