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Applicazioni della spettroscopia NMR in fasesolida e semisolida a problematiche agro-

alimentari ed ambientali

Pellegrino Conte

Dipartimento di Ingegneria e Tecnologie Agro-Forestali (DITAF)

pellegrino.conte@unipa.it

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preparation

pulse

acquisition

Direct detection

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CSA, strong dipolar couplings

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preparation

pulse

acquisition13C

1Hdecoupling

Elimination of the strong dipolar couplings

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Elimination of the Chemical Shift Anisotropy (CSA)

)1cos3(2

2

3

r

SI

S

h

Magic angle spinning (MAS)

= = = =

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Sensitivity enhancement by cross polarization

Cross polarization

pulse

acquisition13C

1Hdecoupling

ContactTime (CT)

ContactTime (CT)

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Static, no decoupling

Static, decoupling

MAS, no decoupling

MAS, decoupling and/or CP

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Uso della Spettroscopia NMR sui solidi: MAS

2.5 mm

4 mm

7 mm

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7 mm, 5000 Hz 4 mm, 13000 Hz

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Uso della Spettroscopia NMR sui semisolidi: HRMAS

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Uso della Spettroscopia NMR sui semisolidi: HRMAS

Aumento della sensibilit e della risoluzione

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Analisi di sostanza organica naturale

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Definition of Natural Organic Matter (NOM)

The decomposition of plant, animaland microbial material in soil andwater produces a variety of complex

organic molecules, collectively callednatural organic matter. Thesecompounds play many importantroles in the environment.

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Roles of Natural Organic Matter (NOM)

NOM is ubiquitous in soils, watersand sediments.

In agriculture, NOM is importantbecause of its positive effects onthe structure, water retentionand nutrient properties of soils.

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Roles of Natural Organic Matter (NOM)

NOM also interacts with metal ionsand minerals to form complexes ofwidely differing chemical and

biological nature.

Solubility, mobility and toxicity ofmany trace metals are stronglycorrelated with the concentration of

natural organic matter in soil andwater.

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Roles of Natural Organic Matter (NOM)

NOM interacts with organicpollutants (i.e. polycyclic aromatic

hydrocarbons, PAH) to reduce theirenvironmental availability and toxicity

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Roles of Natural Organic Matter (NOM)

Natural organic matter createsproblems for the water supplyindustry. It is required its removal inorder to minimize water color andeliminate formation of potentiallyharmful chemical byproducts as aresult of chlorination.

Through a process called "bio-fouling," natural organic matter is

also a major culprit in degrading theperformance of membrane filtrationsystems used for water purificationand desalination.

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Nature of Natural Organic Matter (NOM)

Persistent and universal naturalorganic matter molecules are in theenvironment, they are littleunderstood.

Natural organic matter has no uniquestructure or composition, cannot becrystallized and is extremely difficultto characterize.

Structure proposed by Flaig (1960)

Structure proposed by Stevenson (1982)

Structureproposed byStein (1997)

Structure proposed bySchulten (1993)

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Nature of Natural Organic Matter (NOM)

Natural Organic Matter is a complexmixture of several different self-

aggregatingcompounds formingsupramolecular aggregates.

The size of each molecular systemwithin NOM does not exceed 2000Da.

The new view (by Piccolo, 2001)

Weak dispersive forcesare mainlyinvolved in the conformationalstabilization of NOM

Saccharides

Peptides

Alkyl chains

Aromatic systems

Cations

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Importance of NMR in characterizing NOM

Solid state NMR(CPMAS 13C-NMR)

Liquid state NMR(13C-NMR)

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Hydrophobic core

Hydrophilic moiety

Water

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Study of humic substances by solid state NMR spectroscopy

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ppm (t2)-200-1000100200300400

=

CH

CH

CH

T

CTHT

T

HT

CT

HT

TItI

)(1

exp1)(

exp)(

1)(1

1

1

1

0

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TCH: 1. fast local motionshigh TCHvalues;2. high amount of protonslow TCHvalues

T1(H):proton spin lattice in the rotating frame; fast local motionsshorterT1(H) values. High proton concentrationfaster spin diffusion withshorter T1(H) values.

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Spin diffusion in natural organic matter

SO4-

12

34

56

78

910

1112

OH

O

OHO

CH3

12

34

5

6

78

9

10

CH3

CH3

CH3

CH3

CH3

CH3

Hexamethylbenzene (HMB)

Sodium dodecylsulphonate (SDS)

Ferulic Acid

12

3

45

O

OH

O

6

OH

O

CH2

7O

O

O

*

*

n

Carboxymethylcellulose sodium salt (CMC)

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Chemical Shift (ppm)

-200-1000100200300400

HMB

CMC

SDS

Ferulic Acid

Quaternary Aromatic C

SSB SSB

Methyl C

COO-C1

C2-C6

C1

C2-C9 C10

C11

C12

C9C3-C4

C7

SSB

C1-C2

C5, C6, C8

C10

OH

O

OHO

CH3

12

34

5

6

78

9

10

SO4-

12

34

56

78

910

1112

12

3

45

O

OH

O

6

OH

O

CH2

7O

O

O

*

*

n

CH3

CH3

CH3

CH3

CH3

CH3

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Effect of spin diffusion mediatedby water

CH3

CH3

CH3

CH3CH

3

CH3

CH3

CH3 CH2CH

3

CH3

OHOH

CH3

CH3

OH

OH

OH

OH

SO4

C

H2

CH

2

C

H2

CH

2

C

H2

CH

2

C

H2

CH

2

C

H2

CH

2

CH3

CH3

C

H2

CH

2

C

H2

CH

2

C

H2

CH

2

C

H2

CH

2

C

H2

CH

2

SO4

CH3

C

H2

CH

2

C

H2

CH

2

C

H2

CH

2

C

H2

CH

2

C

H2

CH

2

SO4

O

H

HO

H

H

CH3

CH3

CH3

CH3CH

3

CH3

CH3

CH3

CH2

CH3

CH3

OHOH

CH3

CH3

OH

OH

OH

OH

OH

H

OH

H

CH3

CH3

CH2

CH3

CH3

OHOH

CH3

CH3

OH

OH

OH

OH

OH

H

O

H

H

OH H

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Interactions between natural organic matter and heavy metals(i.e. Fe(III))

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Measurements of the relaxation: VCT experiments

=

CH

CH

CH

T

CTHT

T

HT

CT

HT

TItI

)(1

exp1)(

exp)(

1)(1

1

1

1

0

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Measurements of the relaxation

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Paramagnetic species reduce relaxation times,

thereby favouring reduction of NMR sensitivity.

In order to increase sensitivity and allow aquantitative evaluation of solid state NMR spectra

paramagnetism must be reduced.

The main paramagnetic ion in soils is Fe3+.

A Fe/C

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What does it mean?

(ppm)

-40-20020406080100120140160180200220240

Content of a chemical groupI

i/I

ix 100

Quantitative assessment of CPMAS 13C-NMR spectroscopy

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Standards Humic substances

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Solid state NMR in the differentiation of NOM of different origin

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Pure Lignites

Coal

Lignitestreated withgluconic acids

Fulvic acids

Humic acids

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Analisi di suoli

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Solid state NMR in the analyses of bulk soils

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A Chinese soil

An Italian soil

An Australian soil

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Studio della degradazione dei contaminanti organici

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Spettro CPMAS 13C NMR di un acido

umico da suolo addizionato di

fenantrene marcato al C1

Spettro CPMAS 13C NMR dellacido

umico da suolo

Spettro differenza (b)-(a)

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Il C1 del fenantrene risuona intorno a125 ppm

In presenza di un acido umico e dopo 89

giorni di incubazione, il carbonio

marcato si osserva non solo a 125 ppmma anche a 63, 73, 135, 150, e 190 ppm

La presenza di nuovi segnali indica che

il fenantrene si degradato. La

combinazione tra NMR e tecniche qualila MS consentono di concludere che i

residui di fenantrene si legano

stabilmente alla sostanza organica

naturale

Durante i processi di umificazione

contaminanti organici come gli IPA

perdono la loro tossicit perch

diventano essi stessi parte integrante

della sostanza organica naturale

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I processi di umificazione possonoessere utilizzati per la bonifica di suoli

contaminati

Pi in particolare lavaggio di suoli

contaminati da IPA con sostanzeorganiche naturali pu essere un

efficace metodo per la bonifica rapida

di siti inquinati

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Analisi di sistemi agroalimentari

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Analyses of cheeses by 1H-HRMAS spectroscopy

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soft Kraft

sheep aged mouldy

sheep aged

cow mozzarella

buffalo mozzarella

asiago

parmigiano

-1.5

-1

-0.5

0

0.5

1

-3 -2 -1 0 1 2 3

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Analyses of fruit by 1H-HRMAS spectroscopy

grapes

bananas

peaches

mangoes

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Studio dei processi di germinazione

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Analyses of Radish seeds during germination

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0

10

20

30

40

50

60

70

80

90

100

8-6 ppm 6-5 ppm 4.5-3.0

ppm

3.0-2.5

ppm

2.5-0 ppm

Content(%) 0h

24h

48h

72h

96h

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0

5

10

15

20

25

30

35

40

8-6 ppm 6-5 ppm 4.5-3.0

ppm

3.0-2.5

ppm

2.5-0 ppm

Content(%)

0h

24h

48h96h

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0

10

20

30

40

50

60

70

80

90

8-6 ppm 6-5 ppm 4.5-3.0

ppm

3.0-2.5

ppm

2.5-0 ppm

Cont

ent(%) 0h

24h

48h

72h

96h

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Analyses of fatty acids by GC-MS

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CONCLUSIONS

1. Solid state NMR spectroscopy is a very powerful tool to analyzehardly soluble natural organic matter

2. It is the only way to obtain quantitative information on the chemicalnature of NOM

3. It provides information on the chemical nature of organic matterdirectly in bulk soils

4. It can be used to study the interactions between NOM and theinorganic moieties of soils as well as the interactions withenvironmental pollutants

5. High Resolution MAS spectroscopy is very useful in analyzing

semisolid organic matter. It combines the advantages of solid stateNMR with those of the liquid state technique.

6. HRMAS is particularly powerful for the analyses of foods and planttissues.